Trichoderma enhances salt tolerance in Paeonia ostii ‘Fengdan’ by regulating the rhizosphere soil environment and physiological mechanisms

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Although plant growth-promoting fungi alleviate abiotic stress, the specific mechanisms by which they synergistically enhance salt tolerance through rhizosphere remodeling and physiological regulation remain unclear. Methods We evaluated the efficacy of Trichoderma inoculation on Paeonia ostii ‘Fengdan’ under varying NaCl concentrations (0, 100, 200, and 300 mM) via pot experiments. We comprehensively analyzed plant growth phenotypes, physiological indices, anatomical structures, and rhizosphere soil properties. Results Trichoderma inoculation markedly mitigated salt-induced damage, evidenced by increased biomass, optimized root system architecture (RSA), and enhanced photosynthetic capacity. Physiologically, Trichoderma reduced malondialdehyde (MDA) and ROS content by upregulating antioxidant enzymes and inducing osmolyte accumulation. Anatomically, inoculation maintained the integrity of vascular bundle structures in both roots and stems. Furthermore, Trichoderma colonization significantly improved the rhizosphere microenvironment by decreasing soil pH and electrical conductivity (EC) while stimulating urease, phosphatase, invertase, and dehydrogenase activities, thereby increasing nutrient bioavailability. Conclusions Collectively, Trichoderma confers salt tolerance to P. ostii ‘Fengdan’ through a synergistic mechanism involving the improvement of rhizosphere physicochemical properties, the remodeling of plant anatomical structures, and the activation of physiological metabolism. These findings provide a theoretical basis for cultivating P. ostii ‘Fengdan’ in saline-alkali lands and validate Trichoderma as a high-efficiency bio-inoculant for sustainable agriculture. Paeonia ostii ‘Fengdan’ Trichoderma Physiological characteristics Rhizosphere soil Salt stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Salt stress is one of the primary abiotic stressors limiting global agricultural productivity and ecosystem stability. Currently, approximately one-third of the world's soils are affected by varying degrees of salinization, a trend that is increasingly exacerbated by climate change (Zheng et al., 2020 ). High-salinity environments initially trigger osmotic stress and ion toxicity in roots, subsequently inducing oxidative stress. These processes disrupt metabolic homeostasis and the functionality of photosynthetic systems, leading to stomatal closure and reduced biomass accumulation, which ultimately severely inhibit plant growth and development or even cause mortality (Fu et al., 2023; Han et al., 2020 ; Wang et al., 2022 ). Consequently, exploring eco-friendly and efficient mitigation strategies to enhance crop salt tolerance has become an urgent imperative for sustainable agricultural development. Paeonia suffruticosa Andr. is not only a traditional and precious ornamental flower in China but also serves as the botanical origin of oil peony, an emerging woody oil-bearing crop. Specifically, Paeonia ostii ‘Fengdan’ the dominant cultivar of oil peony, has been certified as a vital novel food resource due to its high seed yield and superior oil quality, which is notably rich in α-linolenic acid (Lu et al., 2022 ). However, with the rapid expansion of the oil peony industry and the increasing scarcity of suitable arable land, cultivation areas are gradually shifting towards marginal soils, such as saline-alkali lands. Nevertheless, P. ostii ‘Fengdan’ is relatively sensitive to salinity. Salt stress-induced issues, such as growth retardation, chlorosis, and wilting, have severely constrained the economic viability of this industry (Shi et al., 2023 ; Wen et al., 2020 ). Therefore, enhancing the adaptive capacity of P. ostii ‘Fengdan’ in saline environments represents a critical issue that urgently requires resolution. Leveraging beneficial soil microorganisms to enhance plant adaptation to stressful environments is an environmentally friendly strategy. As a crucial group of symbiotic microorganisms, endophytic fungi are capable of colonizing host plant tissues and establishing mutualistic relationships, thereby exerting profound impacts on plant growth, development, and environmental adaptability (Nisa et al., 2015 ). Research indicates that these beneficial fungi not only directly promote growth by secreting phytohormones (Li et al., 2024 ) and solubilizing mineral elements (Li et al., 2015 ) but also assist hosts in combating abiotic stresses, such as drought and salinity, by inducing systemic resistance (Cui et al., 2024 ; Ueno et al., 2024 ). Compared with bacteria, filamentous fungi possess unique advantages in improving soil structure and nutrient cycling by forming extensive hyphal networks in the rhizosphere. This characteristic underpins their potential application in the ecological restoration of saline-alkali lands. Trichoderma spp. represents a class of ubiquitous and potent plant growth-promoting fungi (PGPF). Extensive research has demonstrated that Trichoderma can successfully colonize the plant rhizosphere and significantly alleviate salt stress symptoms in crops such as wheat (Zhang et al., 2016b ), rice (Anshu et al., 2022 ), and cucumber (Qi et al., 2013). These beneficial effects are primarily achieved by modulating osmolyte accumulation, activating antioxidant defense systems, and regulating the expression of stress-responsive genes. However, current studies on Trichoderma -mediated salt tolerance have predominantly focused on herbaceous crops or relied solely on physiological and biochemical indicators. There is a paucity of in-depth analyses conducted from a systemic dimension that integrates “rhizosphere soil microenvironment—plant anatomical structure—physiological metabolic function”. In particular, for the woody oil-bearing crop P. ostii ‘Fengdan’, it remains unclear whether Trichoderma can establish a defense against salinity by remodeling the root-stem vascular system structures and activating rhizosphere soil enzyme activities. Accordingly, this study employed P. ostii ‘Fengdan’ seedlings to investigate the multidimensional regulatory mechanisms by which Trichoderma enhances salt tolerance. In addition to a comprehensive assessment of macroscopic growth, photosynthetic gas exchange, and physiological metabolic homeostasis, we utilized microscopic techniques to characterize the adaptive changes in stomatal traits and root-stem anatomical structures. Concurrently, we investigated the response characteristics of rhizosphere soil physicochemical properties and enzyme activities. The findings of this research are intended to offer practical guidance for the ecological cultivation of oil peony in saline-alkali soils and to promote the application of Trichoderma inoculants as a sustainable strategy for alleviating salt stress in forestry and agriculture. Materials and methods Experimental materials Two-year-old seedlings of P. ostii ‘Fengdan’ were obtained from the Germplasm Resource Nursery of Peony and Herbaceous Peony of Shenyang Agricultural University. In April 2025, healthy, pest-free seedlings exhibiting uniform growth were transplanted into pots (14 cm diameter × 16.7 cm height) containing a standard nutrient substrate, with one seedling per pot. The pots were filled to approximately 1 cm below the rim. The baseline physicochemical properties of the soil were as follows: pH 6.99, electrical conductivity of 2.73 mS·cm -1 , ammonium nitrogen of 8.49 mg·kg -1 , available phosphorus of 20.36 mg·kg -1 , urease activity of 0.50 mg·g -1 d -1 , invertase activity of 33.43 mg·g -1 d -1 , phosphatase activity of 22.10 mg·g -1 d -1 , and dehydrogenase activity of 6.58 mg·g -1 d -1 . Subsequently, the potted plants were transferred to a greenhouse at the Botanical Garden of Shenyang Agricultural University for acclimatization and routine management. The greenhouse conditions were maintained at an average temperature of 23°C, a relative humidity of 55%, and a 14 h photoperiod. The compound Trichoderma inoculant was supplied by the Trichoderma and Bacillus Fertilizer Research and Development Team of Shenyang Agricultural University, containing an effective viable count of ≥ 1 × 10 8 CFU/g. Experimental treatments The study was conducted using a randomized block design consisting of 8 treatments (2 inoculation levels × 4 salt concentrations). In May 2025, following acclimatization, uniform P. ostii ‘Fengdan’ seedlings were selected. Inoculation was performed via root irrigation with a Trichoderma suspension (5 g·L -1 , 100 mL per pot), applied three times at 7-day intervals. Control plants received an equivalent volume of water. Seven days post-inoculation, salt stress was imposed using four NaCl gradients: 0, 100, 200, and 300 mM. To mitigate osmotic shock, salt solutions were applied in a stepwise increment manner (100 mL every 3 days) until the target concentrations were reached. Photosynthetic parameters were assessed after 14 days of sustained stress. Immediately thereafter, plant tissues were harvested, flash-frozen, and stored at -80°C for biochemical analysis. Each treatment included 30 seedlings, and the entire experiment was repeated three times to minimize environmental variability. Detailed treatment specifications are listed in Table 1. Table 1 Test treatments Treatment group Treatment reagent 0 mM NaCl CK Distilled water control T Trichoderma 100 mM NaCl CK 100 mM NaCl T Trichoderma +100 mM NaCl 200 mM NaCl CK 100 mM NaCl T Trichoderma +200 mM NaCl 300 mM NaCl CK 100 mM NaCl T Trichoderma +300 mM NaCl Measurements and methods Observation of plant morphology and measurement of growth parameters Following the salt stress treatment, plant morphological changes were observed daily between 8:00 and 9:00 AM. Key indicators, including leaf color, texture, and general condition, were recorded and documented photographically. Upon the conclusion of the experiment, three plants were randomly selected from each treatment. Plant height (vertical distance from the soil surface to the highest point) was measured using a steel tape (precision: 0.1 cm), and stem diameter was measured 2 cm above the soil surface using a Vernier caliper (precision: 0.01 cm). Subsequently, plants were harvested, washed, and surface-dried. The shoots were separated from the roots at the root-shoot junction. The fresh weights (FW) of the shoots and roots were determined using an analytical balance, and the root-to-shoot ratio was calculated. Root morphology was digitized using an Epson Expression 11000 XL scanner (Epson, Japan). The resulting images were analyzed using the WinRHIZO root analysis system (Regent Instruments Inc., Canada) to quantify parameters including total root length, projected area, surface area, average diameter, root volume, and the number of root tips. Root activity was determined using the TTC (2,3,5-triphenyltetrazolium chloride) reduction method (Zhang et al., 2014). Measurement of photosynthetic parameters Between 9:00 and 11:00 am when light was stable, a portable Li-6400 photosynthesis system was used to measure net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO 2 concentration (Ci), and stomatal conductance (Gs). Measurements were conducted under the following conditions: a leaf chamber area of 3 cm 2 , ambient CO 2 concentration, a standard flow rate of 750 μmol/s, and a photosynthetically active radiation (PAR) of 1200 μmol/m 2 ·s -1 provided by a red/blue LED light source (Zhang et al., 2023). Determination of physiological indices Relative electrical conductivity (REC) was measured using a conductivity meter following the method of Lutts et al. (1996). Malondialdehyde (MDA) content was determined via the thiobarbituric acid (TBA) assay (Velikova et al., 2000). Superoxide anion radicals (O 2 − ) and hydrogen peroxide (H 2 O 2 ) were assessed using a combination of qualitative localization and quantitative determination. For qualitative analysis, histochemical staining of leaves was performed using NBT and DAB, respectively, to visualize the in situ accumulation of O 2 − and H 2 O 2 (Yuan et al., 2024). For quantitative analysis, the production rate of O 2 − was determined by the hydroxylamine method (Jiang et al., 2001), while H 2 O 2 content was measured using the titanium sulfate (TiSO 4 ) method (Velikova et al., 2000). Soluble sugar (SS) content was determined using the anthrone colorimetric method. Soluble protein (SP) content was measured via the Coomassie Brilliant Blue G-250 assay. Proline content was determined using the acid ninhydrin colorimetric method (Ikram et al., 2019). Antioxidant enzyme activities were assayed according to the protocols of Wu et al. (2021), with slight modifications. Enzyme extraction: 0.2 g of fresh leaf samples was homogenized in 1 mL of 0.1 mol/L phosphate buffer (PBS, pH 7.0) and made up to a final volume of 2 mL. The homogenate was centrifuged at 10,000 × g for 20 min at 4°C, and the resulting supernatant served as the crude enzyme extract. Superoxide dismutase (SOD) activity was determined by the nitroblue tetrazolium (NBT) photoreduction method. Peroxidase (POD) activity was assayed using the guaiacol colorimetric method. Catalase (CAT) activity was measured by the UV absorption method, monitoring the change in absorbance at 240 nm. Observation of stomata and anatomical structures Three healthy, disease-free mature leaves were randomly selected from each treatment. The abaxial epidermis was peeled off using forceps to prepare temporary wet mounts. Observations were conducted under an optical microscope (Olympus CX-31; Olympus, Japan) using a 40 × objective lens. Ten fields of view were randomly selected and photographed for each treatment. Stomatal parameters, including length, width, density, and aperture, were measured using Motic Images Advanced 3.0 software. The anatomical structures of roots, stems, and leaves were observed using the conventional paraffin sectioning method, following the protocol of Nassar et al. (2020) with modifications. Fresh samples were washed and cut into segments (0.3 × 0.5 cm for roots and stems; 0.5 × 0.5 cm for leaves). The specimens were fixed in FAA fixative for over 24 h. Subsequently, the samples underwent dehydration through a graded ethanol series, clearing with a clearing agent, and infiltration with paraffin via a stepwise temperature process before being embedded. The specimens were sectioned using a microtome, dried at 42°C, stained with Safranin O-Fast Green, and mounted with neutral balsam. Determination of rhizosphere soil properties Plants were carefully removed from the pots, and the loosely adhering soil was shaken off. The rhizosphere soil, defined as the soil tightly adhering to the roots, was collected and mixed to form a composite sample. The sample was divided into two portions: one portion was stored fresh at -80°C for biological analysis, and the other was air-dried for the determination of physicochemical properties. Soil electrical conductivity (EC) and pH were measured using a conductivity meter and the potentiometric method, respectively. Soil available phosphorus (AP) content was determined using the molybdenum-antimony anti-spectrophotometric method (Tao et al., 2022). Soil ammonium nitrogen (NH 4 + -N) content was determined using the indophenol blue colorimetric method (Zhang et al., 2004). Soil urease activity was assayed using the sodium phenol-sodium hypochlorite colorimetric method by measuring absorbance at 578 nm. Soil phosphatase activity was determined using the disodium phenyl phosphate colorimetric method, with absorbance measured at 510 nm. Soil sucrase activity was assessed using the 3,5-dinitrosalicylic acid (DNS) colorimetric method, measuring absorbance at 508 nm (Han et al., 2020). Soil dehydrogenase activity was determined via the triphenyltetrazolium chloride (TTC) colorimetric method by monitoring absorbance at 492 nm (Olmos-Ruiz et al., 2025). Data analysis Data compilation and calculation were performed using Microsoft Excel 2019. Statistical analyses were conducted using IBM SPSS Statistics 22. One-way analysis of variance (ANOVA) was employed, followed by Duncan's multiple range test to determine significant differences among treatments. Figures were generated using GraphPad Prism 9.0. All data are presented as means ± standard deviation (SD). Results Plant growth performance and root system architecture Salt stress induced dose-dependent morphological damage in P. ostii ‘Fengdan’. Plants exposed to low salinity exhibited distinct foliar yellowing, while those under high concentrations displayed severe marginal necrosis, leaf rolling, and loss of turgor (Fig. 1 A). In contrast, Trichoderma inoculation effectively alleviated these symptoms. Plants receiving Trichoderma inoculation exhibited a robust phenotype, characterized by expanded leaf blades, vibrant green foliage, and sustained turgidity. Quantitatively, Trichoderma demonstrated dual efficacy by promoting growth under both non-saline and saline conditions (Fig. 1 B–D). While salt stress significantly inhibited growth metrics in controls, the inoculated group consistently maintained superior performance. Notably, the mitigation effect peaked at 200 mM NaCl, where plant height, stem diameter, and root-to-shoot ratio significantly increased by 27.99%, 23.08%, and 19.30%, respectively, compared to the corresponding control. Regarding root system architecture (RSA), Trichoderma inoculation comprehensively improved root multidimensional traits (Fig. 1 E and Table 2 ). Under non-saline conditions, the fungus elicited a robust direct growth-promoting effect, increasing total root length by 33.73%, root surface area by 69.83%, and root tip number by 51.86%. Furthermore, Trichoderma inoculation significantly thickened the root system, boosting projected area, root volume, and average diameter by 41.79%, 38.59%, and 14.70%, respectively. Under salt stress, the alleviative effect peaked at 100 mM NaCl. At this concentration, inoculated plants exhibited a more developed root architecture than saline controls, with increases of 17.50% in total length, 54.95% in surface area, and 41.45% in root tips. Crucially, the absorptive capacity was preserved, as evidenced by simultaneous enhancements of 31.98% in projected area, 32.70% in root volume, and 13.49% in average diameter. However, this protective efficacy exhibited a physiological threshold, as the beneficial impacts on both shoot and root traits were attenuated under severe salinity at 300 mM NaCl. Table 2 Effects of Trichoderma inoculation on root system parameters of P. ostii ‘Fengdan’ under salt stress Treatments Total root length/cm Projected Area/cm 2 Root surface area/cm 2 Average diameter/mm Volume/mm 3 Tips 0 mM NaCl CK 234.74 ± 8.97 b 18.47 ± 0.92 b 44.12 ± 2.79 c 0.61 ± 0.02 b 10.12 ± 0.22 b 90.67 ± 2.08 c T 313.93 ± 11.31 a 26.19 ± 1.05 a 74.94 ± 3.88 a 0.70 ± 0.01 a 14.02 ± 0.15 a 137.67 ± 1.53 a 100 mM NaCl CK 211.13 ± 6.45 c 14.06 ± 0.83 c 34.35 ± 1.91 d 0.53 ± 0.02 c 7.70 ± 0.66 d 78.00 ± 2.00 d T 248.07 ± 5.82 b 18.55 ± 0.53 b 53.23 ± 4.08 b 0.61 ± 0.02 b 10.21 ± 0.22 b 110.33 ± 1.53 b 200 mM NaCl CK 175.75 ± 9.01 d 10.33 ± 1.20 d 26.18 ± 2.34 e 0.52 ± 0.01 c 6.35 ± 0.61 e 58.67 ± 4.51 e T 204.60 ± 12.50 c 13.09 ± 0.92 c 35.17 ± 1.77 d 0.58 ± 0.02 b 8.51 ± 0.37 c 79.00 ± 1.00 d 300 mM NaCl CK 149.05 ± 5.92 e 8.09 ± 0.32 e 24.18 ± 2.64 e 0.48 ± 0.03 d 4.63 ± 0.20 f 51.67 ± 2.08 f T 174.05 ± 8.71 d 9.53 ± 0.53 de 27.52 ± 1.21 e 0.51 ± 0.01 cd 5.59 ± 0.34 e 61.67 ± 2.52 e Data are presented as means ± SD (n = 3). CK : non-inoculated control; T : Trichoderma -inoculated treatment. Different lowercase letters indicate significant differences among treatments ( P < 0.05) Anatomical adaptations of roots and stems Microscopic analysis illustrated that salt stress caused systemic anatomical atrophy in P. ostii ‘Fengdan’, specifically targeting the vascular transport system. In the stems of the control group, increasing salinity levels led to a progressive thinning of the vascular ring and restricted secondary xylem development. With the escalation of NaCl concentration, vascular tissues gradually exhibited a loose arrangement accompanied by a significant reduction in vessel caliber (Fig. 2 A). Mirroring the stem response, root tissues in the control group exhibited marked stele atrophy and retarded secondary growth, characterized by scattered vessels and diminished xylem area (Fig. 2 B). In contrast, Trichoderma inoculation effectively protected the vascular structure of both stems and roots. Regardless of salinity levels, inoculated plants maintained a robust vascular system. In stems, Trichoderma inoculation induced the formation of broader secondary xylem zones and a continuous, compact xylem ring, even under severe salt stress at 300 mM NaCl. Concurrently, roots of inoculated plants displayed a plump stele with well-developed secondary xylem. A key anatomical feature observed in the inoculated group was the significantly increased number and enlarged caliber of vessels in both stems and roots, which facilitates efficient water transport. Furthermore, histochemical observations showed intensified deep purple-red safranin staining in the xylem vessel walls of inoculated plants, indicating an enhanced degree of lignification and mechanical strength compared to the saline controls. Stomatal characteristics and adaptive regulation Microscopic observations and parametric analysis (Fig. 3 , Table 3 ) indicated that Trichoderma inoculation effectively mitigated salt-induced stomatal distortion. In the control group, salt stress caused pronounced morphological aberrations. Guard cells exhibited severe dehydration and shrinkage, leading to extensive pore closure. This resulted in a significant reduction in the stomatal short axis and an increase in the length-to-width ratio, presenting a distinct "narrow and elongated" closure phenotype. Trichoderma inoculation reversed this trend, increasing the short axis by 13.49%, 14.63%, and 11.48% under 100, 200, and 300 mM NaCl, respectively, compared to corresponding controls. Stomatal aperture was enhanced across salinity gradients from 0 to 300 mM, with increases of 6.80%, 7.63%, 18.46%, and 5.58%, respectively. Furthermore, Trichoderma inoculation exhibited a bidirectional regulation on stomatal density, increasing it by 11.98% under non-saline conditions while reducing it by 9.34%, 10.61%, and 4.88% under salt stress. Table 3 Effects of Trichoderma inoculation on stomatal parameters of P. ostii ‘Fengdan’ under salt stress Treatments Stomatal long axis/µm Stomatal short axis/µm length-to-width ratio Stomatal aperture/µm Stomatal density/mm − 2 Stomatal index/% 0 mM NaCl CK 24.54 ± 0.72 a 15.52 ± 1.27 ab 1.59 ± 0.17 b 3.84 ± 0.08 b 238.97 ± 9.65 d 18.40 ± 0.26 b T 24.63 ± 1.05 a 16.11 ± 0.72 a 1.53 ± 0.02 b 4.10 ± 0.07 a 267.60 ± 3.22 bc 19.47 ± 0.06 a 100 mM NaCl CK 24.31 ± 0.51 a 14.20 ± 0.73 bc 1.71 ± 0.11 a 3.40 ± 0.02 c 261.83 ± 8.80 c 19.28 ± 0.20 a T 24.64 ± 0.05 a 16.12 ± 0.49 a 1.53 ± 0.05 b 3.69 ± 0.05 b 237.37 ± 5.95 d 19.22 ± 0.12 a 200 mM NaCl CK 23.79 ± 0.08 a 13.70 ± 0.61 cd 1.74 ± 0.07 a 2.66 ± 0.24 e 302.20 ± 5.82 a 19.41 ± 0.07 a T 24.44 ± 0.27 a 15.71 ± 0.91 ab 1.56 ± 0.11 b 3.15 ± 0.10 d 270.13 ± 7.74 bc 19.42 ± 0.04 a 300 mM NaCl CK 23.55 ± 0.66 a 12.32 ± 1.03 d 1.92 ± 0.14 a 2.56 ± 0.06 e 295.30 ± 3.60 a 19.37 ± 0.05 a T 24.20 ± 0.63 a 13.74 ± 0.88 cd 1.73 ± 0.14 a 2.69 ± 0.05 e 280.90 ± 7.04 b 19.35 ± 0.07 a Note : Data are presented as means ± SD (n = 3). CK : non-inoculated control; T : Trichoderma -inoculated treatment. Different lowercase letters indicate significant differences among treatments ( P < 0.05) Photosynthetic performance and photosynthetic pigments Salt stress significantly suppressed gas exchange processes, leading to dose-dependent declines in net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO 2 concentration (Ci). However, Trichoderma inoculation effectively alleviated this inhibition (Fig. 4 A–D). Under non-saline conditions, Trichoderma inoculation enhanced physiological performance, increasing Pn, Tr, Gs, and Ci by 23.79%, 21.32%, 31.0%, and 17.92%, respectively. Under salt stress, the alleviation effect peaked at 100 mM NaCl, where Pn, Tr, Gs, and Ci rose by 27.02%, 40.38%, 46.84%, and 19.71%, respectively, relative to the corresponding control. Although the magnitude of improvement diminished slightly with rising salinity, the inoculated group consistently maintained significantly higher indices. Regarding photosynthetic pigments, salt stress induced a significant reduction in chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll (Chl), and carotenoids (Car). Trichoderma inoculation significantly alleviated the reduction in these pigments (Fig. 4 E–H). Under non-saline conditions, basal levels of Chl a, Chl b, Chl, and Car increased by 13.3%, 43.3%, 21.8%, and 14.7%, respectively. Under salt stress, the enhancement effect was most pronounced at 100 mM NaCl, with increases of 32.6%, 18.4%, and 19.8% for Chl b, Chl, and Car, respectively. Even under severe salinity of 300 mM NaCl, where pigment biosynthesis was strongly inhibited, Chl a content in the inoculated group showed a relative increase of 16.9%. Reactive oxygen species accumulation and antioxidant enzyme activities Histochemical in situ staining combined with quantitative analysis revealed the accumulation patterns of reactive oxygen species (ROS) in plants under salt stress. In the control group, the intensity and extent of reddish-brown precipitates (indicating H 2 O 2 ) and blue-black precipitates (indicating O 2 − ) gradually increased with rising salt concentrations (Fig. 5 A, B). Under 200 mM and 300 mM treatments, the leaves exhibited extensive dark staining areas. Consistent with histochemical observations, the H 2 O 2 content and O 2 − production rate in control plants increased significantly with salt concentration (Fig. 5 C, D). Conversely, Trichoderma inoculation significantly reduced ROS accumulation levels. This reduction was most pronounced under low-to-moderate salinity levels of 100 mM and 200 mM NaCl. In these treatments, the H 2 O 2 content in inoculated leaves decreased significantly by 55.08% and 43.42%, respectively, while the O 2 − production rate declined by 22.08% and 21.75%, respectively. Even under severe salinity of 300 mM NaCl, ROS levels in the inoculated group remained significantly lower than those in the control group. Regarding antioxidant defense, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) exhibited a trend of initial increase followed by a subsequent decline with increasing salt concentrations, peaking at 200 mM NaCl (Fig. 5 E–G). Trichoderma inoculation maintained higher levels of enzyme activities across all salinity gradients. The maximal relative increase occurred under the 100 mM NaCl treatment, where the activities of SOD, POD, and CAT increased by 21.92%, 30.68%, and 95.24%, respectively. With a further increase in salinity, although the intrinsic enzyme activities declined, the inoculated group sustained significantly higher levels compared to the control group. Membrane stability and osmotic adjustment Salt stress induced a dose-dependent increase in relative electrical conductivity (REC) and malondialdehyde (MDA) content. However, Trichoderma inoculation maintained these metrics at lower levels compared to the control group (Fig. 6 A, B). Under non-saline conditions, no significant differences were observed in membrane indices between the inoculated and control groups. Under salt stress, Trichoderma inoculation suppressed the elevation of membrane damage indices. Specifically, compared to the corresponding control group, REC in the treated group decreased by 23.79%, 20.22%, and 13.08% at 100, 200, and 300 mM NaCl treatments, respectively. Similarly, MDA content exhibited reductions of 51.89%, 38.31%, and 29.93%, respectively, relative to the control. Regarding osmoregulation, the contents of soluble sugar (SS), soluble protein (SP), and proline (Pro) exhibited a dose-dependent increase as salt concentrations rose. Trichoderma inoculation further elevated the accumulation of these three osmolytes (Fig. 6 C–E). Under non-saline conditions, the basal levels of SS, SP, and Pro in the leaves of the treated group increased by 45.68%, 32.43%, and 32.0%, respectively. Under salt stress, the increase in SS and SP contents peaked at 100 mM NaCl treatment, showing elevations of 91.49% and 41.12% relative to the control. For Pro, the maximum increase of 57.7% was observed at 200 mM NaCl treatment. Even under severe salinity of 300 mM NaCl, the inoculated group maintained significantly higher levels of osmolytes. Rhizosphere soil physicochemical properties and enzyme activities Salt stress significantly elevated soil pH and electrical conductivity (EC), but Trichoderma inoculation consistently mitigated these increases (Fig. 7 A, B). Specifically, the treatment reduced pH by 4.09% and 4.13% under 200 and 300 mM NaCl, respectively, and achieved a maximum EC reduction of 28.63% at 200 mM NaCl. Conversely, Trichoderma inoculation enhanced the contents of ammonium nitrogen (NH 4 + -N) and available phosphorus (AP) (Fig. 7 C, D). Under non-saline conditions, Trichoderma inoculation increased NH 4 + -N and AP by 18.24% and 24.71%, respectively. Under salt stress, the enhancement effect peaked at 200 mM NaCl, rising by 21.19% and 27.45%, respectively, with significantly higher levels maintained even at 300 mM NaCl. Regarding enzyme activities, salt stress inhibited urease and phosphatase in control, causing reductions of 55.09% and 23.39% at 300 mM NaCl, respectively (Fig. 7 E, F). Trichoderma inoculation enhanced basal activities of urease and phosphatase by 29.65% and 29.02% under non-saline conditions. Under salt stress, the increase in urease activity peaked at 34.28% under 200 mM NaCl, while phosphatase activity showed a maximum increase of 33.43% under 100 mM NaCl. Additionally, sucrase activity in the inoculated group peaked at 200 mM NaCl with a 45.14% increase (Fig. 7 G), while dehydrogenase activity showed a rising enhancement trend with salinity, reaching a maximum increase of 37.18% at 300 mM NaCl (Fig. 7 H). Discussion Soil salinization severely limits the cultivation of P. ostii ‘Fengdan’. Our study demonstrates that Trichoderma inoculation effectively mitigates salt-induced systemic damage. Mechanistically, Trichoderma ameliorates the rhizosphere environment by improving soil physicochemical properties and enzyme activities, while simultaneously preserving anatomical integrity. These findings demonstrate that Trichoderma confers salt tolerance to P. ostii ‘Fengdan’ through the coordination of rhizosphere remediation and plant adaptive responses, providing a viable strategy for the cultivation of this crop in saline environments. Beneficial microorganisms are pivotal in enhancing plant resilience to abiotic stress. In this study, Trichoderma inoculation significantly mitigated salt-induced morphometric restrictions in P. ostii ‘Fengdan’, conferring superior plant height, stem diameter, and biomass accumulation. These results corroborate previous findings in sweet sorghum (Wei et al., 2023 ) and wheat (Zhang et al., 2016a ), where Trichoderma colonization promoted robust host growth. Crucially, this vegetative improvement is intrinsically linked to the root system. While salt stress typically compromises root plasticity by inhibiting elongation and lateral branching (Koevoets et al., 2016 ), Trichoderma inoculation effectively reversed these inhibitory effects, significantly increasing total root length, surface area, and root tip number. This optimization of root system architecture (RSA) aligns with Rouphael et al. ( 2017 ), suggesting that the enhanced root expansion facilitates efficient water and nutrient acquisition from deeper soil layers. Mechanistically, this remodeling is likely driven by Trichoderma -induced auxin accumulation in root primordia, which stimulates lateral root development to counteract salt-induced growth stagnation (Contreras-Cornejo et al., 2014 ). While an optimized root architecture facilitates water acquisition, the efficient delivery of these resources to aerial tissues fundamentally relies on the anatomical integrity of the internal vascular system. In this study, Trichoderma inoculation effectively optimized the systemic water transport network connecting roots, stems, and leaves. Specifically, in roots and stems, salt stress caused stele atrophy and narrowed vascular rings, whereas inoculated plants maintained robust steles and broader secondary xylem zones, and possessed larger vessel diameters. This preservation of vascular geometry was likely reinforced by enhanced cell wall lignification, which ensured efficient upward water transport and mechanical resilience against stress-induced negative pressure. These observations align with Basinska-Barczak et al. ( 2020 ), who reported that microbe-induced lignin accumulation mechanically reinforces vessels in wheat. Consistent with the findings of S. Taha et al. ( 2020 ) in lupine and Palupi et al. (2023) in Indian mustard, the maintenance of vascular diameter in inoculated P. ostii ‘Fengdan’ plants is crucial for alleviating physiological drought. Crucially, this secured water supply translated into superior stomatal regulation. Salt stress typically causes guard cells to undergo dehydration and shrinkage, leading to functional closure. However, inoculated plants retained turgid, kidney-shaped guard cells and significantly wider apertures. This is similar to the findings of Jing et al. ( 2023 ), who demonstrated that microbial inoculation mitigates drought-induced stomatal closure in walnut, thereby sustaining gas exchange. Photosynthesis acts as the engine for biomass accumulation but is highly susceptible to salt stress via both stomatal and non-stomatal limitations (Acosta-Motos et al., 2017 ). We observed that salt stress significantly reduced the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO₂ concentration (Ci). However, Trichoderma inoculation alleviated this inhibition, enabling plants to maintain higher levels of gas exchange across all salinity levels. This result is consistent with Han et al. ( 2017 ), who demonstrated that microbial inoculation enhances carbon assimilation capacity by regulating stomatal behavior in Codonopsis pilosula . Beyond gas exchange, pigment stability is vital. In our study, salt stress induced a significant reduction in chlorophyll a, chlorophyll b, and carotenoids. Conversely, Trichoderma inoculation significantly attenuated this pigment degradation. This aligns with Patani et al. ( 2023 ), who found that inoculation with salt-tolerant plant growth-promoting rhizobacteria preserved leaf chlorophyll content in tomato. This protective effect is likely attributed to the ability of the fungal inoculant to maintain chloroplast membrane integrity by mitigating oxidative stress, thereby retarding the oxidative decomposition of photosynthetic pigments. The fundamental mechanism underlying this protection against oxidative stress lies in the modulation of reactive oxygen species (ROS) homeostasis. Salt stress disrupts cellular homeostasis by inducing a burst of ROS, leading to oxidative stress and cell death (Miller et al., 2010 ). In this study, histochemical staining revealed extensive ROS accumulation in stressed control leaves, manifested as reddish-brown and blue-black precipitates. However, Trichoderma inoculation significantly attenuated this oxidative load. Quantitative analysis confirmed that the H 2 O 2 content and O 2 − production rate were significantly reduced in inoculated plants. This ROS scavenging capability is mechanistically attributed to the activation of the antioxidant enzyme system. Our data showed that Trichoderma upregulated the activities of SOD, POD, and CAT, forming an efficient scavenging network. These results align with Chen et al. ( 2019 ) in cucumber and Guler et al. ( 2016 ) in maize, where beneficial microbes mitigated oxidative damage by boosting enzymatic defense. Expanding on this mechanism, Zhang et al. ( 2024 ) and Khan et al. ( 2023 ) further elucidated that such upregulation of antioxidant enzymes serves as a trigger to induce systemic resistance. Consequently, the reduction in ROS levels directly preserved the integrity of the cell membrane system. Salt stress typically exacerbates membrane lipid peroxidation, indicated by elevated relative electrical conductivity (REC) and malondialdehyde (MDA) content. However, Trichoderma inoculation significantly suppressed the rising trend of these indicators, effectively alleviating cell membrane damage. This corroborates the findings of Sofy et al. ( 2022 ) which demonstrated that Trichoderma reduces electrolyte leakage and MDA accumulation by enhancing antioxidant activity. Parallel to enzymatic defense, the accumulation of osmolytes is a critical strategy for maintaining cellular water balance. We found that Trichoderma further promoted the accumulation of soluble sugars, soluble proteins, and proline under salt stress. This enhancement of osmoregulatory capacity aligns with Ahmad et al. ( 2015 ) in mustard, suggesting that Trichoderma -mediated osmolyte accumulation mitigates ion toxicity and stabilizes membrane structures, thereby contributing to improved salt tolerance. The degradation of soil physicochemical properties and the inhibition of rhizosphere microbial functions constitute major constraints on plant growth under saline conditions. In this study, salt stress induced a marked elevation in soil pH and electrical conductivity (EC); however, Trichoderma inoculation effectively counteracted these increases, stabilizing the rhizosphere environment. This amelioration aligns with Wu et al. ( 2024 ), who demonstrated that optimizing the soil physicochemical environment via beneficial microbes enhances water and nutrient uptake. Furthermore, the decline in soil EC lends support to the mechanism proposed by Ruiz-Lozano et al. ( 2012 ), wherein the extensive fungal hyphal network sequesters and compartmentalizes salt ions within vacuoles, thereby lowering the ionic concentration of the rhizosphere soil solution. Beyond these physicochemical improvements, soil enzyme activity—a pivotal indicator of soil quality and metabolic function—was significantly revitalized. Trichoderma inoculation upregulated the activities of soil urease, phosphatase, invertase, and dehydrogenase. This enzymatic enhancement aligns with Chaudhary et al. ( 2025 ) and the findings of Khalifa et al. ( 2021 ) in maize under saline-alkali environments, confirming that microbial inoculants boost metabolic function under stress. Accompanied by this enzymatic activation, we found that the contents of soil ammonium nitrogen (NH 4 + -N) and available phosphorus also increased significantly. These results demonstrate that remodeling the rhizosphere bio-physicochemical environment and enhancing soil nutrient availability constitute key mechanisms by which Trichoderma alleviates salt stress. Conclusion This study demonstrates that Trichoderma inoculation enhances salt tolerance in P. ostii ‘Fengdan’ through the synergistic amelioration of the rhizosphere environment and physiological regulation. Specifically, Trichoderma optimized the soil microenvironment by reducing soil pH and electrical conductivity (EC) while boosting soil enzyme activities. Simultaneously, it mitigated oxidative stress by enhancing antioxidant defenses and stimulating osmolyte accumulation. Anatomically, the treatment preserved the structural integrity of roots, stems, and stomata, thereby securing water transport and photosynthetic efficiency. Collectively, these adaptive changes significantly improved plant growth, providing a theoretical basis for the microbial-assisted cultivation of peonies in saline regions. Declarations Competing Interests On behalf of all authors, the corresponding author states that there is no conflict of interest. Funding This work was supported by the National Natural Science Foundation of China, 32071814 and 31470696. Author Contributions Dairu Jiang contributed toward writing—original draft, visualization, validation, software, resources, project administration, methodology, and data curation. Shixin Guan contributed toward writing—review & editing, and project administration. Xuening Kang contributed toward validation and data curation. Ayimukezi Maimaitizunong contributed toward validation and data curation. Zhong Chen contributed toward validation and data curation. Yanxin Gu contributed toward validation and data curation. 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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-8707138","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":589834139,"identity":"05189b41-214a-414e-8dc3-5b8e4a08a4a2","order_by":0,"name":"Dairu Jiang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dairu","middleName":"","lastName":"Jiang","suffix":""},{"id":589834140,"identity":"e25bdb5c-8e63-4a84-a6ed-46a6c968fc1c","order_by":1,"name":"Shixin Guan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shixin","middleName":"","lastName":"Guan","suffix":""},{"id":589834141,"identity":"ed0cc345-c078-4f8b-bfb0-af220e922073","order_by":2,"name":"Xuening Kang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xuening","middleName":"","lastName":"Kang","suffix":""},{"id":589834142,"identity":"cef37a60-9e88-450d-96fb-cf4ce657d0a9","order_by":3,"name":"Ayimukezi Maimaitizunong","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ayimukezi","middleName":"","lastName":"Maimaitizunong","suffix":""},{"id":589834143,"identity":"6470291f-bf0c-4b69-a9c1-c8010a6439ab","order_by":4,"name":"Zhong Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhong","middleName":"","lastName":"Chen","suffix":""},{"id":589834144,"identity":"5445d232-8d23-423d-8f79-fa3da5defeb6","order_by":5,"name":"Yanxin Gu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yanxin","middleName":"","lastName":"Gu","suffix":""},{"id":589834145,"identity":"77cd5132-8e6f-439c-b059-b00653f50509","order_by":6,"name":"Xiaomei Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYHAC9h8fKiTkoBxm4vRIzjhjY0yaFmnetrTEBqK1yM/ITjDgOXM4ff6M5GcSDBXWiQ3sZw/g1WJw5uyGBImKw7kbbqSZSTCcSU9s4MlLwK+FvXfDAYMzQC0SCWYSjG2HExskeAzwO6yZd2NDYtvhdPkZ6d8kGP8RoYXheO9mhoNtaQkMN3KAtjQQoQXol22MDWdsDDeceVNskXAs3biNJ4eAw2bkbmP+UyEhL9+evvHGhxpr2X72MwQcBgcCCQwMQMTARqR6IOA/QLzaUTAKRsEoGFkAADnpSJr2PqpjAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1782-7341","institution":"Shenyang Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Xiaomei","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2026-01-27 07:32:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8707138/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8707138/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102854731,"identity":"3f78b52b-1f7e-4413-a114-37bea0455d7a","added_by":"auto","created_at":"2026-02-17 14:50:46","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":199213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTrichoderma\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e inoculation on growth phenotype, root system architecture, and growth parameters of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. ostii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ‘Fengdan’ under salt stress. (A) \u003c/strong\u003ePlant phenotype photographs; \u003cstrong\u003e(B) \u003c/strong\u003ePlant height; \u003cstrong\u003e(C) \u003c/strong\u003eDiameter;\u003cstrong\u003e (D) \u003c/strong\u003eRoot-to-shoot ratio; \u003cstrong\u003e(E) \u003c/strong\u003eScanned images of root systems.\u003cstrong\u003e \u003c/strong\u003eData are presented as means ± SD (n=3). \u003cstrong\u003eCK:\u003c/strong\u003enon-inoculated control; \u003cstrong\u003eT:\u003c/strong\u003e \u003cem\u003eTrichoderma\u003c/em\u003e-inoculated treatment. Different lowercase letters indicate significant differences within the CK group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05); Different uppercase letters indicate significant differences within the T group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Asterisks indicate significant differences between CK and T groups at the same salt concentration (ns, not significant; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8707138/v1/8711a7f814aa7057e8b224bb.jpeg"},{"id":102854727,"identity":"b5544320-edca-47e0-a93a-a1888ca8f389","added_by":"auto","created_at":"2026-02-17 14:50:46","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":371734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTrichoderma\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e inoculation on anatomical characteristics of stems and roots of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. ostii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ‘Fengdan’ under salt stress.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Transverse sections of stems; \u003cstrong\u003e(B)\u003c/strong\u003e Transverse sections of roots. \u003cstrong\u003eCK:\u003c/strong\u003e non-inoculated control; \u003cstrong\u003eT:\u003c/strong\u003e \u003cem\u003eTrichoderma\u003c/em\u003e-inoculated treatment\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8707138/v1/78299c7f529b979b14dc5fa3.jpeg"},{"id":102854729,"identity":"fef39f31-3b5d-4850-aa97-63f8d8ccb6d8","added_by":"auto","created_at":"2026-02-17 14:50:46","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":253895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTrichoderma\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003einoculation on leaf stomatal morphology of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. ostii \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e‘Fengdan’ under salt stress\u003c/strong\u003e. \u003cstrong\u003eCK:\u003c/strong\u003e non-inoculated control; \u003cstrong\u003eT:\u003c/strong\u003e \u003cem\u003eTrichoderma\u003c/em\u003e-inoculated treatment\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8707138/v1/7fe590503685c3deaa719c53.jpeg"},{"id":102854728,"identity":"36e4f7e8-3398-4017-b759-69ef197dfd4e","added_by":"auto","created_at":"2026-02-17 14:50:46","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":353671,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTrichoderma\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e inoculation on gas exchange parameters and photosynthetic pigments of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. ostii \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e‘Fengdan’ under salt stress. (A) \u003c/strong\u003eNet photosynthetic rate\u003cstrong\u003e; (B) \u003c/strong\u003eTranspiration rate\u003cstrong\u003e; (C) \u003c/strong\u003eStomatal conductance\u003cstrong\u003e; (D) \u003c/strong\u003eIntercellular CO₂ concentration;\u003cstrong\u003e (E)\u003c/strong\u003e Chlorophyll a content\u003cstrong\u003e; (F) \u003c/strong\u003eChlorophyll b content\u003cstrong\u003e; (H) \u003c/strong\u003eChlorophyll content;\u003cstrong\u003e (G)\u003c/strong\u003e Carotenoids content\u003cstrong\u003e.\u003c/strong\u003eData are presented as means ± SD (n=3). \u003cstrong\u003eCK:\u003c/strong\u003e non-inoculated control; \u003cstrong\u003eT:\u003c/strong\u003e \u003cem\u003eTrichoderma\u003c/em\u003e-inoculated treatment. Different lowercase letters indicate significant differences within the CK group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05); Different uppercase letters indicate significant differences within the T group (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05). Asterisks indicate significant differences between CK and T groups at the same salt concentration (ns, not significant; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8707138/v1/fb051c2631ad919c8a78b9dd.jpeg"},{"id":102854734,"identity":"99a2b2e7-2245-45a6-afdb-95e582153699","added_by":"auto","created_at":"2026-02-17 14:50:46","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":377070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTrichoderma\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e inoculation on ROS accumulation and antioxidant enzyme activities in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. ostii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ‘Fengdan’ leaves under salt stress.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Histochemical staining of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e; \u003cstrong\u003e(B)\u003c/strong\u003e Histochemical staining of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e;\u003cstrong\u003e (C)\u003c/strong\u003e H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e contents; \u003cstrong\u003e(D)\u003c/strong\u003e O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e production rate; \u003cstrong\u003e(E)\u003c/strong\u003e SOD activity; \u003cstrong\u003e(F)\u003c/strong\u003e POD activity; \u003cstrong\u003e(G)\u003c/strong\u003e CAT activity. Data are presented as means ± SD (n=3). \u003cstrong\u003eCK:\u003c/strong\u003e non-inoculated control; \u003cstrong\u003eT:\u003c/strong\u003e \u003cem\u003eTrichoderma\u003c/em\u003e-inoculated treatment. Different lowercase letters indicate significant differences within the CK group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05); Different uppercase letters indicate significant differences within the T group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Asterisks indicate significant differences between CK and T groups at the same salt concentration (ns, not significant; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8707138/v1/69f7fa2ca66f4f770d98427e.jpeg"},{"id":102963605,"identity":"f59e8577-4361-4304-af40-84805087c584","added_by":"auto","created_at":"2026-02-19 04:19:18","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":167292,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTrichoderma\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e inoculation on membrane stability indices and osmolyte contents in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. ostii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ‘Fengdan’ leaves under salt stress. (A) \u003c/strong\u003eRelative electrical conductivity; \u003cstrong\u003e(B) \u003c/strong\u003eMalondialdehyde content;\u003cstrong\u003e(C) \u003c/strong\u003eSoluble sugar;\u003cstrong\u003e (D)\u003c/strong\u003e Soluble protein; \u003cstrong\u003e(E) \u003c/strong\u003eProline content. Data are presented as means ± SD (n=3). \u003cstrong\u003eCK:\u003c/strong\u003e non-inoculated control; \u003cstrong\u003eT:\u003c/strong\u003e \u003cem\u003eTrichoderma\u003c/em\u003e-inoculated treatment. Different lowercase letters indicate significant differences within the CK group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05); Different uppercase letters indicate significant differences within the T group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Asterisks indicate significant differences between CK and T groups at the same salt concentration (ns, not significant; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8707138/v1/87e596171ee279a6c17f6131.jpeg"},{"id":102854735,"identity":"61741762-dc6c-45ad-84a9-42b0c70077ca","added_by":"auto","created_at":"2026-02-17 14:50:46","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":324044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTrichoderma\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e inoculation on physicochemical properties and enzyme activities of rhizosphere soil of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. ostii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ‘Fengdan’ under salt stress. (A) \u003c/strong\u003eSoil pH;\u003cstrong\u003e (B) \u003c/strong\u003eSoil conductivity;\u003cstrong\u003e (C) \u003c/strong\u003eSoil NH4\u003csup\u003e+\u003c/sup\u003e-N content;\u003cstrong\u003e (D) \u003c/strong\u003eSoil phosphorus available content;\u003cstrong\u003e (E)\u003c/strong\u003e Soil urease activity;\u003cstrong\u003e (F) \u003c/strong\u003eSoil phosphatase activity;\u003cstrong\u003e (G) \u003c/strong\u003eSoil sucrase activity;\u003cstrong\u003e (H) \u003c/strong\u003eSoil dehydrogenase activity.\u003cstrong\u003e \u003c/strong\u003eData are presented as means ± SD (n=3). \u003cstrong\u003eCK:\u003c/strong\u003e non-inoculated control; \u003cstrong\u003eT:\u003c/strong\u003e \u003cem\u003eTrichoderma\u003c/em\u003e-inoculated treatment. Different lowercase letters indicate significant differences within the CK group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05); Different uppercase letters indicate significant differences within the T group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Asterisks indicate significant differences between CK and T groups at the same salt concentration (ns, not significant; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8707138/v1/9924fda932deb45ee9499263.jpeg"},{"id":102965054,"identity":"81108e91-e2d3-41c1-a96f-03575b348522","added_by":"auto","created_at":"2026-02-19 04:30:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3627191,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8707138/v1/53dbece7-7a74-453e-bffd-08019acecf3e.pdf"}],"financialInterests":"","formattedTitle":"Trichoderma enhances salt tolerance in Paeonia ostii ‘Fengdan’ by regulating the rhizosphere soil environment and physiological mechanisms","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSalt stress is one of the primary abiotic stressors limiting global agricultural productivity and ecosystem stability. Currently, approximately one-third of the world's soils are affected by varying degrees of salinization, a trend that is increasingly exacerbated by climate change (Zheng et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). High-salinity environments initially trigger osmotic stress and ion toxicity in roots, subsequently inducing oxidative stress. These processes disrupt metabolic homeostasis and the functionality of photosynthetic systems, leading to stomatal closure and reduced biomass accumulation, which ultimately severely inhibit plant growth and development or even cause mortality (Fu et al., 2023; Han et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consequently, exploring eco-friendly and efficient mitigation strategies to enhance crop salt tolerance has become an urgent imperative for sustainable agricultural development.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePaeonia suffruticosa\u003c/em\u003e Andr. is not only a traditional and precious ornamental flower in China but also serves as the botanical origin of oil peony, an emerging woody oil-bearing crop. Specifically, \u003cem\u003ePaeonia ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo; the dominant cultivar of oil peony, has been certified as a vital novel food resource due to its high seed yield and superior oil quality, which is notably rich in α-linolenic acid (Lu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, with the rapid expansion of the oil peony industry and the increasing scarcity of suitable arable land, cultivation areas are gradually shifting towards marginal soils, such as saline-alkali lands. Nevertheless, \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo; is relatively sensitive to salinity. Salt stress-induced issues, such as growth retardation, chlorosis, and wilting, have severely constrained the economic viability of this industry (Shi et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wen et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, enhancing the adaptive capacity of \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo; in saline environments represents a critical issue that urgently requires resolution.\u003c/p\u003e \u003cp\u003eLeveraging beneficial soil microorganisms to enhance plant adaptation to stressful environments is an environmentally friendly strategy. As a crucial group of symbiotic microorganisms, endophytic fungi are capable of colonizing host plant tissues and establishing mutualistic relationships, thereby exerting profound impacts on plant growth, development, and environmental adaptability (Nisa et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Research indicates that these beneficial fungi not only directly promote growth by secreting phytohormones (Li et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and solubilizing mineral elements (Li et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) but also assist hosts in combating abiotic stresses, such as drought and salinity, by inducing systemic resistance (Cui et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ueno et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Compared with bacteria, filamentous fungi possess unique advantages in improving soil structure and nutrient cycling by forming extensive hyphal networks in the rhizosphere. This characteristic underpins their potential application in the ecological restoration of saline-alkali lands.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTrichoderma\u003c/em\u003e spp. represents a class of ubiquitous and potent plant growth-promoting fungi (PGPF). Extensive research has demonstrated that \u003cem\u003eTrichoderma\u003c/em\u003e can successfully colonize the plant rhizosphere and significantly alleviate salt stress symptoms in crops such as wheat (Zhang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e), rice (Anshu et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and cucumber (Qi et al., 2013). These beneficial effects are primarily achieved by modulating osmolyte accumulation, activating antioxidant defense systems, and regulating the expression of stress-responsive genes. However, current studies on \u003cem\u003eTrichoderma\u003c/em\u003e-mediated salt tolerance have predominantly focused on herbaceous crops or relied solely on physiological and biochemical indicators. There is a paucity of in-depth analyses conducted from a systemic dimension that integrates \u0026ldquo;rhizosphere soil microenvironment\u0026mdash;plant anatomical structure\u0026mdash;physiological metabolic function\u0026rdquo;. In particular, for the woody oil-bearing crop \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo;, it remains unclear whether \u003cem\u003eTrichoderma\u003c/em\u003e can establish a defense against salinity by remodeling the root-stem vascular system structures and activating rhizosphere soil enzyme activities.\u003c/p\u003e \u003cp\u003eAccordingly, this study employed \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo; seedlings to investigate the multidimensional regulatory mechanisms by which \u003cem\u003eTrichoderma\u003c/em\u003e enhances salt tolerance. In addition to a comprehensive assessment of macroscopic growth, photosynthetic gas exchange, and physiological metabolic homeostasis, we utilized microscopic techniques to characterize the adaptive changes in stomatal traits and root-stem anatomical structures. Concurrently, we investigated the response characteristics of rhizosphere soil physicochemical properties and enzyme activities. The findings of this research are intended to offer practical guidance for the ecological cultivation of oil peony in saline-alkali soils and to promote the application of \u003cem\u003eTrichoderma\u003c/em\u003e inoculants as a sustainable strategy for alleviating salt stress in forestry and agriculture.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eExperimental materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo-year-old seedlings of \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo; were obtained from the Germplasm Resource Nursery of Peony and Herbaceous Peony of Shenyang Agricultural University. In April 2025, healthy, pest-free seedlings exhibiting uniform growth were transplanted into pots (14 cm diameter \u0026times; 16.7 cm height) containing a standard nutrient substrate, with one seedling per pot. The pots were filled to approximately 1 cm below the rim. The baseline physicochemical properties of the soil were as follows: pH 6.99, electrical conductivity of 2.73 mS\u0026middot;cm\u003csup\u003e-1\u003c/sup\u003e, ammonium nitrogen of 8.49 mg\u0026middot;kg\u003csup\u003e-1\u003c/sup\u003e, available phosphorus of 20.36 mg\u0026middot;kg\u003csup\u003e-1\u003c/sup\u003e, urease activity of 0.50 mg\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e, invertase activity of 33.43 mg\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e, phosphatase activity of 22.10 mg\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e, and dehydrogenase activity of 6.58 mg\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e. Subsequently, the potted plants were transferred to a greenhouse at the Botanical Garden of Shenyang Agricultural University for acclimatization and routine management. The greenhouse conditions were maintained at an average temperature of 23\u0026deg;C, a relative humidity of 55%, and a 14 h photoperiod.\u003c/p\u003e\n\u003cp\u003eThe compound \u003cem\u003eTrichoderma\u003c/em\u003e inoculant was supplied by the \u003cem\u003eTrichoderma\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e Fertilizer Research and Development Team of Shenyang Agricultural University, containing an effective viable count of\u0026nbsp;\u0026ge;\u0026nbsp;1\u0026nbsp;\u0026times;\u0026nbsp;10\u003csup\u003e8\u0026nbsp;\u003c/sup\u003eCFU/g.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted using a randomized block design consisting of 8 treatments (2 inoculation levels \u0026times; 4 salt concentrations). In May 2025, following acclimatization, uniform \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo; seedlings were selected. Inoculation was performed via root irrigation with a \u003cem\u003eTrichoderma\u003c/em\u003e suspension (5 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e, 100 mL per pot), applied three times at 7-day intervals. Control plants received an equivalent volume of water. Seven days post-inoculation, salt stress was imposed using four NaCl gradients: 0, 100, 200, and 300 mM. To mitigate osmotic shock, salt solutions were applied in a stepwise increment manner (100 mL every 3 days) until the target concentrations were reached. Photosynthetic parameters were assessed after 14 days of sustained stress. Immediately thereafter, plant tissues were harvested, flash-frozen, and stored at -80\u0026deg;C for biochemical analysis. Each treatment included 30 seedlings, and the entire experiment was repeated three times to minimize environmental variability. Detailed treatment specifications are listed in Table 1.\u003c/p\u003e\n\u003cp\u003eTable 1 Test treatments\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 198px;\"\u003e\n \u003cp\u003eTreatment group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eTreatment reagent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003eDistilled water control\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 104px;\"\u003e\n \u003cp\u003e100 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003e100 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma\u003c/em\u003e +100 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 104px;\"\u003e\n \u003cp\u003e200 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003e100 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma\u003c/em\u003e +200 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 104px;\"\u003e\n \u003cp\u003e300 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003e100 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 340px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma\u003c/em\u003e +300 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurements and methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObservation of plant morphology and measurement of growth parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the salt stress treatment, plant morphological changes were observed daily between 8:00 and 9:00 AM. Key indicators, including leaf color, texture, and general condition, were recorded and documented photographically. Upon the conclusion of the experiment, three plants were randomly selected from each treatment. Plant height (vertical distance from the soil surface to the highest point) was measured using a steel tape (precision: 0.1 cm), and stem diameter was measured 2 cm above the soil surface using a Vernier caliper (precision: 0.01 cm). Subsequently, plants were harvested, washed, and surface-dried. The shoots were separated from the roots at the root-shoot junction. The fresh weights (FW) of the shoots and roots were determined using an analytical balance, and the root-to-shoot ratio was calculated.\u003c/p\u003e\n\u003cp\u003eRoot morphology was digitized using an Epson Expression 11000 XL scanner (Epson, Japan). The resulting images were analyzed using the WinRHIZO root analysis system (Regent Instruments Inc., Canada) to quantify parameters including total root length, projected area, surface area, average diameter, root volume, and the number of root tips. Root activity was determined using the TTC (2,3,5-triphenyltetrazolium chloride) reduction method (Zhang et al., 2014).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of photosynthetic parameters\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBetween 9:00 and 11:00 am when light was stable, a portable Li-6400 photosynthesis system was used to measure net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration (Ci), and stomatal conductance (Gs).\u0026nbsp;Measurements were conducted under the following conditions: a leaf chamber area of 3 cm\u003csup\u003e2\u003c/sup\u003e, ambient CO\u003csub\u003e2\u003c/sub\u003e concentration, a standard flow rate of 750 \u0026mu;mol/s, and a photosynthetically active radiation (PAR) of 1200 \u0026mu;mol/m\u003csup\u003e2\u003c/sup\u003e\u0026middot;s\u003csup\u003e-1\u003c/sup\u003e provided by a red/blue LED light source\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(Zhang et al., 2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of physiological indices\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRelative electrical conductivity (REC) was measured using a conductivity meter following the method of Lutts et al. (1996).\u0026nbsp;Malondialdehyde (MDA) content was determined via the thiobarbituric acid (TBA) assay\u0026nbsp;(Velikova et al., 2000).\u0026nbsp;Superoxide anion radicals (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) were assessed using a combination of qualitative localization and quantitative determination. For qualitative analysis, histochemical staining of leaves was performed using NBT and DAB, respectively, to visualize the in situ accumulation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Yuan et al., 2024).\u0026nbsp;For quantitative analysis, the production rate of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e was determined by the hydroxylamine method\u0026nbsp;(Jiang et al., 2001), while H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content was measured using the titanium sulfate (TiSO\u003csub\u003e4\u003c/sub\u003e) method\u0026nbsp;(Velikova et al., 2000).\u003c/p\u003e\n\u003cp\u003eSoluble sugar (SS) content was determined using the anthrone colorimetric method. Soluble protein (SP) content was measured via the Coomassie Brilliant Blue G-250 assay. Proline content was determined using the acid ninhydrin colorimetric method (Ikram et al., 2019).\u003c/p\u003e\n\u003cp\u003eAntioxidant enzyme activities were assayed according to the protocols of Wu et al. (2021), with slight modifications. Enzyme extraction: 0.2 g of fresh leaf samples was homogenized in 1 mL of 0.1 mol/L phosphate buffer (PBS, pH 7.0) and made up to a final volume of 2 mL. The homogenate was centrifuged at 10,000\u0026nbsp;\u0026times;\u0026nbsp;g for 20 min at 4\u0026deg;C, and the resulting supernatant served as the crude enzyme extract. Superoxide dismutase (SOD) activity was determined by the nitroblue tetrazolium (NBT) photoreduction method. Peroxidase (POD) activity was assayed using the guaiacol colorimetric method. Catalase (CAT) activity was measured by the UV absorption method, monitoring the change in absorbance at 240 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObservation of stomata and anatomical structures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree healthy, disease-free mature leaves were randomly selected from each treatment. The abaxial epidermis was peeled off using forceps to prepare temporary wet mounts. Observations were conducted under an optical microscope (Olympus CX-31; Olympus, Japan) using a 40\u0026nbsp;\u0026times;\u0026nbsp;objective lens. Ten fields of view were randomly selected and photographed for each treatment. Stomatal parameters, including length, width, density, and aperture, were measured using Motic Images Advanced 3.0 software.\u003c/p\u003e\n\u003cp\u003eThe anatomical structures of roots, stems, and leaves were observed using the conventional paraffin sectioning method, following the protocol of Nassar et al. (2020) with modifications. Fresh samples were washed and cut into segments (0.3\u0026nbsp;\u0026times;\u0026nbsp;0.5 cm for roots and stems; 0.5\u0026nbsp;\u0026times;\u0026nbsp;0.5 cm for leaves). The specimens were fixed in FAA fixative for over 24 h. Subsequently, the samples underwent dehydration through a graded ethanol series, clearing with a clearing agent, and infiltration with paraffin via a stepwise temperature process before being embedded. The specimens were sectioned using a microtome, dried at 42\u0026deg;C, stained with Safranin O-Fast Green, and mounted with neutral balsam.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of rhizosphere soil properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlants were carefully removed from the pots, and the loosely adhering soil was shaken off. The rhizosphere soil, defined as the soil tightly adhering to the roots, was collected and mixed to form a composite sample. The sample was divided into two portions: one portion was stored fresh at -80\u0026deg;C for biological analysis, and the other was air-dried for the determination of physicochemical properties.\u003c/p\u003e\n\u003cp\u003eSoil electrical conductivity (EC) and pH were measured using a conductivity meter and the potentiometric method, respectively. Soil available phosphorus (AP) content was determined using the molybdenum-antimony anti-spectrophotometric method (Tao et al., 2022). Soil ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) content was determined using the indophenol blue colorimetric method (Zhang et al., 2004).\u003c/p\u003e\n\u003cp\u003eSoil urease activity was assayed using the sodium phenol-sodium hypochlorite colorimetric method by measuring absorbance at 578 nm. Soil phosphatase activity was determined using the disodium phenyl phosphate colorimetric method, with absorbance measured at 510 nm. Soil sucrase activity was assessed using the 3,5-dinitrosalicylic acid (DNS) colorimetric method, measuring absorbance at 508 nm (Han et al., 2020). Soil dehydrogenase activity was determined via the triphenyltetrazolium chloride (TTC) colorimetric method by monitoring absorbance at 492 nm (Olmos-Ruiz et al., 2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData compilation and calculation were performed using Microsoft Excel 2019. Statistical analyses were conducted using IBM SPSS Statistics 22. One-way analysis of variance (ANOVA) was employed, followed by Duncan\u0026apos;s multiple range test to determine significant differences among treatments. Figures were generated using GraphPad Prism 9.0. All data are presented as means \u0026plusmn; standard deviation (SD).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePlant growth performance and root system architecture\u003c/h2\u003e \u003cp\u003eSalt stress induced dose-dependent morphological damage in \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo;. Plants exposed to low salinity exhibited distinct foliar yellowing, while those under high concentrations displayed severe marginal necrosis, leaf rolling, and loss of turgor (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In contrast, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation effectively alleviated these symptoms. Plants receiving Trichoderma inoculation exhibited a robust phenotype, characterized by expanded leaf blades, vibrant green foliage, and sustained turgidity. Quantitatively, \u003cem\u003eTrichoderma\u003c/em\u003e demonstrated dual efficacy by promoting growth under both non-saline and saline conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;D). While salt stress significantly inhibited growth metrics in controls, the inoculated group consistently maintained superior performance. Notably, the mitigation effect peaked at 200 mM NaCl, where plant height, stem diameter, and root-to-shoot ratio significantly increased by 27.99%, 23.08%, and 19.30%, respectively, compared to the corresponding control.\u003c/p\u003e \u003cp\u003eRegarding root system architecture (RSA), \u003cem\u003eTrichoderma\u003c/em\u003e inoculation comprehensively improved root multidimensional traits (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Under non-saline conditions, the fungus elicited a robust direct growth-promoting effect, increasing total root length by 33.73%, root surface area by 69.83%, and root tip number by 51.86%. Furthermore, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation significantly thickened the root system, boosting projected area, root volume, and average diameter by 41.79%, 38.59%, and 14.70%, respectively. Under salt stress, the alleviative effect peaked at 100 mM NaCl. At this concentration, inoculated plants exhibited a more developed root architecture than saline controls, with increases of 17.50% in total length, 54.95% in surface area, and 41.45% in root tips. Crucially, the absorptive capacity was preserved, as evidenced by simultaneous enhancements of 31.98% in projected area, 32.70% in root volume, and 13.49% in average diameter. However, this protective efficacy exhibited a physiological threshold, as the beneficial impacts on both shoot and root traits were attenuated under severe salinity at 300 mM NaCl.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of \u003cem\u003eTrichoderma\u003c/em\u003e inoculation on root system parameters of \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo; under salt stress\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal root length/cm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProjected\u003c/p\u003e \u003cp\u003eArea/cm\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRoot surface\u003c/p\u003e \u003cp\u003earea/cm\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003cp\u003ediameter/mm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eVolume/mm\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTips\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0 mM NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e234.74\u0026thinsp;\u0026plusmn;\u0026thinsp;8.97 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.79 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e90.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08 c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e313.93\u0026thinsp;\u0026plusmn;\u0026thinsp;11.31 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e74.94\u0026thinsp;\u0026plusmn;\u0026thinsp;3.88 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e137.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e100 mM NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e211.13\u0026thinsp;\u0026plusmn;\u0026thinsp;6.45 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e78.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e248.07\u0026thinsp;\u0026plusmn;\u0026thinsp;5.82 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53.23\u0026thinsp;\u0026plusmn;\u0026thinsp;4.08 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e110.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e200 mM NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e175.75\u0026thinsp;\u0026plusmn;\u0026thinsp;9.01 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.34 e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e58.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.51 e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e204.60\u0026thinsp;\u0026plusmn;\u0026thinsp;12.50 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e79.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e300 mM NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e149.05\u0026thinsp;\u0026plusmn;\u0026thinsp;5.92 e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.64 e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e51.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08 f\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e174.05\u0026thinsp;\u0026plusmn;\u0026thinsp;8.71 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53 de\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21 e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e61.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52 e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eData are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). \u003cb\u003eCK\u003c/b\u003e: non-inoculated control; \u003cb\u003eT\u003c/b\u003e: \u003cem\u003eTrichoderma\u003c/em\u003e-inoculated treatment. Different lowercase letters indicate significant differences among treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnatomical adaptations of roots and stems\u003c/h2\u003e \u003cp\u003eMicroscopic analysis illustrated that salt stress caused systemic anatomical atrophy in \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo;, specifically targeting the vascular transport system. In the stems of the control group, increasing salinity levels led to a progressive thinning of the vascular ring and restricted secondary xylem development. With the escalation of NaCl concentration, vascular tissues gradually exhibited a loose arrangement accompanied by a significant reduction in vessel caliber (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Mirroring the stem response, root tissues in the control group exhibited marked stele atrophy and retarded secondary growth, characterized by scattered vessels and diminished xylem area (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn contrast, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation effectively protected the vascular structure of both stems and roots. Regardless of salinity levels, inoculated plants maintained a robust vascular system. In stems, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation induced the formation of broader secondary xylem zones and a continuous, compact xylem ring, even under severe salt stress at 300 mM NaCl. Concurrently, roots of inoculated plants displayed a plump stele with well-developed secondary xylem. A key anatomical feature observed in the inoculated group was the significantly increased number and enlarged caliber of vessels in both stems and roots, which facilitates efficient water transport. Furthermore, histochemical observations showed intensified deep purple-red safranin staining in the xylem vessel walls of inoculated plants, indicating an enhanced degree of lignification and mechanical strength compared to the saline controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStomatal characteristics and adaptive regulation\u003c/h2\u003e \u003cp\u003eMicroscopic observations and parametric analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) indicated that \u003cem\u003eTrichoderma\u003c/em\u003e inoculation effectively mitigated salt-induced stomatal distortion. In the control group, salt stress caused pronounced morphological aberrations. Guard cells exhibited severe dehydration and shrinkage, leading to extensive pore closure. This resulted in a significant reduction in the stomatal short axis and an increase in the length-to-width ratio, presenting a distinct \"narrow and elongated\" closure phenotype. \u003cem\u003eTrichoderma\u003c/em\u003e inoculation reversed this trend, increasing the short axis by 13.49%, 14.63%, and 11.48% under 100, 200, and 300 mM NaCl, respectively, compared to corresponding controls. Stomatal aperture was enhanced across salinity gradients from 0 to 300 mM, with increases of 6.80%, 7.63%, 18.46%, and 5.58%, respectively. Furthermore, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation exhibited a bidirectional regulation on stomatal density, increasing it by 11.98% under non-saline conditions while reducing it by 9.34%, 10.61%, and 4.88% under salt stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of \u003cem\u003eTrichoderma\u003c/em\u003e inoculation on stomatal parameters of \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo; under salt stress\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStomatal long axis/\u0026micro;m\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStomatal short axis/\u0026micro;m\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003elength-to-width ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eStomatal aperture/\u0026micro;m\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eStomatal density/mm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eStomatal index/%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0 mM NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e238.97\u0026thinsp;\u0026plusmn;\u0026thinsp;9.65 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e267.60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22 bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e19.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e100 mM NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73 bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e261.83\u0026thinsp;\u0026plusmn;\u0026thinsp;8.80 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e19.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e237.37\u0026thinsp;\u0026plusmn;\u0026thinsp;5.95 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e19.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e200 mM NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e302.20\u0026thinsp;\u0026plusmn;\u0026thinsp;5.82 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e19.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e270.13\u0026thinsp;\u0026plusmn;\u0026thinsp;7.74 bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e19.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e300 mM NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e295.30\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e19.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e280.90\u0026thinsp;\u0026plusmn;\u0026thinsp;7.04 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e19.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003cb\u003eNote\u003c/b\u003e: Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). \u003cb\u003eCK\u003c/b\u003e: non-inoculated control; \u003cb\u003eT\u003c/b\u003e: \u003cem\u003eTrichoderma\u003c/em\u003e-inoculated treatment. Different lowercase letters indicate significant differences among treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePhotosynthetic performance and photosynthetic pigments\u003c/h2\u003e \u003cp\u003eSalt stress significantly suppressed gas exchange processes, leading to dose-dependent declines in net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration (Ci). However, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation effectively alleviated this inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;D). Under non-saline conditions, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation enhanced physiological performance, increasing Pn, Tr, Gs, and Ci by 23.79%, 21.32%, 31.0%, and 17.92%, respectively. Under salt stress, the alleviation effect peaked at 100 mM NaCl, where Pn, Tr, Gs, and Ci rose by 27.02%, 40.38%, 46.84%, and 19.71%, respectively, relative to the corresponding control. Although the magnitude of improvement diminished slightly with rising salinity, the inoculated group consistently maintained significantly higher indices.\u003c/p\u003e \u003cp\u003eRegarding photosynthetic pigments, salt stress induced a significant reduction in chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll (Chl), and carotenoids (Car). \u003cem\u003eTrichoderma\u003c/em\u003e inoculation significantly alleviated the reduction in these pigments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026ndash;H). Under non-saline conditions, basal levels of Chl a, Chl b, Chl, and Car increased by 13.3%, 43.3%, 21.8%, and 14.7%, respectively. Under salt stress, the enhancement effect was most pronounced at 100 mM NaCl, with increases of 32.6%, 18.4%, and 19.8% for Chl b, Chl, and Car, respectively. Even under severe salinity of 300 mM NaCl, where pigment biosynthesis was strongly inhibited, Chl a content in the inoculated group showed a relative increase of 16.9%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eReactive oxygen species accumulation and antioxidant enzyme activities\u003c/h2\u003e \u003cp\u003eHistochemical \u003cem\u003ein situ\u003c/em\u003e staining combined with quantitative analysis revealed the accumulation patterns of reactive oxygen species (ROS) in plants under salt stress. In the control group, the intensity and extent of reddish-brown precipitates (indicating H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and blue-black precipitates (indicating O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) gradually increased with rising salt concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Under 200 mM and 300 mM treatments, the leaves exhibited extensive dark staining areas. Consistent with histochemical observations, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e production rate in control plants increased significantly with salt concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Conversely, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation significantly reduced ROS accumulation levels. This reduction was most pronounced under low-to-moderate salinity levels of 100 mM and 200 mM NaCl. In these treatments, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content in inoculated leaves decreased significantly by 55.08% and 43.42%, respectively, while the O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e production rate declined by 22.08% and 21.75%, respectively. Even under severe salinity of 300 mM NaCl, ROS levels in the inoculated group remained significantly lower than those in the control group.\u003c/p\u003e \u003cp\u003eRegarding antioxidant defense, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) exhibited a trend of initial increase followed by a subsequent decline with increasing salt concentrations, peaking at 200 mM NaCl (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE\u0026ndash;G). \u003cem\u003eTrichoderma\u003c/em\u003e inoculation maintained higher levels of enzyme activities across all salinity gradients. The maximal relative increase occurred under the 100 mM NaCl treatment, where the activities of SOD, POD, and CAT increased by 21.92%, 30.68%, and 95.24%, respectively. With a further increase in salinity, although the intrinsic enzyme activities declined, the inoculated group sustained significantly higher levels compared to the control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMembrane stability and osmotic adjustment\u003c/h2\u003e \u003cp\u003eSalt stress induced a dose-dependent increase in relative electrical conductivity (REC) and malondialdehyde (MDA) content. However, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation maintained these metrics at lower levels compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Under non-saline conditions, no significant differences were observed in membrane indices between the inoculated and control groups. Under salt stress, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation suppressed the elevation of membrane damage indices. Specifically, compared to the corresponding control group, REC in the treated group decreased by 23.79%, 20.22%, and 13.08% at 100, 200, and 300 mM NaCl treatments, respectively. Similarly, MDA content exhibited reductions of 51.89%, 38.31%, and 29.93%, respectively, relative to the control.\u003c/p\u003e \u003cp\u003eRegarding osmoregulation, the contents of soluble sugar (SS), soluble protein (SP), and proline (Pro) exhibited a dose-dependent increase as salt concentrations rose. \u003cem\u003eTrichoderma\u003c/em\u003e inoculation further elevated the accumulation of these three osmolytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u0026ndash;E). Under non-saline conditions, the basal levels of SS, SP, and Pro in the leaves of the treated group increased by 45.68%, 32.43%, and 32.0%, respectively. Under salt stress, the increase in SS and SP contents peaked at 100 mM NaCl treatment, showing elevations of 91.49% and 41.12% relative to the control. For Pro, the maximum increase of 57.7% was observed at 200 mM NaCl treatment. Even under severe salinity of 300 mM NaCl, the inoculated group maintained significantly higher levels of osmolytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRhizosphere soil physicochemical properties and enzyme activities\u003c/h2\u003e \u003cp\u003eSalt stress significantly elevated soil pH and electrical conductivity (EC), but \u003cem\u003eTrichoderma\u003c/em\u003e inoculation consistently mitigated these increases (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). Specifically, the treatment reduced pH by 4.09% and 4.13% under 200 and 300 mM NaCl, respectively, and achieved a maximum EC reduction of 28.63% at 200 mM NaCl. Conversely, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation enhanced the contents of ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) and available phosphorus (AP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). Under non-saline conditions, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation increased NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and AP by 18.24% and 24.71%, respectively. Under salt stress, the enhancement effect peaked at 200 mM NaCl, rising by 21.19% and 27.45%, respectively, with significantly higher levels maintained even at 300 mM NaCl.\u003c/p\u003e \u003cp\u003eRegarding enzyme activities, salt stress inhibited urease and phosphatase in control, causing reductions of 55.09% and 23.39% at 300 mM NaCl, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F). \u003cem\u003eTrichoderma\u003c/em\u003e inoculation enhanced basal activities of urease and phosphatase by 29.65% and 29.02% under non-saline conditions. Under salt stress, the increase in urease activity peaked at 34.28% under 200 mM NaCl, while phosphatase activity showed a maximum increase of 33.43% under 100 mM NaCl. Additionally, sucrase activity in the inoculated group peaked at 200 mM NaCl with a 45.14% increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG), while dehydrogenase activity showed a rising enhancement trend with salinity, reaching a maximum increase of 37.18% at 300 mM NaCl (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSoil salinization severely limits the cultivation of \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo;. Our study demonstrates that \u003cem\u003eTrichoderma\u003c/em\u003e inoculation effectively mitigates salt-induced systemic damage. Mechanistically, \u003cem\u003eTrichoderma\u003c/em\u003e ameliorates the rhizosphere environment by improving soil physicochemical properties and enzyme activities, while simultaneously preserving anatomical integrity. These findings demonstrate that \u003cem\u003eTrichoderma\u003c/em\u003e confers salt tolerance to \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo; through the coordination of rhizosphere remediation and plant adaptive responses, providing a viable strategy for the cultivation of this crop in saline environments.\u003c/p\u003e \u003cp\u003eBeneficial microorganisms are pivotal in enhancing plant resilience to abiotic stress. In this study, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation significantly mitigated salt-induced morphometric restrictions in \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo;, conferring superior plant height, stem diameter, and biomass accumulation. These results corroborate previous findings in sweet sorghum (Wei et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and wheat (Zhang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e), where \u003cem\u003eTrichoderma\u003c/em\u003e colonization promoted robust host growth. Crucially, this vegetative improvement is intrinsically linked to the root system. While salt stress typically compromises root plasticity by inhibiting elongation and lateral branching (Koevoets et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), \u003cem\u003eTrichoderma\u003c/em\u003e inoculation effectively reversed these inhibitory effects, significantly increasing total root length, surface area, and root tip number. This optimization of root system architecture (RSA) aligns with Rouphael et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), suggesting that the enhanced root expansion facilitates efficient water and nutrient acquisition from deeper soil layers. Mechanistically, this remodeling is likely driven by \u003cem\u003eTrichoderma\u003c/em\u003e-induced auxin accumulation in root primordia, which stimulates lateral root development to counteract salt-induced growth stagnation (Contreras-Cornejo et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). While an optimized root architecture facilitates water acquisition, the efficient delivery of these resources to aerial tissues fundamentally relies on the anatomical integrity of the internal vascular system. In this study, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation effectively optimized the systemic water transport network connecting roots, stems, and leaves. Specifically, in roots and stems, salt stress caused stele atrophy and narrowed vascular rings, whereas inoculated plants maintained robust steles and broader secondary xylem zones, and possessed larger vessel diameters. This preservation of vascular geometry was likely reinforced by enhanced cell wall lignification, which ensured efficient upward water transport and mechanical resilience against stress-induced negative pressure. These observations align with Basinska-Barczak et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who reported that microbe-induced lignin accumulation mechanically reinforces vessels in wheat. Consistent with the findings of S. Taha et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) in lupine and Palupi et al. (2023) in Indian mustard, the maintenance of vascular diameter in inoculated \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo; plants is crucial for alleviating physiological drought. Crucially, this secured water supply translated into superior stomatal regulation. Salt stress typically causes guard cells to undergo dehydration and shrinkage, leading to functional closure. However, inoculated plants retained turgid, kidney-shaped guard cells and significantly wider apertures. This is similar to the findings of Jing et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), who demonstrated that microbial inoculation mitigates drought-induced stomatal closure in walnut, thereby sustaining gas exchange.\u003c/p\u003e \u003cp\u003ePhotosynthesis acts as the engine for biomass accumulation but is highly susceptible to salt stress via both stomatal and non-stomatal limitations (Acosta-Motos et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). We observed that salt stress significantly reduced the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO₂ concentration (Ci). However, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation alleviated this inhibition, enabling plants to maintain higher levels of gas exchange across all salinity levels. This result is consistent with Han et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), who demonstrated that microbial inoculation enhances carbon assimilation capacity by regulating stomatal behavior in \u003cem\u003eCodonopsis pilosula\u003c/em\u003e. Beyond gas exchange, pigment stability is vital. In our study, salt stress induced a significant reduction in chlorophyll a, chlorophyll b, and carotenoids. Conversely, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation significantly attenuated this pigment degradation. This aligns with Patani et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), who found that inoculation with salt-tolerant plant growth-promoting rhizobacteria preserved leaf chlorophyll content in tomato. This protective effect is likely attributed to the ability of the fungal inoculant to maintain chloroplast membrane integrity by mitigating oxidative stress, thereby retarding the oxidative decomposition of photosynthetic pigments. The fundamental mechanism underlying this protection against oxidative stress lies in the modulation of reactive oxygen species (ROS) homeostasis. Salt stress disrupts cellular homeostasis by inducing a burst of ROS, leading to oxidative stress and cell death (Miller et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In this study, histochemical staining revealed extensive ROS accumulation in stressed control leaves, manifested as reddish-brown and blue-black precipitates. However, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation significantly attenuated this oxidative load. Quantitative analysis confirmed that the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e production rate were significantly reduced in inoculated plants. This ROS scavenging capability is mechanistically attributed to the activation of the antioxidant enzyme system. Our data showed that \u003cem\u003eTrichoderma\u003c/em\u003e upregulated the activities of SOD, POD, and CAT, forming an efficient scavenging network. These results align with Chen et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) in cucumber and Guler et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) in maize, where beneficial microbes mitigated oxidative damage by boosting enzymatic defense. Expanding on this mechanism, Zhang et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and Khan et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) further elucidated that such upregulation of antioxidant enzymes serves as a trigger to induce systemic resistance. Consequently, the reduction in ROS levels directly preserved the integrity of the cell membrane system. Salt stress typically exacerbates membrane lipid peroxidation, indicated by elevated relative electrical conductivity (REC) and malondialdehyde (MDA) content. However, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation significantly suppressed the rising trend of these indicators, effectively alleviating cell membrane damage. This corroborates the findings of Sofy et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) which demonstrated that \u003cem\u003eTrichoderma\u003c/em\u003e reduces electrolyte leakage and MDA accumulation by enhancing antioxidant activity. Parallel to enzymatic defense, the accumulation of osmolytes is a critical strategy for maintaining cellular water balance. We found that \u003cem\u003eTrichoderma\u003c/em\u003e further promoted the accumulation of soluble sugars, soluble proteins, and proline under salt stress. This enhancement of osmoregulatory capacity aligns with Ahmad et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) in mustard, suggesting that \u003cem\u003eTrichoderma\u003c/em\u003e-mediated osmolyte accumulation mitigates ion toxicity and stabilizes membrane structures, thereby contributing to improved salt tolerance.\u003c/p\u003e \u003cp\u003eThe degradation of soil physicochemical properties and the inhibition of rhizosphere microbial functions constitute major constraints on plant growth under saline conditions. In this study, salt stress induced a marked elevation in soil pH and electrical conductivity (EC); however, \u003cem\u003eTrichoderma\u003c/em\u003e inoculation effectively counteracted these increases, stabilizing the rhizosphere environment. This amelioration aligns with Wu et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), who demonstrated that optimizing the soil physicochemical environment via beneficial microbes enhances water and nutrient uptake. Furthermore, the decline in soil EC lends support to the mechanism proposed by Ruiz-Lozano et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), wherein the extensive fungal hyphal network sequesters and compartmentalizes salt ions within vacuoles, thereby lowering the ionic concentration of the rhizosphere soil solution. Beyond these physicochemical improvements, soil enzyme activity\u0026mdash;a pivotal indicator of soil quality and metabolic function\u0026mdash;was significantly revitalized. \u003cem\u003eTrichoderma\u003c/em\u003e inoculation upregulated the activities of soil urease, phosphatase, invertase, and dehydrogenase. This enzymatic enhancement aligns with Chaudhary et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and the findings of Khalifa et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) in maize under saline-alkali environments, confirming that microbial inoculants boost metabolic function under stress. Accompanied by this enzymatic activation, we found that the contents of soil ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) and available phosphorus also increased significantly. These results demonstrate that remodeling the rhizosphere bio-physicochemical environment and enhancing soil nutrient availability constitute key mechanisms by which \u003cem\u003eTrichoderma\u003c/em\u003e alleviates salt stress.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that \u003cem\u003eTrichoderma\u003c/em\u003e inoculation enhances salt tolerance in \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Fengdan\u0026rsquo; through the synergistic amelioration of the rhizosphere environment and physiological regulation. Specifically, \u003cem\u003eTrichoderma\u003c/em\u003e optimized the soil microenvironment by reducing soil pH and electrical conductivity (EC) while boosting soil enzyme activities. Simultaneously, it mitigated oxidative stress by enhancing antioxidant defenses and stimulating osmolyte accumulation. Anatomically, the treatment preserved the structural integrity of roots, stems, and stomata, thereby securing water transport and photosynthetic efficiency. Collectively, these adaptive changes significantly improved plant growth, providing a theoretical basis for the microbial-assisted cultivation of peonies in saline regions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eOn behalf of all authors, the corresponding author states that there is no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China, 32071814 and 31470696.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eDairu Jiang contributed toward writing\u0026mdash;original draft, visualization, validation, software, resources, project administration, methodology, and data curation. Shixin Guan contributed toward writing\u0026mdash;review \u0026amp; editing, and project administration. Xuening Kang contributed toward validation and data curation. Ayimukezi Maimaitizunong contributed toward validation and data curation. Zhong Chen contributed toward validation and data curation. Yanxin Gu contributed toward validation and data curation. Xiaomei Sun contributed toward writing\u0026mdash;review \u0026amp;editing, supervision, conceptualization, and funding acquisition.\u003c/p\u003e\u003ch2\u003eData Availability statement\u003c/h2\u003e \u003cp\u003eThe data that support the results are included in this article. Other relevant materials are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcosta-Motos JR, Ortu\u0026ntilde;o MF, Bernal-Vicente A, Diaz-Vivancos P, Sanchez-Blanco MJ, Hernandez JA (2017) Plant responses to salt stress: adaptive mechanisms. 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International Journal of Agriculture and Biology 24: 319-332. doi: https://doi.org/10.17957/ijab/15.1441.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Paeonia ostii ‘Fengdan’, Trichoderma, Physiological characteristics, Rhizosphere soil, Salt stress","lastPublishedDoi":"10.21203/rs.3.rs-8707138/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8707138/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eAims\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSalt stress severely constrains the yield of oil peony. Although plant growth-promoting fungi alleviate abiotic stress, the specific mechanisms by which they synergistically enhance salt tolerance through rhizosphere remodeling and physiological regulation remain unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe evaluated the efficacy of \u003cem\u003eTrichoderma\u003c/em\u003e inoculation on \u003cem\u003ePaeonia ostii\u003c/em\u003e ‘Fengdan’ under varying NaCl concentrations (0, 100, 200, and 300 mM) via pot experiments. We comprehensively analyzed plant growth phenotypes, physiological indices, anatomical structures, and rhizosphere soil properties.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTrichoderma\u003c/em\u003einoculation markedly mitigated salt-induced damage, evidenced by increased biomass, optimized root system architecture (RSA), and enhanced photosynthetic capacity. Physiologically, \u003cem\u003eTrichoderma\u003c/em\u003e reduced malondialdehyde (MDA) and ROS content by upregulating antioxidant enzymes and inducing osmolyte accumulation. Anatomically, inoculation maintained the integrity of vascular bundle structures in both roots and stems. Furthermore, \u003cem\u003eTrichoderma\u003c/em\u003ecolonization significantly improved the rhizosphere microenvironment by decreasing soil pH and electrical conductivity (EC) while stimulating urease, phosphatase, invertase, and dehydrogenase activities, thereby increasing nutrient bioavailability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollectively, \u003cem\u003eTrichoderma\u003c/em\u003e confers salt tolerance to \u003cem\u003eP. ostii\u003c/em\u003e ‘Fengdan’ through a synergistic mechanism involving the improvement of rhizosphere physicochemical properties, the remodeling of plant anatomical structures, and the activation of physiological metabolism. These findings provide a theoretical basis for cultivating \u003cem\u003eP. ostii\u003c/em\u003e ‘Fengdan’ in saline-alkali lands and validate \u003cem\u003eTrichoderma\u003c/em\u003e as a high-efficiency bio-inoculant for sustainable agriculture.\u003c/p\u003e","manuscriptTitle":"Trichoderma enhances salt tolerance in Paeonia ostii ‘Fengdan’ by regulating the rhizosphere soil environment and physiological mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 14:50:38","doi":"10.21203/rs.3.rs-8707138/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-02-12T01:33:51+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-11T19:27:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2026-02-10T00:39:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-10T00:19:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2026-02-03T04:02:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"139c29d6-479d-4781-b94c-c44c460a96e9","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-17T14:50:39+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 14:50:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8707138","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8707138","identity":"rs-8707138","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}