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Learn more: PMC Disclaimer | PMC Copyright Notice BMC Plant Biol . 2026 Mar 4;26:662. doi: 10.1186/s12870-026-08471-4 Search in PMC Search in PubMed View in NLM Catalog Add to search Ectomycorrhizal fungi increase phosphorus uptake in Pinus sylvestris var. mongolica and the mechanism controlling Sphaeropsis sapinea Chuyao Huang Chuyao Huang 1 College of Forestry, Shenyang Agricultural University, Shenyang, 110866 People’s Republic of China Find articles by Chuyao Huang 1 , Dachuan Yin Dachuan Yin 1 College of Forestry, Shenyang Agricultural University, Shenyang, 110866 People’s Republic of China Find articles by Dachuan Yin 1, ✉ Author information Article notes Copyright and License information 1 College of Forestry, Shenyang Agricultural University, Shenyang, 110866 People’s Republic of China ✉ Corresponding author. Received 2025 Nov 3; Accepted 2026 Feb 25; Collection date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13069821 PMID: 41782091 Abstract Background Shoot blight caused by Sphaeropsis sapinea is a major threat to introduced Pinus sylvestris var. mongolica plantations, and effective biocontrol strategies are urgently needed. This study investigated the mechanism by which the ectomycorrhizal fungus (ECMF) Suillus luteus enhances resistance to this disease. We hypothesized that S. luteus inoculation would increase plant phosphorus (P) uptake and modulate the activity of antioxidant enzymes, thereby reducing disease severity. Results Compared with non-mycorrhizal plants, S. luteus -inoculated S. sapinea- infected seedlings resulted in significant differences in antioxidant enzyme activity and tissue phosphorus content. The mycorrhizal plants also showed lower disease rates and lower disease severity indices. Thus, S. luteus inoculation improves the resistance of P. sylvestris var. mongolica seedlings to shoot blight by activating the antioxidant enzyme system in infected plants and regulating internal phosphorus distribution, thereby reducing seedling senescence and damage from shoot blight. Compared with the control, fungi and plant roots significantly decreased soil pH, and the release of acid phosphatase and organic acids led to a significant ( P < 0.05) increase in soil active P content. Moreover, ECMF increased the P content in the roots and leaves of P. sylvestris var. mongolica ( P < 0.05). Correlation analysis revealed a significant negative relationship between root P content and disease severity. Conclusions S. luteus enhances the resistance of P. sylvestris var. mongolica to shoot blight by synergistically improving P acquisition and regulating internal antioxidant defenses. This study provides a new theoretical basis and biological control strategy for the sustainable management of P. sylvestris var. mongolica plantations. Keywords: Suillus luteus , Pinus sylvestris var. mongolica , Sphaeropsis sapinea , Phosphorus Introduction Pinus sylvestris var. mongolica is a geographical variant of P. sylvestris native to the Far East [ 1 , 2 ]. Owing to its strong adaptability, it has emerged as a key species in China’s Three-North Shelterbelt Forest Program [ 3 ]. Shoot blight of P. sylvestris var. mongolica is a typical host-dominant disease that progresses as a tree’s vigor decreases [ 4 ]. Sphaeropsis sapinea- induced shoot blight is currently the leading cause of large-scale declines in P. sylvestris var. mongolica plantations [ 5 ]. Shoot blight caused by S. sapinea is extremely severe in China, with a vast affected area (exceeding 330,000 hectares) and catastrophic economic and ecological losses. This disease is a typical host-dominated disease whose occurrence depends on host growth vigor. Enhancing host nutrient absorption by applying probiotics is an effective strategy for preventing and controlling this disease [ 6 ]. Phosphorus (P) is the second most essential nutrient in plants after nitrogen and is needed for plant growth and development [ 7 , 8 ]. Most metabolic activities in plants, such as cell proliferation, photosynthesis, and signal transduction, require phosphorus [ 9 ]. In addition to its role in energy metabolism and nucleic acid synthesis, P status is crucial for plant defense signaling. P deficiency can impair the salicylic acid (SA)-mediated defense pathway, thereby increasing plant susceptibility to biotrophic and hemibiotrophic pathogens. Conversely, an adequate P supply has been shown to increase lignin deposition and the synthesis of defense-related proteins [ 10 ]. Phosphorus levels are crucial for enhancing plant vigor, enabling robust growth and better defense against host-dominant diseases, which is important for disease control [ 11 ]. Ectomycorrhizal fungi (ECMF) constitute a major fungal group in forests. Extramatrical hyphae, mantles, Hartig nets, and rhizomorphs are morphological features that help plant roots absorb and retain nutrients from the soil while reducing pathogen exposure [ 12 ]. A mycorrhizal structure can increase the absorption area of plant roots, allowing them to absorb more inorganic nutrients. Hyphae can absorb various inorganic elements from the soil and deliver them to plants, thereby increasing plant biomass [ 13 ]. ECMF enhances host P acquisition primarily by extending the root absorption area via extraradical hyphae. More importantly, they secrete protons, organic acids (e.g., citrate and oxalate), and acid phosphatases into the soil, which mobilize insoluble P forms (e.g., Fe–P, Al-P, and Ca-P) and make them available for plant uptake [ 14 , 15 ]. In recent years, the vitality of P. sylvestris var. mongolica plantations has steadily declined, primarily because of severe outbreaks of shoot blight [ 16 – 18 ]. Studies have demonstrated that ectomycorrhizal fungi can increase nutrient uptake in plants, increase host plant vigor, and increase resistance to environmental stress and pests [ 19 ]. This study investigated how Suillus luteus , a natural ectomycorrhizal fungus associated with pines, can increase phosphorus uptake and mitigate declines in P. sylvestris var. mongolica forests. Furthermore, this study aimed to explore the intrinsic relationships among phosphorus, S. luteus , and shoot blight disease in P. sylvestris var. mongolica . These findings provide new insights for enhancing the ecological security of P. sylvestris var. mongolica forests in northern sandy regions and developing effective shoot blight disease management strategies. Based on the above findings, we hypothesized that inoculating P. sylvestris var. mongolica with its native symbiotic fungus, S. luteus (isolated from the same region to ensure ecological relevance), would alleviate shoot blight by improving P nutrition and strengthening the antioxidant system. The objectives of this study were to (1) evaluate the effect of S. luteus on the incidence and severity of S. sapinea -induced shoot blight; (2) assess the changes in antioxidant enzyme activities in response to dual inoculation ( S. luteus + S. sapinea ); and (3) quantify the activation of soil P and its uptake and distribution within plant tissues through the ‘Three-Chamber’ system. Materials and methods Test materials P. sylvestris var. mongolica seedlings were obtained by seed solidification. The seeds were collected from the Zhangwu County Forest Farm of Shenyang Agricultural University in Liaoning Province, China (42°48′N, 122°29′E). The test strains were S. luteus and S. sapinea isolated from Zhanggutai Experimental Forestry, Zhangwu County, Liaoning Province (42°35′–42°47′N, 12°23′–122°40′E). S. luteus was isolated from fruiting bodies growing naturally in a Pinus sylvestris forest; S. sapinea was isolated from diseased needles of P. sylvestris var. mongolica . All these fungal strains were preserved in the Forest Pathology Laboratory at Shenyang Agricultural University. S. luteus and S. sapinea cultured for 20 days on potato dextrose agar (PDA) medium at pH 6.6 were drilled into three pieces of fungal cakes with a 1 cm diameter sterile punch and inoculated in triangular flasks (500 mL) containing 200 mL of potato dextrose (PD) liquid medium. The cultures were shaken on a shaker (25 °C, 170 r/min) for 30 days to obtain the liquid bacterial agent. Before use, the bacterial agent was pulverized, and the mycelium was stirred to produce PD liquid and water at a 1:3 ratio for seedling inoculation [ 3 ]. The seed surfaces were sterilized with 0.5% (v/v) potassium permanganate solution for 30 min and then rinsed three times with sterile water for about 7 days. After the seedlings germinated, they were moved to pots (diameter 20 cm × height 14 cm, 20–30 seeds per pot) and planted in a soil mixture that had been sterilized for 2 h in an autoclave (2:1:1 v/v/v mixture of charcoal, vermiculite, and river sand) at 121 °C. The seedlings were inoculated with S. luteus by placing them in pots under greenhouse conditions (day/night temperature difference of 23/19 ± 2 °C, 14 h light/10 h dark photoperiod) and watering every three days for one year [ 18 ]. Test design The experiment followed a factorial design with two factors: (1) S. luteus inoculation (+ Sl) and (2) the Con group (-Sl). The Con group (control) was inoculated with an equal volume of a 1:3 mixture of PD medium and water. All the plants in the main experiment were inoculated with S. sapinea (+ Ss) to assess disease resistance. The test design was intended primarily to verify the relationship between the presence or absence of mycorrhizae and the disease resistance of P. sylvestris var. mongolica ; thus, the design is not fully factorial for disease assessment. The main objective of this study was to elucidate how the combination of S. luteus and seedlings enhances host resistance under pathogen stress. Therefore, a direct comparison between the “pathogen only” (+ Ss) group and the “mycorrhiza + pathogen” (+ Sl + Ss) group can effectively reveal the specific role of mycorrhizae in the disease resistance process, avoiding interference from other factors under pathogen-free conditions. S. luteus was inoculated into the roots of one-year-old P. sylvestris var. mongolica plants via the root drenching method. Each group contained six replicates, with each seedling receiving 50 mL of the corresponding inoculum. Two months after ectomycorrhizal inoculation, the pine seedlings were inoculated with the fungal pathogen S. sapinea . We selected eight sets of needles from each seedling for pathogen inoculation, each consisting of five bundles (10 needles). Each needle was punctured ten times with sterilized insect pins. We used a sterile punch to extract fungal plugs of S. sapinea from the edge of the culture plates and subsequently applied them to the wounded needles. After wounding, the seedlings were covered with transparent plastic bags for 48 h to maintain high humidity (> 70%) and promote infection, after which they were transferred to a nursery at an average temperature of 25 °C for 30 days of disease development [ 19 ]. Measurement of key indicators Determination of the incidence rate and disease severity index in P. sylvestris var. mongolica . Leaf blight disease was graded according to a standard based on the appearance of disease spots after 30 days of inoculation (see Table 1 ) [ 20 ]. Table 1. Classification of the severity of leaf blight disease in P. sylvestris var. mongolica seedlings Disease severity Grading standards Central value I Less than 10% of the lesion area 0 II The lesion area accounts for 10%–20% 1 III The lesion area accounts for 20%–30% 2 IV The lesion area accounts for 30%–40% 3 V The lesion area accounts for 40%–50% 4 Open in a new tab The incidence rate (IR) and disease severity index (DSI) were calculated using the following formulas [ 20 ]: 1 2 Determination of antioxidant enzyme activities in P. sylvestris var. mongolica The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were determined using commercial assay kits (Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions. Briefly, SOD activity was measured based on its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT). POD activity was assayed by monitoring the increase in absorbance at 420 nm due to the catalysis of guaiacol oxidation by hydrogen peroxide (H 2 O 2 ). CAT activity was evaluated by measuring the decomposition rate of H 2 O 2 at 240 nm over a fixed time period. P. sylvestris var. mongolica roots, stems, and leaves (0.1 g) were pulverized into powder using liquid nitrogen. We added 10 mL of phosphate buffer (pH 7.8, 50 mmol/L) to this powder and centrifuged the suspension at 10,000 rpm for 20 min at 4 °C. The resulting supernatant was used for enzymatic activity analysis. SOD activity was determined in triplicate using a Nanjing Jiancheng assay kit [ 8 ]. The preparation process was identical to that described above. POD and CAT activities were measured in triplicate using a Nanjing Jiancheng assay kit [ 11 ]. Determination of the phosphorus content in diseased P. sylvestris var. mongolica tissues The plants were subsequently uprooted, rinsed, and placed in an oven at 100 °C to deactivate the enzymes. After 30 min, the temperature was readjusted to 75 °C for drying. The dried samples were ground into powder, and 0.3 g of sample material per treatment was digested with H 2 SO 4 -H 2 O 2 . We transferred 4 mL of the digested solution to a 50-mL volumetric flask, diluted it to 30 mL with water, added 2 drops of the dinitrophenol indicator, and adjusted the pH to 3. Afterward, we added 5 mL of molybdenum-antimony colorimetric reagent, diluted it to 50 mL with distilled water, and allowed the mixture to stand. After 30 min, we measured the absorbance at 880 nm and calculated the phosphorus content in the seedling tissues using a standard curve and the following formula: 3 where C is the phosphorus concentration derived from the regression equation (mg/L), V1 is the total volume of the digest solution (mL), V2 is the volume of the digest solution taken for measurement (mL), V3 is the volume of the colorimetric solution (mL), and m is the dry sample mass (g). Effects of S. sapinea stress on P. sylvestris var. mongolica The effects of S. sapinea inoculation on P. sylvestris var. mongolica were comprehensively evaluated via a membership function with the following formula [ 9 ]: 4 where R (X i ) is the membership function value of indicator i for the incidence rate and disease severity index; X i represents the original value of indicator i; X min indicates the minimum value of the i indicator; X max denotes the maximum value of indicator i. The membership function values for the incidence rate and disease severity index of P. sylvestris var. mongolica were calculated for all treatments, with the arithmetic mean serving as the average. The higher this value, the better the comprehensive evaluation and the lower the risk of infection with S. sapinea . An experiment to promote P activation and absorption was performed using a “three-chamber culture system” (Fig. 1 ) [ 8 ], which can effectively limit seedling root growth space, hence eliminating the effect of the root system on the soil between the mycelia, to quantitatively analyze the effect of the extended hyphae of S. luteus on rhizosphere soil. The “three-chamber cultivation system” was made of acrylic plates, and nylon mesh with a pore size of 30 μm was used to separate the device into a plant growth chamber (10 × 10 × 15 cm), a buffer chamber (4 × 10 × 15 cm), and a mycelium chamber (6 × 10 × 15 cm). Those obtained from the root compartment of Sl subjected to the combined action of S. luteus and the plant were mycorrhizal soils, those harvested from the mycelial compartment were hyphal soils, and those obtained from the root systems of the Con plants were root soils. Fig. 1. Open in a new tab Schematic illustration of the compartmentalized culture device. Note: The specifications and related data of the device are shown in the diagram Mycorrhizal inoculation was performed via perforated root irrigation, where a suspension culture of the fungus (100 mL) was added to the root system of each plant. The plants in the Con treatment were combined with the same volume of sterile medium. Each treatment included 10 pots, each with 5 plants. Each experiment had five replicates, which were randomly selected from the plants [ 8 ]. Measurement of the soil pH and organic acid content The mycelium chamber and the outer acrylic plate of the root chamber of the partition net device were disassembled, and after 2 cm of soil was removed from the surface layer, a PVC plate (1 cm thick) was inserted into the left side of the buffer chamber. The soil in the mycelium chamber of the root chamber was pushed to the right and left. The soil of the root chamber and the mycelium chamber was cut longitudinally, the soil of the cut-down intermycelial and mycelial intervals was mixed thoroughly, and the soil samples were placed in an air-drying process at a temperature not higher than 40 °C after being mixed and then sieved through a 2 mm sieve. A 5 mL air-dried soil sample was placed in a 50 mL triangular flask, and 25 mL of distilled water was added. A mechanical shaker was used to shake the mixture for 60 min, after which it was allowed to rest for 2 h; care was taken to prevent air from entering during this period. At 20 ± 2 °C, the suspension was stirred to ensure that the soil particles were relatively uniformly distributed without air entrapment, and the suspension was then immediately measured using a pH meter [ 21 ]. Fresh soil samples were collected by removing root fragments and impurities, weighing 5 g into a 50 mL conical flask, adding 25 mL of 0.1% H 3 PO 4 solution, stirring with a glass rod to uniformly mix the soil, and then oscillating in a reciprocating shaker for 2 h. Next, the solution was centrifuged at 10,000 r/min for 10 min, and the supernatant was filtered through a 0.22 μm aqueous filter membrane. The oxalic and citric acid levels were measured using an Agilent 1290 Infinity II ultraperformance liquid chromatograph [ 8 ]. Determination of acid phosphatase activity and effective P in the soil Soil acid phosphatase (ACP) activity was measured according to the method described by Baar (2000) [ 22 ]. ACP activity was assayed using the colorimetric method of disodium benzoate phosphate and expressed as the mass of p-nitrophenol per gram of soil per unit of time in mg/g/h. The soil effective P (SEP) level was determined by the molybdenum antimony colorimetric method as follows: 2.50 g of each air-dried soil sample was weighed through a 1 mm sieve in a 150 mL dry triangular flask. Afterward, 50 mL of distilled water was added, the temperature of the liquid was maintained at 25 °C, the samples were oscillated for 30 min at 180 rpm using an oscillator, and the samples were subsequently filtered into a dry 150 mL triangular flask using dry filter paper that did not contain P. First, 10 mL of filtrate was aspirated, 5 mL of molybdenum antimony anticolorant was added, and the mixture was shaken slowly so that the CO 2 molecules escaped. Afterward, 10 mL of distilled water was added, the mixture was shaken well, the remaining CO 2 was exhausted, the mixture was incubated at room temperature for 30 min, the absorbance was measured at 700 nm, and a blank solution was used as a reference. The standard curve and the following formula were used to calculate the effective P content in the soil [ 11 ]. 5 where C is the concentration of effective P from the regression equation (mg/L) and where 20 is the liquid-soil ratio during leaching. Phosphorus determination in plant tissues and P energy spectrum analysis in root sections The harvested seedlings were placed in an oven at 105 °C for 30 min and then dried at 80 °C until a constant weight was reached. The samples were pulverized, 0.3 g of sample was weighed for each treatment, and H 2 SO 4 -H 2 O 2 was used [ 23 ]. Next, 4 mL of the decoction solution was added to a 50-mL volumetric flask and diluted to about 30 mL with water, after which 2 drops of the dinitrophenol indicator were added. The solution pH was subsequently adjusted to 3. Of the molybdenum antimony anticolorant, 5 mL was added; the mixture was diluted to 50 mL with distilled water, incubated for 30 min, and the absorbance was measured at 880 nm, with a blank solution used as a reference. The tissue P (TP) content of the seedlings was calculated by combining the standard curve and the following formula. 6 where C is the concentration of P (mg/L) from the regression equation, V 1 is the volume of the decoction solution fixed (mL), V 2 is the volume of the decoction solution determined by aspiration (mL), V 3 is the volume of color-developing liquid (mL), and m is the mass of the dry sample (g). The seedlings were harvested by washing the root system with distilled water and severing it under a body mirror. Next, the sample was placed in a glass vial containing the fixative, the vial was evacuated, and the samples were kept in a freezer at 4 °C. The P-energy spectra of the root sections were analyzed using a Hitachi Regulus 8100 scanning electron microscope. Data processing and analysis All the data were subjected to one-way or two-way analysis of variance (ANOVA) using SPSS 23.0. Before ANOVA, the homogeneity of variance was tested using Levene’s test, and the data were transformed when necessary. Significantly different means ( P < 0.05) were separated using Tukey’s honestly significant difference (HSD) post hoc test. Pearson correlation analysis was performed to examine the relationships between the soil parameters and P availability, with correlation coefficients (r) and P values reported. The graphs were plotted using Origin 2021. Results Mycorrhizal colonization and morphology observation The morphological features of the mycorrhizae observed under a microscope are illustrated in Fig. 2 . The root system of P. sylvestris var. mongolica after symbiosis with S. luteus had a bifurcated and branched mycorrhizal structure (Fig. 2 A & B). The root tips were enlarged, short, and thick, whereas the root tips of the non-inoculated seedlings were small, and no mycorrhizal formation was observed (Fig. 2 C & D). Fig. 2. Open in a new tab Mycorrhizal forms. A & B : Inoculation of S. luteus . C & D : Experimental control group. Bars = 2 mm Effects of Suillus luteus inoculation on the occurrence of Pinus sylvestris var. mongolica shoot blight Following inoculation with S. sapinea , P. sylvestris var. mongolica gradually developed disease symptoms. The needle inoculation sites progressively turned chlorotic, with a bronze hue that slowly transitioned to reddish-brown or even grayish-black. The shoot tips also gradually curved, wilted, and died, with the disease spreading to the branches and trunk, ultimately killing the entire plant. As shown in Table 2 , inoculation with S. luteus significantly reduced both the incidence rate and the disease severity index of pine shoot blight ( P < 0.01). The incidence rate of mycorrhizal plants was 31.67%, and the disease severity index was 8.33. In contrast, the control group had a substantially higher incidence rate (45%) and a disease severity index (27). Visual assessment revealed a clear reduction in disease symptoms in the Sl group compared with those in the Con group. (Fig. 3 ). Table 2. Incidence rates and disease indices of P. sylvestris var. mongolica shoot blight Treatment Incidence rate(%) Disease Severity index Con 45.00 ± 2.65a 27.00 ± 2.65a Sl 31.67 ± 2.08b 8.33 ± 1.44b Open in a new tab SI Inoculation of S. luteus . Con: Experimental control group. The values of the phosphorus content are presented as the means ± SEMs; n = 5. Different letters in each column represent significant differences between treatments ( P < 0.05) Fig. 3. Open in a new tab Incidence of needle leaves. Sl: Inoculation of S. luteus . Con: Experimental control group. Bars = 1 cm The membership function can determine the degree of plant susceptibility to disease. A higher average membership function value indicates reduced plant susceptibility, whereas a lower average value indicates increased susceptibility. As shown in Table 3 , the average membership function value of the treated group (0.61) was greater than that of the control group (0.50), indicating that inoculation with S. luteus can reduce the susceptibility of P. sylvestris var. mongolica seedlings to shoot blight. Table 3. Evaluation of the incidence of P. sylvestris var. mongolica shoot blight under various treatments Treatment Membership function value Phosphorus content Incidence rate Disease Severity index average value Con 0.40 0.60 0.50 0.61 ± 0.15b Sl 0.56 0.67 0.61 0.94 ± 0.59a Open in a new tab SI Inoculation of S. luteus . Con: Experimental control group. The values of the phosphorus content are presented as the means ± SEMs; n = 5. Different letters in each column represent significant differences between treatments ( P < 0.05) Effect of Suillus luteus inoculation on antioxidant enzyme activity in Pinus sylvestris var. mongolica seedlings under Sphaeropsis sapinea stress S. luteus significantly increased the CAT activity in all parts of P. sylvestris var. mongolica infected with shoot blight ( P < 0.01). The root CAT activity increased by 1.48 times, whereas the stem CAT activity in infected plants inoculated with S. luteus was 2.12 times greater than that in non-mycorrhizal plants (Fig. 4 ). Compared with that in the control group, the leaf CAT activity in the experimental group was 15.57 times greater. Fig. 4. Open in a new tab Effect of ECMF on the CAT content in Pinus sylvestris var. mongolica A : Roots, B : Stems, and C : Leaves. The values indicate the means ± SEMs, n = 5, and different letters represent significant differences between treatments ( P < 0.05) S. luteus had varying effects on POD activity in different parts of diseased P. sylvestris var. mongolica seedlings. Ectomycorrhizal inoculation increased POD activity in the roots and stems ( P < 0.01) of infected plants by 6.26% and 24.57%, respectively. However, in the needles, ectomycorrhizal inoculation significantly reduced POD activity by 5.7% compared with that in the control group (Fig. 5 ). Fig. 5. Open in a new tab Effect of ECMF on POD activity in Pinus sylvestris var. mongolica A : Roots, B : Stems, and C : Leaves. The values indicate the means ± SEMs, n = 5, and different letters represent significant differences between treatments ( P < 0.05) In contrast to the results for CAT activity and POD activity, the root, stem, and leaf tissues of P. sylvestris var. mongolica seedlings inoculated with S. luteus presented lower SOD activity than those in the control group. SOD activity levels decreased significantly, by 43.74% and 58.68%, in the roots and stems, respectively ( P < 0.01), whereas leaf SOD activity decreased by 17.84% ( P < 0.05) (Fig. 6 ). Fig. 6. Open in a new tab Effect of ECMF on the SOD activity of Pinus sylvestris var. mongolica A : Roots, B : Stems, and C : Leaves. The values indicate the means ± SEMs, n = 5, and different letters represent significant differences between treatments ( P < 0.05) Soil ACP, pH, and organic acid content under different inoculation treatments Two-way ANOVA revealed that fungal and rhizosphere interactions did not significantly affect ACP vigor, soil pH, or the content of organic acids in the soil. Still, the presence or absence of brown cyclic lactobacilli significantly affected ACP vigor, soil pH, or the content of citric acid (Table 4 ). Compared with that in the rhizosphere soil without ECMF inoculation, the soil ACP vigor in the rhizosphere soil with mycorrhizae increased by 256.45%. In contrast, the ACP vigor increased by 43.95% compared with that in the mycorrhizal soil, and both values decreased by 4.88% and 26.54%, respectively, compared with those in the rhizosphere soil alone. The difference between the citric acid content in the mycorrhizal and rhizosphere soils was not significant, and the citric acid content in the mycorrhizal soil was 20.28% and 25.07% greater than the citric acid content in both samples. Table 4. Effects of S. luteus inoculation on soil ACP, soil pH, and organic acid content Indicators\Treatment Mycorrhiza soil Hyphal soil Root soil Blank soil ACP(μmol/h/g) 23.57 ± 12.12a 16.38 ± 1.07b 6.61 ± 3.48c 1.97 ± 0.97d pH 4.87 ± 0.19b 4.76 ± 0.19b 5.12 ± 0.44b 6.48 ± 0.16a Citrate(μg/mL) 46.69 ± 1.51a 38.82 ± 4.75b 37.33 ± 4.27b 29.63 ± 1.39c Oxalic acid(μg/mL) 6.90 ± 0.60a 6.52 ± 0.16ab 6.75 ± 0.13ab 5.82 ± 0.33b Open in a new tab Sl inoculation of S. luteus . Con: Experimental control group. The values indicate the means ± SEMs; n = 5. Different letters in each row represent significant differences between treatments ( P < 0.05) Effective soil phosphorus and tissue phosphorus contents under different inoculation treatments Plant roots and ECMF can substantially increase the effective P content of soil, and this effect is maximized when the two symbiotically form ectomycorrhizae. Compared with those of the rooted soil without S. luteus inoculation , the effective P contents of the mycorrhizal and mycelial interstices increased by 63% and 45%, respectively (Fig. 7 ). Fig. 7. Open in a new tab Effects of S. luteus inoculation on the effective P content in soil. The values indicate the means ± SEMs, n = 5, and different letters represent significant differences between treatments ( P < 0.05) As shown in Fig. 8 , S. luteus inoculation increased the P content of the P. sylvestris var. mongolica root system and the leaf parts but did not significantly affect the P content of the stem, with significant differences between the Sl and Con leaf P contents. Compared with the Con group, the P content of the roots and leaves of the Sl group increased by 22% and 137.5%, respectively. Fig. 8. Open in a new tab Effects of S. luteus inoculation on the P content in different tissues of P. sylvestris var. mongolica. The values indicate the means ± SEMs, n = 5, and different letters represent significant differences between treatments ( P < 0.05); ns represents no significant difference Scanning electron microscopy and energy spectrum analysis The energy spectrum of the root system segment surface was analyzed using the line analysis method (Fig. 9 ). First, a straight line was drawn along the root section, then it was scanned. The lengths of the two sets of delineated straight lines were the same. After scanning, 1809 points of the root segment from the surface to the center were analyzed to obtain the ROI values (region of interest, which is a representation of the observed element level in the region) for the energy spectrum analysis of each point on the line segment (Fig. 10 ). Two of the adjacent points were spaced 0.2 μm apart, and the total number of line sections analyzed was 361.6 μm. Fig. 9. Open in a new tab Phosphorus element energy spectrum analysis of the P. sylvestris var. mongolica root system segment surface (line analysis) A : Con; B : Inoculated with S. luteus Fig. 10. Open in a new tab Line sweep of P elemental energy spectrum analysis of root sections. Con: Control; Sl: Suillus luteus . The values indicate the means ± SEMs, n = 5, and different letters represent significant differences between treatments ( P < 0.05) Inoculation with S. luteus increased the ROI value of the energy spectrum analysis of the root segments of P. sylvestris var . mongolica seedlings (Fig. 11 ). A highly significant increase of 30.22% in ROI was observed in the Sl group compared with the Con group ( P < 0.01). Fig. 11. Open in a new tab Semiquantitative analysis of P in the root systems under different treatments Analysis of the correlations between various indicators and the occurrence of shoot blight S. luteus significantly increased phosphorus levels in the roots of P. sylvestris var. mongolica . Correlation analysis (Fig. 12 ) revealed a highly significant negative correlation between the root phosphorus content and both the incidence and severity of shoot blight in P. sylvestris var. mongolica , demonstrating that phosphorus absorption strongly inhibits disease. Fig. 12. Open in a new tab Correlations between phosphorus content and P. sylvestris var. mongolica shoot blight Following pathogen infection, plants regulate the rate and distribution of phosphorus transport within their tissues, prioritizing infected areas to ensure adequate phosphorus reserves for active protein phosphorylation, high ATP metabolic consumption, and other resistance-related physiological activities. Ectomycorrhizal fungi regulate phosphorus distribution within plants while suppressing pathogens, minimizing the damage caused by S. sapinea to P. sylvestris var. mongolica . This explains why compared with mycorrhizal plants, infected plants not inoculated with S. luteus presented higher phosphorus levels in their stems and leaves. The soil effective P content was strongly significantly positively related to ACP (r = 0.84 ** ), strongly negatively related to soil pH (r = –0.95***), extremely significantly positively correlated with the citric acid content (r = 0.77**), and significantly positively correlated with the oxalic acid content (r = 0.60*), as shown in Fig. 13 . ECMF decreases soil pH and releases acid phosphatase by releasing protons and organic acids, such as citric and oxalic acids, which further activate insoluble P in the soil. Fig. 13. Open in a new tab Correlation analysis of the available P in the soil and physiological indices under different inoculation treatments Discussion Under adverse growth conditions, plant cells produce high levels of reactive oxygen species (ROS) and repress specific ROS-scavenging functions [ 24 – 26 ]. The accumulated ROS cause further damage to plant cell components such as nucleic acids and cell membranes, triggering the plant ROS scavenging system. The ROS-scavenging system primarily includes CAT, POD, and SOD [ 27 – 29 ]. The coordinated increase in CAT (which detoxifies H 2 O 2 ) alongside a decrease in SOD (which produces H 2 O 2 from O 2– ) in mycorrhizal plants may indicate a more efficient and less disruptive H 2 O 2 flux, potentially serving as a signal for defense activation while minimizing cellular damage. After inoculation with S. luteus , CAT activity in various parts of susceptible plants increased several-fold, demonstrating that ectomycorrhizae can improve disease resistance in P. sylvestris var. mongolica by regulating CAT activity [ 30 , 31 ]. Measurements of the antioxidant enzyme activities in the diseased plants in this work revealed that S. luteus treatment reduced SOD activity more effectively in the root, stem, and leaf tissues of P. sylvestris var. mongolica seedlings. These findings demonstrate that ectomycorrhizal fungi can mitigate the severity of P. sylvestris var. mongolica shoot blight by regulating the activity of multiple antioxidant enzymes. Disease resistance in plants relies heavily on balanced nutritional components. A plant’s ability to resist disease is substantially influenced by the availability and limitations of specific elements during distinct growth stages and under varying environmental conditions [ 32 – 34 ]. The application of phosphorus fertilizer has a significant positive effect on seedling disease susceptibility, primarily by stimulating root growth, which enhances plant resistance to pathogens. In experiments involving phosphorus application to various rapeseed cultivars to assess Sclerotinia resistance, phosphorus consistently improved resistance when the variety and environmental factors were held constant [ 35 – 37 ]. These findings suggest that increasing phosphorus absorption by roots significantly enhances plant disease resistance, consistent with this study's findings. The mobility of P in soil is low, and most soil P exists in the form of inorganic PO 4 3– and organic matter that is easily immobilized by metal ions such as Ca 2+ , Fe 3+ , and Al 3+ or chelated by soil colloids. This greatly decreases the amount of effective P available to plants, and only 20% of soil P can be absorbed and used by plants [ 38 , 39 ]. ECMF helps effectively overcome this limitation. In this study, the effective P contents of the intermycorrhizal and interfuborrhizal soils increased by 63% and 45%, respectively, compared with those of the rhizosphere soil not inoculated with S. luteus , and the difference was significant, as determined by the ANOVA results (Fig. 7 ). Some researchers have conducted liquid culture of Entoloma clypeatum and reported that the effective P content in the treatment group under different refractory P sources increased by 4–11 times compared with that in the non-inoculated group [ 35 , 40 ], further verifying the conclusion that ECMF can activate refractory P into effective forms. In this study, S. luteus significantly decreased the soil pH from 6.48 (blank soil value) to 4.76 (intermycelial value) and 4.87 (intermycorrhizal value) (Table 2 ). The effect of a decrease in soil pH is due mainly to the following factors. An acidic environment favors the growth of S. luteus , and the optimal growth pH of S. luteus was found to be about 4.5 in another study [ 41 ], which revealed that the ECMF first releases protons into the soil when it is planted to reduce the soil pH and create an environment suitable for its survival. The release of protons increases the solubilization of insoluble phosphates [ 42 ] and provides the optimal pH for acid phosphatase to function; the secretion of organic acids, such as citric acid and oxalic acid, is an important factor for the solubilization of insoluble phosphates and the release of protons [ 10 , 43 ]. The solubilization of insoluble organic P relies mainly on phosphatase activity. The solubilization effect of insoluble P in soil is positively correlated with the activity of phosphatase in soil [ 44 ]. ECMF decreases soil pH and releases acid phosphatase by releasing protons and organic acids, further activating insoluble P in the soil. In this study, the P content of the seedlings was quantitatively analyzed, and the root and leaf P contents were greater in the treatment group inoculated with S. luteus than in the control group. The results of the semiquantitative analysis shown in Fig. 12 also revealed that S. luteus inoculation significantly increased the ROI of the root system. In this study, the N and P contents of the leaves of P. sylvestris var . mongolica inoculated with S. luteus were significantly greater than those of the control plants under both moisture conditions, indicating that S. luteus inoculation increased the P content of the leaves. Some researchers have reported that inoculation with ECMF can significantly increase the P content in plant roots, stems, and leaves. Inoculation with Calvatia uiacina and Cantharelles cibarius Fr. also increased the P content of all tissues of Rosa sterilis by about one-fold. [ 45 , 46 ] These findings indicate that inoculation with S. luteus promotes the longitudinal turnover of P from roots to leaves in seedlings. The results revealed that inoculation with ECMF increased the P content in P. sylvestris var. mongolica seedlings and promoted P turnover in the soil [ 47 , 48 ]. Importantly, this study was conducted under controlled greenhouse conditions. Future field trials are necessary to validate the efficacy of S. luteus as a biocontrol agent in natural forest settings. Additionally, while we focused on P and antioxidant enzymes, other mechanisms, such as the induction of defense-related genes or the modulation of phytohormones (e.g., JA and SA) by S. luteus, warrant further investigation. Conclusion Our study provides compelling evidence that the ectomycorrhizal fungus S. luteus significantly enhances the resistance of P. sylvestris var. mongolica to S. sapinea -induced shoot blight. This protective effect is mediated through two mechanisms: (1) enhanced activation and uptake of phosphorus from the soil and (2) systemic modulation of the host’s antioxidant enzyme system upon pathogen challenge (Fig. 14 ). Fig. 14. Open in a new tab Diagrammatic representation of S. luteus enhancing resistance to P. sylvestris var. mongolica shoot blight This study involved experiments in which S. sapinea was inoculated onto P. sylvestris var. mongolica , with and without S. luteus application. Disease incidence, disease severity index, antioxidant enzyme activity, and phosphorus content in plant tissues were measured. The results revealed that inoculation with S. luteus could increase the vigor of P. sylvestris var. mongolica by increasing the average phosphorus content of the plants and regulating their antioxidant enzyme activities. This improvement increases plant resistance to shoot blight, decreasing the disease incidence and severity index of P. sylvestris var. mongolica shoot blight. Thus, S. luteus can increase the resistance of P. sylvestris var. mongolica to S. sapinea by promoting phosphorus absorption. Acknowledgements none Authors’ contributions Huang Chuyao Conceptualization, Methodology, Investigation, Data analysis, and Writing Original Draft.Yin Dachuan Supervision, Project administration, and Writing-Review&Editing. All authors have read and approved the final manuscript. Funding This study was part of the National Natural Science Foundation of China (32572061;31800542) and supported by the Liaoning Science Foundation (Z20250060). Data availability The authors confirmed that experimental research on plants, including the collection of plant material, complies with relevant institutional, national, and international guidelines and legislation. The plant material used in the study was not a wild species, and permission for the use of the plant material was obtained. The voucher specimen was deposited in the Herbarium of Shenyang Agricultural University, with the catalog number ZZS001, and was identified by Yin Dachuan. All data supporting the findings of this study are presented within the article; no external data repository was used. Data are available from the corresponding author upon reasonable request. All data supporting the findings of this study are presented within the article; no external data repository was used. Data are available from the corresponding author upon reasonable request. All data supporting the findings of this study are presented within the article; no external data repository was used. Data are available from the corresponding author upon reasonable request. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. 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All data supporting the findings of this study are presented within the article; no external data repository was used. Data are available from the corresponding author upon reasonable request. All data supporting the findings of this study are presented within the article; no external data repository was used. Data are available from the corresponding author upon reasonable request. All data supporting the findings of this study are presented within the article; no external data repository was used. Data are available from the corresponding author upon reasonable request. 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