Effects of Suillus luteus on growth promotion and root phosphorus activation and absorption of Pinus sylvestris var. Mongolica

Effects of Suillus luteus on growth promotion and root phosphorus activation and absorption of Pinus sylvestris var. 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Mongolica huang chuyao, qu zhongxue, he yingqian, chen yongyue, yin dachuan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3914697/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aims : Pinus sylvestris var. Mongolica is a major afforestation tree species in northern China. However, the soil in the natural growth area of P. sylvestris var. Mongolica often lacks nutrients. Applying ectomycorrhizal fungi (ECMF) to seedlings can effectively enhance their root system's absorption of phosphorus. However, how does mycorrhizal fungi activate insoluble phosphorus elements in the rhizosphere? The purpose of this study is to explain how ectomycorrhizal fungi activate insoluble phosphorus in the rhizosphere of seedlings into soluble phosphorus, which is supplied to seedlings for absorption. Methods： P. sylvestris var. Mongolica seedlings were inoculated with Suillus luteus to study the efficacy of ECMF on the performance of P. sylvestris var. Mongolica seedlings. At the same time, the effects of S. luteus on the activation of P in the interrhizosphere and the absorption of P in the rhizosphere of P. sylvestris var. Mongolica were studied by using a space-division grid device. Results： The outcome expressed that S. luteus treatment could enhance the seedling growth. The content of cellulose and lignin increased by 9.36% and 31.16%; And the root growth was significantly improved. Inoculation with ECMF significantly reduced soil pH, and the release of acid phosphatase and organic acids led to a significant increase in soil active phosphorus content. Meanwhile, ECMF could increase P content in root and leaf of P. sylvestris var. Mongolica . Conclusion： S. luteus can activate soil phosphorus, promote the absorption and turnover of P by P. sylvestris var. Mongolica , and improve growth potential. Ectomycorrhizal fungi Pinus sylvestris var. Mongolica Growth promotion Dephosphorization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Phosphorus (P) is the second most critical nutrient element in plants and the main component of energy storage and transport in plants. It is also a constituent of membrane phospholipids and plays an important role in maintaining membrane structural stability (Crous et al. 2015 ) Furthermore, phosphorus is also a major restrictive factor for plant thriving. Although the soil is rich in phosphorus (about 0.05%), most of the phosphorus is in the form of insoluble phosphate. As reported previously, As mentioned earlier, about 80% of the soil phosphorus is not available for plant uptake and utilization (Suriyagoda et al. 2011). Therefore, in agriculture and forestry production, people usually increase the amount of phosphate fertilizer to meet the phosphorus demand of crops. However, phosphate fertilizer is easy to combine with Ca 2+ , Fe 3+ , Al 3+ in soil and form insoluble phosphate (Ji et al. 2021 ; Chen et al. 2021 ). Pinus sylvestris var. Mongolica is a species naturally occurring in northern Greater Khingan Chinese, the Hulun Buir Sands and parts of Russia and Mongolia(Zhu et al. 2006 ;Song et al. 2018 ), Because of its superior qualities like hardiness, drought resistance and barrenness tolerance, it has been brought in and cultivated on a huge scale in the “Three north” sandy region, its plantation has many ecological benefits, such as windbreak, sand fixation, water conservation, agricultural production increase, carbon fixation and oxygen release. At the present time, the area of Pinus sylvestris var. Mongolica plantation in sandy land of China reaches 4.17×10 5 hm 2 (Song et al. 2017 ).Plant element analysis and fertilization experiments show that P. sylvestris var. Mongolica is generally deficient in phosphorus, and phosphorus fertilizer could significantly promote the growth of P. sylvestris var. Mongolica (Song et al. 2003 ; Xie et al. 2004 ).Phosphorus deficiency in soil can decrease the vigour of P. sylvestris var. Mongolica and make it more susceptible to shoot blight (Zhang et al. 2004 ). The healthy growth of P. sylvestris var. Mongolica depends on its symbiotic relationship with ectomycorrhizal fungi (ECMF)(Ren et al. 2023 ). Ectomycorrhizal fungi (ECMF), as one of the most important mycorrhizal fungi, are involved in constituting forest ecosystems (Dai et al., 2021 ).They symbiotically form ectomycorrhizal fungi with tree roots, promoting the absorption of low concentration and weakly mobile nutrients in the soil by tree roots (Tang, 2014 ; Guo, 2014; Li et al., 2014 ), which can alter soil quality and facilitate plant growth (Courty et al., 2008 ; Zhao et al., 2022 ).The mycelium formed by mycorrhizal fungi can not only enter the phosphorus deficient area of plant roots, expand the contact surface with soil phosphorus, but also extend into very fine soil particle gaps, improving the spatial utilization efficiency of plants for soil phosphorus (Bago, 2000 ).Organic acids and protons are secreted by ECMF to dissolve insoluble phosphates in soil and promote plant uptake of phosphorus (Mei et al., 2022 ).For organic phosphorus in soil, the phosphatase secreted during ECMF metabolism can effectively mineralize it (Sun et al., 2022 ).In an additional study, it was found that phosphorus in mycorrhizal fungi mainly comes in two forms: phosphate and polyphosphate, while in mycorrhizal fungi there is only one form of phosphate, indicating that ECMF promotes the absorption of polyphosphate by plant roots (MacFall et al, 1992 ).The ability of ECMF greatly improves the problem of phosphorus deficiency in the plant's inter-root soil and difficulty in absorbing soil phosphorus. In this study, we investigated the effect of ECMF inoculation on the growth of P inus sylvestris var. Mongolica seedlings by inoculating them with Suillus luteus . Simultaneously exploring the improvement effect of S. luteus on phosphorus absorption in P. sylvestris var. Mongolica seedlings. This study helps to further reveal the mechanism by which the S. luteus promotes the growth of P. sylvestris var. Mongolica , providing a reliable theoretical basis for afforestation and ecological management in poor soil, and improving the survival rate of P. sylvestris var. Mongolica afforestation. Materials and Methods Test materials Pinus sylvestris var. Mongolica seedlings were obtained by seed solidification. The test strain was Suillus luteus isolated from Zhanggutai Experimental Forestry, Zhangwu County, Liaoning Province. (42°35’–42°47’N, 12°23’–122°40’E). Test Preparation Fungal spores, which had been cultured for 20 days on PDA medium at pH 6.6, were drilled into 3 pieces of fungal cakes with a 1 cm diameter sterile punch and inoculated into triangular flasks (500 mL) containing 200 mL of PD liquid medium. (500 mL) in a triangular flask containing 200 mL of PD liquid medium. The cultures were shaken on a shaker (25 ℃, 170 r/min) for 30 days to obtain the liquid bacterial agent. Before use, the bacterial agent was poured into a pulverizer, and the mycelium was stirred to make homogenization, and then the bacterial liquid was mixed according to the ratio of homogenization liquid and water 1:3, for inoculation of seedlings(Yin et al., 2020 ).Seeds were surface sterilized with (0.5%, v/v) potassium permanganate solution for 30 min and then rinsed three times with sterile water, then wrapped in sterilized wet gauze and placed in a thermostat at 25°C to germinate for about 7d. Once the seedlings had germinated, move them 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 hours in a 121°C autoclave. (2:1:1v/v/v mixture of charcoal, vermiculite and river sand) with basic parameters of N550 mg/kg, P 9.2 mg/kg, K 85 mg/kg and pH 7.2. Seedlings were inoculated with the fungus by placing pots under greenhouse conditions (day/night temperature difference of 23/9 ± 2°C, 14 h light/10 h dark photoperiod) and watering every 3days for 1 year. Experimental design The experiment consisted two treatments: (1) S. luteus (Sl) and (2) untreated control (CK).The experiment to promote phosphorus activation and absorption was carried out using a “three-chamber culture system”, which can effectively limit the seedling root growth space, hence the elimination of the effect of the root system on the soil between the mycelium, so as to quantitatively analyzing the impact of ECM's extended hyphae on rhizosphere soil. The “three-chamber cultivation system” was made of acrylic plates, and the nylon mesh with a pore size of 30 µm was used to separate the device into a plant growth chamber(10×10×15cm), a buffer chamber(4×10×15cm),and a mycelium chamber(6×10×15cm). Mycorrhizal inoculation was carried out by perforated root irrigation, where a suspension culture of the fungus (100 mL) was added to the root system of each plant. At the same time, the CK treatment was incorporated into the same volume of sterile medium. Each treatment was planted in 10 pots of 5 plants each. Each experiment had 5 replicates, randomly selected among the plants. Experimental methodology Mycorrhizal percentage of colonization Three months after vaccination, seedlings were carefully gathered without destroying the root system and the soil was rinsed. Five seedlings were randomly selected on the basis of mycorrhizal morphology (under stereomicroscope). Determination of mycorrhizal colonization rate using statistical sampling method. Mycorrhizal percentage of colonization was calculated using the following formula: Mycorrhizal percentage of colonization (%) = Number of root segments that colonized by mycorrhiza/Total amount of root segments ×100% Measurement of growth indicators Five seedlings were serially selected at random from each treatment, and the attached soil was washed off the roots under running water. Seedling height and ground diameter were measured using a straightedge and vernier calipers, and their total root length, volume, and surface area were measured in a WinRHIZO root scanning system (WinRHIZO 2012b, Regent Instruments Canada INC., Montreal, Canada). Determination of biomass was done by measuring dry and fresh weights. The fresh weight of the seedlings was measured with an electronic analytical balance, then placed in an oven at 80°C for 30 min and then transferred to 60°C, dried to a constant weight, and weighed the dry weight of the seedlings. Typical ectomycorrhizae were hand-sectioned and photographed with a digital microscope (Phenix XSP-36) to observe mycorrhizal production. Cellulose lignin measurement Three plants were taken from each of the CK and Sl groups, dried, ground and sieved through a 30-mesh sieve, and weighed 0.05 to 0.10 g. The samples were decocted using a mixture of nitric and acetic acids and sulfuric acid, respectively, and the cellulose and lignin contents of the decocted residues were determined by titration using sodium thiosulfate (Xiong et al., 2005 ). Measurement of 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 removing 2 cm of soil from the surface layer, a PVC plate with a thickness of 1 cm was inserted into the left side of the buffer chamber and the soil in the mycelium chamber of the root chamber was pushed out to the right and left, respectively, and the soil of the root chamber and the mycelium chamber was cut longitudinally, and then the soil of the cut-down intermycelial and mycelial intervals was mixed thoroughly respectively, and the soil samples were placed in an air-drying process at a temperature of not higher than 40°C after being mixed, and then sieved through a 2 mm sieve. Processing. 5 ml of air dried soil sample was taken in a 50 ml triangular flask and 25 ml of distilled water was added. A mechanical shaker was used and the mixture was shaken for 60 min and then allowed to rest for 2 h, avoiding the entry of air during this period. At 20 ± 2°C, the suspension was stirred in order to allow the soil particles to be relatively uniformly distributed in the suspension without air entrapment, and then immediately measured using a pH meter (Li et al., 2007 ). Fresh soil samples were collected by removing root fragments and impurities, weighing 5 g in a 50 mL triangular conical flask, adding 25 mL of 0.1% H 3 PO 4 solution, stirring with a glass rod to make the soil solution uniformly mixed, and then oscillating in a reciprocating shaker for 2 h. Afterwards, the solution was centrifuged in a centrifuge at 10000 r/min for 10 min and the supernatant was filtered through a 0.22 µm aqueous filter membrane. Oxalic and citric acid levels were measured using an Agilent 1290 Infinity II ultra performance liquid chromatograph. Determination of acid phosphatase activity and soil effective phosphorus Soil acid phosphatase activity(ACP)was measured with reference to Bao Shidan's "Soil Agrochemical Analysis": ACP activity was assayed by 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. Soil effective phosphorus level was assayed by molybdenum antimony colorimetric ,method as follows: Weigh 2.50 g of air-dried soil samples through a 1 mm sieve in a 150 mL dry triangular flask, add 50 mL of distilled water, keep the liquid temperature at 25 ℃, oscillate for 30 min on a 180 RPM oscillator, and then filter the samples into a dry 150 mL triangular flask using a dry filter paper that does not contain phosphorus. Aspirate 10 mL of filtrate, add 5 mL of molybdenum antimony anticolorant, shake slowly, so that the CO 2 fully escaped, then add 10 mL of distilled water, shake well, exhaust the remaining CO 2 , placed at room temperature for 30 min, placed at 700 nm to measure its absorbance, zeroed with a blank solution as a reference. Combined with the standard curve and the following formula to calculate the soil effective phosphorus content. Soil effective phosphorus (mg/kg) = C×20 C: the concentration of effective phosphorus from the regression equation (mg/L) 20: Liquid-soil ratio during leaching Phosphorus determination in plant tissues and phosphorus energy spectrum analysis in root sections The harvested seedlings were placed in an oven at 105°C for 30 min, followed by drying at 80°C until constant weight, and the samples were pulverized and weighed 0.3g for each treatment and H 2 SO 4 -H 2 O 2 was used (Li., 2020). Absorb 4 mL of the decoction solution in a 50 mL volumetric flask, dilute with water to about 30 mL plus 2 drops of dinitrophenol indicator, adjust the solution pH to 3, then add 5 mL of molybdenum antimony anticolorant, fixed with distilled water to 50 mL, placed for 30 min, measured its absorbance at 880 nm, with a blank solution zeroed as a reference. Tissue phosphorus content of seedlings was calculated by combining the standard curve and the following formula. Tissue phosphorus content P (%) = c × V 3 × (V 1 /V 2 ) × 10 − 3 /m C: concentration of phosphorus (mg/L) from the regression equation; and V 1 : volume of decoction solution fixed (mL); V 2 : volume of decoction solution determined by aspiration (mL); V 3 : volume of color developing liquid (mL); m: mass of dry sample (g). Seedlings were harvested by washing the root system with distilled water and severing the root system under a body mirror. Next, the sample was placed in a glass vial containing the fixative and the air was evacuated from the vial, and the samples were kept in a freezer at 4℃. Root sections were analyzed by phosphorus energy spectrum using a Hitachi Regulus 8100 scanning electron microscope. Data analysis Analysis of variance (ANOVA) was performed using SPSS 13.0 software and differences were tested at P = 0.05, P = 0.01, and P = 0.001. Graphs were plotted using Origin2021. Results Mycorrhizal morphology and infestation rate Figure 2 shows the morphology of mycorrhizae observed under microscope, it can be seen that the root system of P. sylvestris var. Mongolica after symbiosis with S. luteus had a clear bifurcated and branched mycorrhizal structure (Fig. 2 A&B), and the mycorrhizal infestation rate was measured to be 77.92%. The root tips were enlarged and short and thick, while the root tips of the non-inoculated seedlings were small and no mycorrhizal formation was observed (Fig. 2C&D). P.sylvestris var. Mongolica seedling growth S. luteus improved the growth indexes of host plant. the Sl treatment increased the seedling height and diameter of the ground by 25.93% and 25.85%, respectively, relative to the control group (Fig. 3 A); and the fresh and dry weights of S. luteus -inoculated P. sylvestris var. Mongolica seedlings increased by 81.03% and 59.52%, respectively, relative to the blank control group (Fig. 3 B). increased by 81.03% and 59.52%, respectively (Fig. 3 B). None of the above four parts of data reached the level of significant difference (P > 0.05) S. luteus boosted the cellulose lignin content of P. sylvestris var. Mongolica and improved the quality of wood. Sl treatment increased cellulose content by 9.36% and lignin content by 31.16% relative to the control. However, none of the differences were significant (P > 0.05) (Fig. 4) The error bar represents mean ± standard deviation; NS means no significant difference(P > 0.05). S. luteus inoculation treatment significantly improved the root growth of P. sylvestris var. Mongolica seedlings, as reflected by a series of root parameters such as root length, surface area, projected area, root volume, number of root tips and number of forks. After analysis of variance (Table 1), it can be seen that the differences between the different parameters of the root system of the seedlings generating ectomycorrhizae and those of the control group were extremely significant (P < 0.01); all the parameters of the inoculated Sl group, except for the average diameter of the roots, were significantly better than CK group. Inoculated Sl seedlings showed 77.04% increase in root length;73.54% increase in root surface area; 73.54% increase in root projected area; 69.58% increase in root volume; 69.20% increase in the number of root tips; and 82.70% increase in the number of forks compared with the control group. Only the mean root diameter decreased by 16.95% compared to the control group. According to Fig. 5, S. luteus promoted plant shape growth. Tabel 1 Effects of Suillus luteus on root system of Pinus sylvestris var. Mongolica seedlings Treatment Root length (cm) Root surface area(cm 2 ) Root projection area(cm 2 ) Root diameter (mm) Root volume(cm 3 ) Root tip number Number of bifurcations CK 330.46 ± 146.10B 71.13 ± 28.68B 22.64 ± 9.13B 0.69 ± 0.03A 1.22 ± 0.45B 490.20 ± 192.22B 870.80 ± 402.45B Sl 1439.23 ± 666.1A 268.81 ± 122.74A 85.57 ± 39.07A 0.59 ± 0.05B 4.01 ± 1.83A 1591.60 ± 666.47A 5032.20 ± 2085.59A Note: Values are mean ± standard deviation, and different letters in each column represent significant differences between treatments ( P < 0.01). Effect of S. luteus on soil pH, organic acid content, and phosphatase activity After two-way ANOVA analysis, fungi and rhizosphere interaction did not significantly affect acid phosphatase activity (ACP vigor), soil pH, and content of organic acids in the soil, but the presence or absence of brown cyclic lactobacilli significantly affected ACP vigor, soil pH, and content of citric acid as shown in Table 2 . Soil ACP vigor in the mycorrhizae was increased by 256.45% compared to the rhizosphere soil without ECMF inoculation, and ACP vigor was increased by 43.95% compared to the mycorrhizal soil; the difference between the mycorrhizal pH and mycorrhizal pH was not significant, and both of them decreased by 4.88% and 26.54%, respectively, compared to the soil with only rhizosphere; 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 had 20.28% and 25.07% higher citric acid content than both, respectively. Table 2 Effects of Suillus luteus inoculation on soil Acid phosphatase activity, 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 ± 3.48b 1.97 ± 0.97c pH 4.87 ± 0.19a 4.76 ± 0.19a 5.12 ± 0.44a 6.48 ± 0.16b 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 ± 0.60 6.52 ± 0.16 6.75 ± 0.13 5.82 ± 0.33 Note: Values are mean ± standard deviation, and different letters in each column represent significant differences between treatments ( P < 0.05) Soil effective phosphorus and plant tissue phosphorus content Both plant roots and ECMF can dramatically improve effective phosphorus content of the soil, and this ability is maximized when the two symbiotically form ectomycorrhizae. According to Fig. 6 it can be seen that compared to the rooted soil without inoculation of S. luteus , the effective phosphorus content of mycorrhizal and mycelial interstices increased by 63% and 45%, respectively. According to Fig. 7, S. luteus inoculation increased the phosphorus content of P. sylvestris var. Mongolica root system as well as leaf parts, and had no significant effect on stem phosphorus content, with significant differences between Sl and CK leaf phosphorus content. Compared with the CK group, the root phosphorus and leaf phosphorus contents of the Sl group increased by 22% and 137.5%, respectively. The error bar represents mean ± standard deviation; different lowercase letters on the error bars indicate significant differences in ANOVA (P < 0.05). Mycorrhizal soil means that the soil contains both S. luteus and roots; Hyphal soil means that the soil contains only S. luteus and no roots; Root soil means that the soil contains only roots and no S. luteus ; and Blank soil means that the soil contains neither S. luteus nor roots. The error bar represents mean ± standard deviation; different lowercase letters on the error bars indicate significant differences in ANOVA (P 0.05) Energy spectrum analysis of phosphorus in root section under different treatments The energy spectrum of the root system segmental surface was analyzed using the line analysis method (Fig. 8). A straight line was first delineated for the root section, and then the line was scanned. The length of the two sets of delineated straight lines was the same, and after the scanning was completed, 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 elemental level in the region) for the energy spectrum analysis of each point on the line segment.(Fig. 9)Two of the adjacent points were spaced 0.2 μm apart, and the total number of the line sections analyzed was 361.6 μm. A: Con; B: Inoculated with Suillus luteus Inoculation with Lactobacillus marinus increases the Roi value of energy spectrum analysis of root segments of Sphagnum pine seedlings (Fig. 10 ) There was a highly remarkable increase of 30.22% in Roi values in Sl group as against CK group (P < 0.01). The error bar represents mean ± standard deviation;Different majuscule represented different treatments with significant difference (P<0.01) Discussion S. luteus promotes seedling growth and improves wood quality. P. sylvestris var. Mongolica inoculated with S. luteus could see obvious mycorrhizal structures in the roots and the mycorrhizal production rate could reach 77.92%, which indicated that S. luteus and P. sylvestris var. Mongolica had a good fit. In the present study Sl group seedling height and diameter increased by 25.93% and 25.85%, respectively, compared to the control. This is similar to the results of the previous study in which the height of Quercus variabilis seedlings from Beijing and Henan sources inoculated with ECMF was increased by 19.5% and 18.9%, and the diameter of the ground was increased by 14.9% and 14.4%, respectively, compared with the control (Gao et al., 2022 ). When forming mycorrhizal structures with plants, ectomycorrhizal fungi obtain 10–20% of carbohydrates from the host plant, while providing 70% of nutrients, such as nitrogen and phosphorus, to the host (Finlay & Soderstrom 1992 ), and the uptake of these nutrients helped the host plant to grow more luxuriantly. In addition, ectomycorrhizal plants have a greater ability to sequester carbon compared to non-mycorrhizal plants, and the larger leaf area also allows for a higher photosynthetic rate and photosynthesis area, which make up for the organic matter consumed by the ectomycorrhizal fungi, ultimately resulting in the host plant outgrowing the uninoculated control. Sl group in this experiment showed 81.03% and 59.52% increase in fresh and dry weights, respectively, relative to the control. This is congruous with the findings of the study on the growth-promoting effects of different ectomycorrhizal fungi on Tilia amurensis seedlings, in which it was found that the different inoculation treatments increased the biomass of T. amurensis seedlings. In the optimal treatment group, the biomass of the above-ground and below-ground portions of the T. amurensis seedlings added by 38.9 and 31.1%, respectively, compared to the control group (Liu, et al. 2023).ECMF retains the gene for the restricted plant cell wall degrading enzyme PCWDEs, which can regulate the secretion of phytocellulose degrading enzymes by ECMF, resulting in a reduction in the cell wall cellulose content of the host when the fungus is colonized, which is not a complete degradation but mainly lies in symbiosis. S. luteus inoculation increased P. sylvestris var. Mongolica cellulose by 9.36% in this experiment, which is different from the above conclusion. This may be due to the secretion of cellulose degrading enzymes by ECMF mainly during the colonization phase of the fungus, and after the formation of mycorrhizal structures ECMF promotes enhanced growth potential of the host plant and increased cellulose synthesis. Plant symbiotic fungi can promote lignin deposition in host vascular tissues (Yadav et al. 2023 ), in the present study lignin content of Sl was increased by 31.16% compared to CK group, as found in the previous study inoculated with Amanita vaginata , Suillus bovinus , the two ECMF-treated Pinus tabuliformis , the lignin content was elevated by 37.95% and 3.64%, respectively (Chu, 2017), which is consistent with the findings of this experiment. S. luteus promotes root growth of P. sylvestris var. Mongolica .Fungi can influence the root morphology of the host without forming mycorrhizae by secreting phytohormones, such as growth hormone (IAA) and ethylene (ET), to the outside world. For example, fungi can alter the dynamic equilibrium of plant endogenous IAA by secreting IAA, which inhibits the growth of primary roots and increases the branching of lateral roots, a result that facilitates mycorrhizal root formation by fungal colonization (Yu &Yuan, 2023 ). The morphology of the root system was further affected by the formation of mycorrhizal structures, and in this study, a series of indexes, such as root length, root surface area, root projection area, root volume, number of root tips, and number of forks, were highly significantly increased in the treatment group. The results of the previous study using S. luteus , Trichoderma virens and two ECMF composite inoculation of P. sylvestris var. Mongolica proved that all the root growth indexes increased by more than 50.00%, except for the average root diameter, which increased by a small amount. (Qi & Song, 2018 ). Some scholars inoculated Pinus massoniana seedlings with Suillus placidus , Scleroderma citrinum , and the results proved that the 2 mycorrhizal fungi treatments significantly increased the indicators of root length, root tip number, and average diameter of seedlings under normal moisture (P < 0.05). S. placidusk treatment under drought stress could increase root volume, number of root tips, and number of branches by 11.1%, 167%, and 55.8%; and S. citrinum treatment significantly promoted the rest of the indexes except for the number of root tips (Li et al., 2022 ). This shows that ECMF inoculation can promote host root growth. However, in this experiment, the average diameter of roots after inoculation with S. luteus was 16.95% lower than CK group, which may be due to the promotion of the finer lateral root branches and the inhibition of the growth of the thicker primary root by S. luteus , which resulted in a highly significant increase in the number of roots, and a relative decrease in the average diameter of the root system. Secondly, after ECMF colonization, mycelial sheaths or harzian webs are formed mainly around the root tips, limiting the growth of absorbing roots, and ectomycorrhizal species rely mainly on epiphytic mycelium to increase the absorbing area and range of the root system, whereas thick root diameters increase the cost of root construction, and the increased thickness of the cortex affects the lateral transportation of nutrients by the root system, making the construction of thicker root diameters not necessary (Druebert et al. 2009 ). Fine root diameters also lead to higher mycelial foraging precision in ectomycorrhizal tree species (Han et al., 2023 ). Root length, surface area and volume are important parameters to measure the extent of root distribution of a plant, the wider the root distribution of a plant, the larger the area that can be absorbed into the nutrient; Number of root tips and bifurcation is an essential parameter to measure the efficacy of root uptake.The inoculation of S. luteus not only enlarged the area of nutrient uptake, but also increased the efficiency of uptake, which provided a good opportunity to increase the nutrient uptake efficiency for the organic matter in the seedlings of P. sylvestris var. Mongolica seedlings for organic matter formation and biomass accumulation. The mobility of phosphorus in soil is low, and most of the soil phosphorus 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+ , Al 3+ , or chelated by soil colloids. This greatly reduces the amount of effective phosphorus available to plants, and only 20% of soil phosphorus can be absorbed and utilized by plants (Yang et al., 1999 ). And the presence of ECMF will make this situation effectively alleviated. In this study, the effective phosphorus content of inter-mycorrhizal and inter-mycorrhizal soils was increased by 63% and 45%, respectively, compared with the rhizosphere soil not inoculated with S. luteus , and the difference was significant after ANOVA test. When researchers conducted liquid culture of Entoloma clypeatum, they found that the effective phosphorus content of the treatment group under different refractory phosphorus sources increased by 4–11 times compared with that of the control group (Hao et al., 2023 ), which also verified the conclusion that ECMF can activate refractory phosphorus into effective forms. In this study, S. luteus diminished soil pH from 6.48 (blank soil value) to 4.76 (intermycelial value) to 4.87 (intermycorrhizal value) (Table 2 ) significantly. The effect of soil pH decrease is mainly in the following aspects, firstly the acidic environment is favorable for the growth of S. luteus , and the optimal growth pH of S. luteus was found to be around 4.5 in the previous study (Liu, 2008), which shows 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 own survival. The release of protons enhances the solubilization of insoluble phosphates (Illmer et al., 1995 ), and provides the optimal pH for acid phosphatase to function, and the secretion of organic acids, such as citric acid and oxalic acid, is an important factor for the solubilization of insoluble phosphates furthermore the release of protons (Dai et al., 2020 ). For the solubilization of insoluble organic phosphorus, it mainly relies on phosphatase. Previous research found that the solubilization effect of insoluble phosphorus in soil was positively correlated with the activity of phosphatase in soil (Zhong, 2004 ). According to Fig. 11 , it can be seen that soil effective phosphorus content showed highly significant positive relationship with acid phosphatase activity (r = 0.84***)(r = 0.84***) and highly negative relationship with soil pH (r=-0.95***), extremely significantly positively correlated with citric acid content (r = 0.77**), and significantly positively correlated with oxalic acid content (r = 0.60*). It can be seen that ECMF reduces soil pH and releases acid phosphatase by releasing protons and organic acids such as citric acid and oxalic acid, thus further activating insoluble phosphorus in the soil. Previous studies have also analyzed the relationship between pH, phosphatase, organic acids and the dissolution of insoluble phosphorus, such as (Liu et al., 2010 ) showed that the titratable acidity and pH of the fermentation broths of four species of mycorrhizal fungi and their dissolved phosphorus amount showed a highly significant positive correlation (r = 0.991**) and a highly significant negative correlation (r=-0.939**), respectively. were significantly positively correlated (r = 0.998*). Some scholars used six ECMF strains cultured under various insoluble phosphorus sources, and found that acid phosphatase activity was significantly and positively correlated with phosphorus solubilization rate of the strains when calcium phytate and lecithin were used as the phosphorus sources (r = 0.50–0.95); the phosphorus solubilization rate was significantly and positively correlated with citric acid when aluminum phosphorus was used as the phosphorus source (r = 0.55); and the solubilization rate was significantly and negatively correlated with the pH value (r=-0.939**) for all four types of insoluble phosphorus sources. negative correlation (r=-0.88~-0.70) (Jiang et al., 2023 ). It can be seen that ECMF lowers soil pH and releases acid phosphatase by releasing protons and organic acids, thus further activating insoluble phosphorus in soil. The process of activating insoluble phosphorus in soil does not only rely on the secretion of effective substances by ECMF. In the present study, the secretion of phosphatase and organic acid in the mycorrhizal zone was not simply the result of the superposition of root secretion and mycelial zone. This process may be related to the fact that the mycorrhizal interstice is a bacterial community, and it has been found that fructose secreted by mycorrhizal fungi can promote bacterial growth and act as a signaling molecule to stimulate the expression of bacterial phosphatase, phytase, and genes related to the secretion system, which in turn improves the bacterial ability to detoxify phosphorus (Mei et al., 2022 ). After a series of activation by ECMF, the effective phosphorus content of soil was significantly enhanced (Fig. 6 ). S.luteus promotes phosphorus uptake turnover in P.sylvestris var. Mongolica seedlings phosphorus uptake turnover. Many reports have indicated that the uptake and utilization of phosphorus by the host is significantly enhanced by the symbiosis of plant root systems with ECMF (Cao. et al., 2011). Quantitative analysis of this half of the root cross-section energy spectrum in Fig. 9 shows that a decreasing trend of phosphorus from the inter-root surface to the root center was formed in the control root system, whereas in the Sl group, the phosphorus content was relatively stable from the root surface to the root center. This phenomenon reflects that S. luteus has the ability to promote P. sylvestris var. Mongolica ' lateral absorption of phosphorus at the root surface. In this experiment, the phosphorus content of seedlings was quantitatively analyzed, and it can be seen that the treatment group inoculated with S. luteus had their seedlings' root and leaf phosphorus content (Fig. 7) increased compared with the control group, and the semi-quantitative analysis in Fig. 10 also shows that the inoculation of S. luteus can make the Roi value of the root system increase very significantly. Pinus massoniana ectomycorrhizal fungi In this study, it was shown that the N and P contents of the leaves of P. massoniana seedlings inoculated with S. luteus were significantly higher than those of the control under two moisture conditions (Hao, 2023 ), indicating that S. luteus inoculation increased the P content of plant leaves. Some scholars found that inoculation of ECMF can significantly increase the phosphorus content in plant roots, stems and leaves, in which the mixed inoculation of Calvatia uiacina and Cantharelles cibarius Fr. can increase the phosphorus content of all tissues of Rosa sterilis by about 1 times. This shows that inoculation of S. luteus promotes the longitudinal turnover of phosphorus from roots to leaves in seedlings. The results showed that inoculation with ECMF promoted the phosphorus content of P. sylvestris var. Mongolica seedlings seedlings uptake and turnover of phosphorus in soil (Zhang et al., 2016 ). Conclusion The correlation results of this study indicate that P. sylvestris var. Mongolica seedlings and S. luteus form a mycorrhizal structure that increases the area of the plant for nutrient uptake by promoting root growth, improves physiological indices such as seedling height, diameter, fresh weight dry weight, promotes plant growth and improves the quality of wood by promoting the formation of cellulosic lignin. S. luteus secretes organic acids, protons, and phosphatases to make P. sylvestris var. Mongolica seedlings to activate inter-root soil phosphorus and increase the soil effective phosphorus content to facilitate the phosphorus turnover of P. sylvestris var. Mongolica seedlings for phosphorus uptake turnover. The results of this study revealed that in the process of nutrient uptake by plants, S. luteus externally promotes soil phosphorus activation and internally improves the ability of plants to take up and turn over nutrients, which is conducive to P. sylvestris var. Mongolica seedlings growth in poor soil. 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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-3914697","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":272143715,"identity":"2d101c49-70a8-4048-8974-7ae8ed07259f","order_by":0,"name":"huang chuyao","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"huang","middleName":"","lastName":"chuyao","suffix":""},{"id":272143716,"identity":"23936877-eece-45da-b360-1a4c5651caaf","order_by":1,"name":"qu zhongxue","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"qu","middleName":"","lastName":"zhongxue","suffix":""},{"id":272143717,"identity":"b76a89bd-104f-4e5b-a6b8-6bc979894cf0","order_by":2,"name":"he yingqian","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"he","middleName":"","lastName":"yingqian","suffix":""},{"id":272143718,"identity":"fc529bb8-b100-47b2-b13e-2b4dcc6976f4","order_by":3,"name":"chen yongyue","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"chen","middleName":"","lastName":"yongyue","suffix":""},{"id":272143719,"identity":"e93dc0ba-22a5-4ef0-b5fb-0c483a835537","order_by":4,"name":"yin dachuan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYDACZgYGCQaGAyDWAahQAtFa2GBKCWlhgGvhMSBOi8Fx3oM3ftTcSeyf3fP5M0/NYQZ+9hwDhp878Gg5zJds2XPsWeKMO2e3SfMcO8wg2fPGgLH3DD4tPGYSvA2HExtu5G5j5mE7zGBwI8eAmbENvxbJv0At82/kPP7M8+8wgz0xWqRBtmy4kcMgzdsGtEWCgBbJwzzG1jLHDhtvvJFmJjm3L51H4syzgoO9eLTwnT9jePNNzWHZeTeSH394881ajr89eeODn3i0KBxA4jDxAGMHxDiAVS0UyDcgcRh/4FM6CkbBKBgFIxYAAAcJVv6UZpSmAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2217-0671","institution":"Shenyang Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"yin","middleName":"","lastName":"dachuan","suffix":""}],"badges":[],"createdAt":"2024-01-31 17:53:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3914697/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3914697/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51036056,"identity":"850f4cab-b6d9-489e-9012-51569a03e28b","added_by":"auto","created_at":"2024-02-13 05:07:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":24792,"visible":true,"origin":"","legend":"\u003cp\u003eCompartmentalized culture device schematic(Li,1992)\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3914697/v1/91b7a6af766e237ffc808a58.jpg"},{"id":51036059,"identity":"249b3738-e9b7-4b60-a43f-a3eecb31c4d3","added_by":"auto","created_at":"2024-02-13 05:07:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":353946,"visible":true,"origin":"","legend":"\u003cp\u003eMycorrhizal forms.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3914697/v1/4df0b24194ce9bb864595a8f.png"},{"id":51036228,"identity":"e782413d-4e81-4669-9121-0444e51cf6b1","added_by":"auto","created_at":"2024-02-13 05:15:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":210369,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ECMF on growth indicators of \u003cem\u003ePinus sylvestris\u003c/em\u003evar.\u003cem\u003e Mongolica\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA: Seedling height and ground diameter of the plant B: Dry weight fresh weight of the plant\u003cem\u003e \u003c/em\u003e\u0026nbsp;Error bars denote mean ±standard deviation; NS denotes no significant difference (P\u0026gt;0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3914697/v1/5d86638dc1c84bfffb6c9d41.png"},{"id":51036057,"identity":"6314f1c9-e8e5-477e-bafd-f543cbaac0a1","added_by":"auto","created_at":"2024-02-13 05:07:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":70688,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ECMF on the content of cellulose and lignin in \u003cem\u003ePinus sylvestris\u003c/em\u003e var.\u003cem\u003e Mongolica\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3914697/v1/927fae14d95dd130c6e85f6d.png"},{"id":51036067,"identity":"990f0854-7ec0-4a6f-8a85-905e6b66bef6","added_by":"auto","created_at":"2024-02-13 05:07:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":160586,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ECMF on \u003cem\u003ePinus sylvestris\u003c/em\u003e var.\u003cem\u003eMongolicaon\u003c/em\u003e seedlings\u003c/p\u003e\n\u003cp\u003eSl:\u003cem\u003e Suillus luteus\u003c/em\u003e; CK: Control group. Bars=5 cm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3914697/v1/dceb661014c93b7a8f243085.png"},{"id":51036229,"identity":"e5db900b-1af2-42a6-a1c5-1e0944d643a9","added_by":"auto","created_at":"2024-02-13 05:15:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":67121,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eSuillus luteus\u003c/em\u003e inoculation on available phosphorus content in soil\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3914697/v1/9870e221f16d9ebb0adf0219.png"},{"id":51036060,"identity":"66463808-bc58-4b4d-9c21-290b985e4b54","added_by":"auto","created_at":"2024-02-13 05:07:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":43064,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eSuillus luteus\u003c/em\u003e inoculation on phosphorus content in different tissues of \u003cem\u003ePinus sylvestris \u003c/em\u003evar.\u003cem\u003e Mongolica\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3914697/v1/7f5cd1e6d13e841b062fad23.png"},{"id":51036063,"identity":"f791c3fd-6b7d-4b62-84a1-23aa82e1b773","added_by":"auto","created_at":"2024-02-13 05:07:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":831417,"visible":true,"origin":"","legend":"\u003cp\u003ePhosphorus elemental energy spectrum analysis of \u003cem\u003ePinus sylvestris\u003c/em\u003e var. \u003cem\u003emongolica \u003c/em\u003eroot system segment surface（Line analysis）\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3914697/v1/70bb9578f9339a9c4b6ba690.png"},{"id":51036062,"identity":"95091fba-e0c5-4169-a36f-5ba4803ee2d2","added_by":"auto","created_at":"2024-02-13 05:07:37","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":415391,"visible":true,"origin":"","legend":"\u003cp\u003eLine sweep of phosphorus elemental energy spectrum analysis of root sections\u003c/p\u003e\n\u003cp\u003eCK: Control; Sl: \u003cem\u003eSuillus luteus\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3914697/v1/48e6a952dcd1e59bc54c20b5.png"},{"id":51036061,"identity":"700fa07d-73aa-4fa8-9a76-46f23edd773e","added_by":"auto","created_at":"2024-02-13 05:07:37","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":29165,"visible":true,"origin":"","legend":"\u003cp\u003eSemi-quantitative analysis of phosphorus in root systems of different treatments\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-3914697/v1/50c378f84bee5dece9878b41.png"},{"id":51036064,"identity":"1efdfb65-4a2e-44a1-94f7-751cb25b0895","added_by":"auto","created_at":"2024-02-13 05:07:37","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":34055,"visible":true,"origin":"","legend":"\u003cp\u003ecorrelation analysis of soil available phosphorus and physiological indexes under different inoculation treatments\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-3914697/v1/7e5a76a8a3c4531667abe9f0.png"},{"id":51036065,"identity":"76ea36a4-1fae-42dd-b33e-2086c6670d1c","added_by":"auto","created_at":"2024-02-13 05:07:37","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":100517,"visible":true,"origin":"","legend":"\u003cp\u003eActivation and uptake patterns of soil phosphorus by\u003cem\u003e S. luteus\u003c/em\u003e\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-3914697/v1/093c018ffe4091403bc30d10.png"},{"id":53225862,"identity":"92da8620-c9b4-4d9a-a63c-b26fb505799d","added_by":"auto","created_at":"2024-03-22 06:26:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2287312,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3914697/v1/12c82a35-3d6f-4c1f-b688-5dcbbdbd3d72.pdf"}],"financialInterests":"","formattedTitle":"Effects of Suillus luteus on growth promotion and root phosphorus activation and absorption of Pinus sylvestris var. Mongolica","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePhosphorus (P) is the second most critical nutrient element in plants and the main component of energy storage and transport in plants. It is also a constituent of membrane phospholipids and plays an important role in maintaining membrane structural stability (Crous et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) Furthermore, phosphorus is also a major restrictive factor for plant thriving. Although the soil is rich in phosphorus (about 0.05%), most of the phosphorus is in the form of insoluble phosphate. As reported previously, As mentioned earlier, about 80% of the soil phosphorus is not available for plant uptake and utilization (Suriyagoda et al. 2011). Therefore, in agriculture and forestry production, people usually increase the amount of phosphate fertilizer to meet the phosphorus demand of crops. However, phosphate fertilizer is easy to combine with Ca\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e in soil and form insoluble phosphate (Ji et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePinus sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e is a species naturally occurring in northern Greater Khingan Chinese, the Hulun Buir Sands and parts of Russia and Mongolia(Zhu et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2006\u003c/span\u003e;Song et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), Because of its superior qualities like hardiness, drought resistance and barrenness tolerance, it has been brought in and cultivated on a huge scale in the \u0026ldquo;Three north\u0026rdquo; sandy region, its plantation has many ecological benefits, such as windbreak, sand fixation, water conservation, agricultural production increase, carbon fixation and oxygen release. At the present time, the area of \u003cem\u003ePinus sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e plantation in sandy land of China reaches 4.17\u0026times;10\u003csup\u003e5\u003c/sup\u003e hm\u003csup\u003e2\u003c/sup\u003e(Song et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).Plant element analysis and fertilization experiments show that \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e is generally deficient in phosphorus, and phosphorus fertilizer could significantly promote the growth of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e (Song et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).Phosphorus deficiency in soil can decrease the vigour of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e and make it more susceptible to shoot blight (Zhang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The healthy growth of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e depends on its symbiotic relationship with ectomycorrhizal fungi (ECMF)(Ren et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Ectomycorrhizal fungi (ECMF), as one of the most important mycorrhizal fungi, are involved in constituting forest ecosystems (Dai et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).They symbiotically form ectomycorrhizal fungi with tree roots, promoting the absorption of low concentration and weakly mobile nutrients in the soil by tree roots (Tang, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Guo, 2014; Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), which can alter soil quality and facilitate plant growth (Courty et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).The mycelium formed by mycorrhizal fungi can not only enter the phosphorus deficient area of plant roots, expand the contact surface with soil phosphorus, but also extend into very fine soil particle gaps, improving the spatial utilization efficiency of plants for soil phosphorus (Bago, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).Organic acids and protons are secreted by ECMF to dissolve insoluble phosphates in soil and promote plant uptake of phosphorus (Mei et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).For organic phosphorus in soil, the phosphatase secreted during ECMF metabolism can effectively mineralize it (Sun et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).In an additional study, it was found that phosphorus in mycorrhizal fungi mainly comes in two forms: phosphate and polyphosphate, while in mycorrhizal fungi there is only one form of phosphate, indicating that ECMF promotes the absorption of polyphosphate by plant roots (MacFall et al, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1992\u003c/span\u003e).The ability of ECMF greatly improves the problem of phosphorus deficiency in the plant's inter-root soil and difficulty in absorbing soil phosphorus.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the effect of ECMF inoculation on the growth of P\u003cem\u003einus sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings by inoculating them with \u003cem\u003eSuillus luteus\u003c/em\u003e. Simultaneously exploring the improvement effect of \u003cem\u003eS. luteus\u003c/em\u003e on phosphorus absorption in \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings. This study helps to further reveal the mechanism by which the \u003cem\u003eS. luteus\u003c/em\u003e promotes the growth of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e, providing a reliable theoretical basis for afforestation and ecological management in poor soil, and improving the survival rate of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e afforestation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTest materials\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePinus sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings were obtained by seed solidification. The test strain was \u003cem\u003eSuillus luteus\u003c/em\u003e isolated from Zhanggutai Experimental Forestry, Zhangwu County, Liaoning Province. (42\u0026deg;35\u0026rsquo;\u0026ndash;42\u0026deg;47\u0026rsquo;N, 12\u0026deg;23\u0026rsquo;\u0026ndash;122\u0026deg;40\u0026rsquo;E).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTest Preparation\u003c/h2\u003e \u003cp\u003eFungal spores, which had been cultured for 20 days on PDA medium at pH 6.6, were drilled into 3 pieces of fungal cakes with a 1 cm diameter sterile punch and inoculated into triangular flasks (500 mL) containing 200 mL of PD liquid medium. (500 mL) in a triangular flask containing 200 mL of PD liquid medium. The cultures were shaken on a shaker (25 ℃, 170 r/min) for 30 days to obtain the liquid bacterial agent. Before use, the bacterial agent was poured into a pulverizer, and the mycelium was stirred to make homogenization, and then the bacterial liquid was mixed according to the ratio of homogenization liquid and water 1:3, for inoculation of seedlings(Yin et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).Seeds were surface sterilized with (0.5%, v/v) potassium permanganate solution for 30 min and then rinsed three times with sterile water, then wrapped in sterilized wet gauze and placed in a thermostat at 25\u0026deg;C to germinate for about 7d. Once the seedlings had germinated, move them to pots (diameter 20 cm \u0026times; height 14 cm, 20\u0026ndash;30 seeds per pot) and planted in a soil mixture that had been sterilized for 2 hours in a 121\u0026deg;C autoclave. (2:1:1v/v/v mixture of charcoal, vermiculite and river sand) with basic parameters of N550 mg/kg, P 9.2 mg/kg, K 85 mg/kg and pH 7.2. Seedlings were inoculated with the fungus by placing pots under greenhouse conditions (day/night temperature difference of 23/9\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 14 h light/10 h dark photoperiod) and watering every 3days for 1 year.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eThe experiment consisted two treatments: (1) \u003cem\u003eS. luteus\u003c/em\u003e (Sl) and (2) untreated control (CK).The experiment to promote phosphorus activation and absorption was carried out using a \u0026ldquo;three-chamber culture system\u0026rdquo;, which can effectively limit the seedling root growth space, hence the elimination of the effect of the root system on the soil between the mycelium, so as to quantitatively analyzing the impact of ECM's extended hyphae on rhizosphere soil. The \u0026ldquo;three-chamber cultivation system\u0026rdquo; was made of acrylic plates, and the nylon mesh with a pore size of 30 \u0026micro;m was used to separate the device into a plant growth chamber(10\u0026times;10\u0026times;15cm), a buffer chamber(4\u0026times;10\u0026times;15cm),and a mycelium chamber(6\u0026times;10\u0026times;15cm).\u003c/p\u003e \u003cp\u003eMycorrhizal inoculation was carried out by perforated root irrigation, where a suspension culture of the fungus (100 mL) was added to the root system of each plant. At the same time, the CK treatment was incorporated into the same volume of sterile medium. Each treatment was planted in 10 pots of 5 plants each. Each experiment had 5 replicates, randomly selected among the plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExperimental methodology\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eMycorrhizal percentage of colonization\u003c/h2\u003e \u003cp\u003eThree months after vaccination, seedlings were carefully gathered without destroying the root system and the soil was rinsed. Five seedlings were randomly selected on the basis of mycorrhizal morphology (under stereomicroscope). Determination of mycorrhizal colonization rate using statistical sampling method. Mycorrhizal percentage of colonization was calculated using the following formula:\u003c/p\u003e \u003cp\u003eMycorrhizal percentage of colonization (%)\u0026thinsp;=\u0026thinsp;Number of root segments that colonized by mycorrhiza/Total amount of root segments \u0026times;100%\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of growth indicators\u003c/h2\u003e \u003cp\u003eFive seedlings were serially selected at random from each treatment, and the attached soil was washed off the roots under running water. Seedling height and ground diameter were measured using a straightedge and vernier calipers, and their total root length, volume, and surface area were measured in a WinRHIZO root scanning system (WinRHIZO 2012b, Regent Instruments Canada INC., Montreal, Canada). Determination of biomass was done by measuring dry and fresh weights. The fresh weight of the seedlings was measured with an electronic analytical balance, then placed in an oven at 80\u0026deg;C for 30 min and then transferred to 60\u0026deg;C, dried to a constant weight, and weighed the dry weight of the seedlings. Typical ectomycorrhizae were hand-sectioned and photographed with a digital microscope (Phenix XSP-36) to observe mycorrhizal production.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eCellulose lignin measurement\u003c/h2\u003e \u003cp\u003eThree plants were taken from each of the CK and Sl groups, dried, ground and sieved through a 30-mesh sieve, and weighed 0.05 to 0.10 g. The samples were decocted using a mixture of nitric and acetic acids and sulfuric acid, respectively, and the cellulose and lignin contents of the decocted residues were determined by titration using sodium thiosulfate (Xiong et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of soil pH and organic acid content\u003c/h2\u003e \u003cp\u003eThe mycelium chamber and the outer acrylic plate of the root chamber of the partition net device were disassembled, and after removing 2 cm of soil from the surface layer, a PVC plate with a thickness of 1 cm was inserted into the left side of the buffer chamber and the soil in the mycelium chamber of the root chamber was pushed out to the right and left, respectively, and the soil of the root chamber and the mycelium chamber was cut longitudinally, and then the soil of the cut-down intermycelial and mycelial intervals was mixed thoroughly respectively, and the soil samples were placed in an air-drying process at a temperature of not higher than 40\u0026deg;C after being mixed, and then sieved through a 2 mm sieve. Processing.\u003c/p\u003e \u003cp\u003e5 ml of air dried soil sample was taken in a 50 ml triangular flask and 25 ml of distilled water was added. A mechanical shaker was used and the mixture was shaken for 60 min and then allowed to rest for 2 h, avoiding the entry of air during this period. At 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, the suspension was stirred in order to allow the soil particles to be relatively uniformly distributed in the suspension without air entrapment, and then immediately measured using a pH meter (Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFresh soil samples were collected by removing root fragments and impurities, weighing 5 g in a 50 mL triangular conical flask, adding 25 mL of 0.1% H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e solution, stirring with a glass rod to make the soil solution uniformly mixed, and then oscillating in a reciprocating shaker for 2 h. Afterwards, the solution was centrifuged in a centrifuge at 10000 r/min for 10 min and the supernatant was filtered through a 0.22 \u0026micro;m aqueous filter membrane. Oxalic and citric acid levels were measured using an Agilent 1290 Infinity II ultra performance liquid chromatograph.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of acid phosphatase activity and soil effective phosphorus\u003c/h2\u003e \u003cp\u003eSoil acid phosphatase activity(ACP)was measured with reference to Bao Shidan's \"Soil Agrochemical Analysis\": ACP activity was assayed by 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.\u003c/p\u003e \u003cp\u003eSoil effective phosphorus level was assayed by molybdenum antimony colorimetric ,method as follows: Weigh 2.50 g of air-dried soil samples through a 1 mm sieve in a 150 mL dry triangular flask, add 50 mL of distilled water, keep the liquid temperature at 25 ℃, oscillate for 30 min on a 180 RPM oscillator, and then filter the samples into a dry 150 mL triangular flask using a dry filter paper that does not contain phosphorus. Aspirate 10 mL of filtrate, add 5 mL of molybdenum antimony anticolorant, shake slowly, so that the CO\u003csub\u003e2\u003c/sub\u003e fully escaped, then add 10 mL of distilled water, shake well, exhaust the remaining CO\u003csub\u003e2\u003c/sub\u003e, placed at room temperature for 30 min, placed at 700 nm to measure its absorbance, zeroed with a blank solution as a reference. Combined with the standard curve and the following formula to calculate the soil effective phosphorus content.\u003c/p\u003e \u003cp\u003eSoil effective phosphorus (mg/kg)\u0026thinsp;=\u0026thinsp;C\u0026times;20\u003c/p\u003e \u003cp\u003eC: the concentration of effective phosphorus from the regression equation (mg/L)\u003c/p\u003e \u003cp\u003e20: Liquid-soil ratio during leaching\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhosphorus determination in plant tissues and phosphorus energy spectrum analysis in root sections\u003c/h2\u003e \u003cp\u003eThe harvested seedlings were placed in an oven at 105\u0026deg;C for 30 min, followed by drying at 80\u0026deg;C until constant weight, and the samples were pulverized and weighed 0.3g for each treatment and H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e-H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was used (Li., 2020). Absorb 4 mL of the decoction solution in a 50 mL volumetric flask, dilute with water to about 30 mL plus 2 drops of dinitrophenol indicator, adjust the solution pH to 3, then add 5 mL of molybdenum antimony anticolorant, fixed with distilled water to 50 mL, placed for 30 min, measured its absorbance at 880 nm, with a blank solution zeroed as a reference. Tissue phosphorus content of seedlings was calculated by combining the standard curve and the following formula.\u003c/p\u003e \u003cp\u003eTissue phosphorus content P (%)\u0026thinsp;=\u0026thinsp;c \u0026times; V\u003csub\u003e3\u003c/sub\u003e \u0026times; (V\u003csub\u003e1\u003c/sub\u003e/V\u003csub\u003e2\u003c/sub\u003e) \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e/m\u003c/p\u003e \u003cp\u003eC: concentration of phosphorus (mg/L) from the regression equation; and\u003c/p\u003e \u003cp\u003eV\u003csub\u003e1\u003c/sub\u003e: volume of decoction solution fixed (mL);\u003c/p\u003e \u003cp\u003eV\u003csub\u003e2\u003c/sub\u003e: volume of decoction solution determined by aspiration (mL);\u003c/p\u003e \u003cp\u003eV\u003csub\u003e3\u003c/sub\u003e: volume of color developing liquid (mL);\u003c/p\u003e \u003cp\u003em: mass of dry sample (g).\u003c/p\u003e \u003cp\u003eSeedlings were harvested by washing the root system with distilled water and severing the root system under a body mirror. Next, the sample was placed in a glass vial containing the fixative and the air was evacuated from the vial, and the samples were kept in a freezer at 4℃. Root sections were analyzed by phosphorus energy spectrum using a Hitachi Regulus 8100 scanning electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eAnalysis of variance (ANOVA) was performed using SPSS 13.0 software and differences were tested at P\u0026thinsp;=\u0026thinsp;0.05, P\u0026thinsp;=\u0026thinsp;0.01, and P\u0026thinsp;=\u0026thinsp;0.001. Graphs were plotted using Origin2021.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eMycorrhizal morphology and infestation rate\u003c/h2\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows the morphology of mycorrhizae observed under microscope, it can be seen that the root system of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e after symbiosis with \u003cem\u003eS. luteus\u003c/em\u003e had a clear bifurcated and branched mycorrhizal structure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026amp;B), and the mycorrhizal infestation rate was measured to be 77.92%. The root tips were enlarged and short and thick, while the root tips of the\u0026nbsp;non-inoculated seedlings were small and no mycorrhizal formation was observed (Fig. 2C\u0026amp;D).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP.sylvestris\u003c/strong\u003e \u003cstrong\u003evar.\u003c/strong\u003e \u003cstrong\u003eMongolica\u003c/strong\u003e \u003cstrong\u003eseedling growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. luteus\u003c/em\u003e improved the growth indexes of host plant. the Sl treatment increased the seedling height and diameter of the ground by 25.93% and 25.85%, respectively, relative to the control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA); and the fresh and dry weights of \u003cem\u003eS. luteus\u003c/em\u003e-inoculated \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings increased by 81.03% and 59.52%, respectively, relative to the blank control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). increased by 81.03% and 59.52%, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). None of the above four parts of data reached the level of significant difference (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05)\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. luteus\u003c/em\u003e boosted the cellulose lignin content of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e and improved the quality of wood. Sl treatment increased cellulose content by 9.36% and lignin content by 31.16% relative to the control. However, none of the differences were significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;4)\u003c/p\u003e\n\u003cp\u003eThe error bar represents mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation; NS means no significant difference(P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. luteus\u003c/em\u003e inoculation treatment significantly improved the root growth of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings, as reflected by a series of root parameters such as root length, surface area, projected area, root volume, number of root tips and number of forks. After analysis of variance (Table\u0026nbsp;1), it can be seen that the differences between the different parameters of the root system of the seedlings generating ectomycorrhizae and those of the control group were extremely significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01); all the parameters of the inoculated Sl group, except for the average diameter of the roots, were significantly better than CK group.\u003c/p\u003e\n\u003cp\u003eInoculated Sl seedlings showed 77.04% increase in root length;73.54% increase in root surface area; 73.54% increase in root projected area; 69.58% increase in root volume; 69.20% increase in the number of root tips; and 82.70% increase in the number of forks compared with the control group. Only the mean root diameter decreased by 16.95% compared to the control group. According to Fig.\u0026nbsp;5, \u003cem\u003eS. luteus\u003c/em\u003e promoted plant shape growth.\u003c/p\u003e\n\u003cp\u003eTabel 1 Effects of \u003cem\u003eSuillus luteus\u003c/em\u003e on root system of \u003cem\u003ePinus sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTreatment\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRoot length\u003c/p\u003e\n\u003cp\u003e(cm)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRoot surface area(cm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRoot projection area(cm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRoot diameter\u003c/p\u003e\n\u003cp\u003e(mm)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRoot volume(cm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eRoot tip number\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNumber of bifurcations\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCK\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e330.46\u0026thinsp;\u0026plusmn;\u0026thinsp;146.10B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e71.13\u0026thinsp;\u0026plusmn;\u0026thinsp;28.68B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22.64\u0026thinsp;\u0026plusmn;\u0026thinsp;9.13B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e490.20\u0026thinsp;\u0026plusmn;\u0026thinsp;192.22B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e870.80\u0026thinsp;\u0026plusmn;\u0026thinsp;402.45B\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSl\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1439.23\u0026thinsp;\u0026plusmn;\u0026thinsp;666.1A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e268.81\u0026thinsp;\u0026plusmn;\u0026thinsp;122.74A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e85.57\u0026thinsp;\u0026plusmn;\u0026thinsp;39.07A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1591.60\u0026thinsp;\u0026plusmn;\u0026thinsp;666.47A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5032.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2085.59A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNote: Values are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, and different letters in each column represent significant differences between treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of\u003c/strong\u003e \u003cstrong\u003eS. luteus\u003c/strong\u003e \u003cstrong\u003eon soil pH, organic acid content, and phosphatase activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter two-way ANOVA analysis, fungi and rhizosphere interaction did not significantly affect acid phosphatase activity (ACP vigor), soil pH, and content of organic acids in the soil, but the presence or absence of brown cyclic lactobacilli significantly affected ACP vigor, soil pH, and content of citric acid as shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Soil ACP vigor in the mycorrhizae was increased by 256.45% compared to the rhizosphere soil without ECMF inoculation, and ACP vigor was increased by 43.95% compared to the mycorrhizal soil; the difference between the mycorrhizal pH and mycorrhizal pH was not significant, and both of them decreased by 4.88% and 26.54%, respectively, compared to the soil with only rhizosphere; 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 had 20.28% and 25.07% higher citric acid content than both, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eEffects of \u003cem\u003eSuillus luteus\u003c/em\u003e inoculation on soil Acid phosphatase activity, pH and organic acid content\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIndicators\\\u003c/p\u003e\n\u003cp\u003eTreatment\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMycorrhiza soil\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eHyphal soil\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRoot soil\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBlank soil\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eACP(\u0026micro;mol/h/g)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e23.57\u0026thinsp;\u0026plusmn;\u0026thinsp;12.12a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.61\u0026thinsp;\u0026plusmn;\u0026thinsp;3.48b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97c\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16b\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCitrate(\u0026micro;g/ml)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e46.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e38.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.75b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e37.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.27b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e29.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39c\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOxalic acid(\u0026micro;g/ml)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eNote: Values are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, and different letters in each column represent significant differences between treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eSoil effective phosphorus and plant tissue phosphorus content\u003c/h2\u003e\n\u003cp\u003eBoth plant roots and ECMF can dramatically improve effective phosphorus content of the soil, and this ability is maximized when the two symbiotically form ectomycorrhizae. According to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e it can be seen that compared to the rooted soil without inoculation of \u003cem\u003eS. luteus\u003c/em\u003e, the effective phosphorus content of mycorrhizal and mycelial interstices increased by 63% and 45%, respectively.\u003c/p\u003e\n\u003cp\u003eAccording to Fig.\u0026nbsp;7, \u003cem\u003eS. luteus\u003c/em\u003e inoculation increased the phosphorus content of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e root system as well as leaf parts, and had no significant effect on stem phosphorus content, with significant differences between Sl and CK leaf phosphorus content. Compared with the CK group, the root phosphorus and leaf phosphorus contents of the Sl group increased by 22% and 137.5%, respectively.\u003c/p\u003e\n\u003cp\u003eThe error bar represents mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation; different lowercase letters on the error bars indicate significant differences in ANOVA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Mycorrhizal soil means that the soil contains both \u003cem\u003eS. luteus\u003c/em\u003e and roots; Hyphal soil means that the soil contains only \u003cem\u003eS. luteus\u003c/em\u003e and no roots; Root soil means that the soil contains only roots and no \u003cem\u003eS. luteus\u003c/em\u003e; and Blank soil means that the soil contains neither \u003cem\u003eS. luteus\u003c/em\u003e nor roots.\u003c/p\u003e\n\u003cp\u003eThe error bar represents mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation; different lowercase letters on the error bars indicate significant differences in ANOVA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). NS indicates no significant difference.(P\u0026thinsp;\u0026gt;\u0026thinsp;0.05)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eEnergy spectrum analysis of phosphorus in root section under different treatments\u003c/h2\u003e\n\u003cp\u003eThe energy spectrum of the root system segmental surface was analyzed using the line analysis method (Fig. 8). A straight line was first delineated for the root section, and then the line was scanned. The length of the two sets of delineated straight lines was the same, and after the scanning was completed, 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 elemental level in the region) for the energy spectrum analysis of each point on the line segment.(Fig. 9)Two of the adjacent points were spaced 0.2 \u0026mu;m apart, and the total number of the line sections analyzed was 361.6 \u0026mu;m.\u003c/p\u003e\n\u003cp\u003eA: Con; B: Inoculated with \u003cem\u003eSuillus luteus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eInoculation with Lactobacillus marinus increases the Roi value of energy spectrum analysis of root segments of Sphagnum pine seedlings (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e) There was a highly remarkable increase of 30.22% in Roi values in Sl group as against CK group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe error bar represents mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation;Different majuscule represented different treatments with significant difference (P\u0026lt;0.01)\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eS. luteus\u003c/em\u003e promotes seedling growth and improves wood quality.\u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e inoculated with \u003cem\u003eS. luteus\u003c/em\u003e could see obvious mycorrhizal structures in the roots and the mycorrhizal production rate could reach 77.92%, which indicated that \u003cem\u003eS. luteus\u003c/em\u003e and \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e had a good fit. In the present study Sl group seedling height and diameter increased by 25.93% and 25.85%, respectively, compared to the control. This is similar to the results of the previous study in which the height of \u003cem\u003eQuercus variabilis\u003c/em\u003e seedlings from Beijing and Henan sources inoculated with ECMF was increased by 19.5% and 18.9%, and the diameter of the ground was increased by 14.9% and 14.4%, respectively, compared with the control (Gao et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). When forming mycorrhizal structures with plants, ectomycorrhizal fungi obtain 10\u0026ndash;20% of carbohydrates from the host plant, while providing 70% of nutrients, such as nitrogen and phosphorus, to the host (Finlay \u0026amp; Soderstrom \u003cspan class=\"CitationRef\"\u003e1992\u003c/span\u003e), and the uptake of these nutrients helped the host plant to grow more luxuriantly. In addition, ectomycorrhizal plants have a greater ability to sequester carbon compared to non-mycorrhizal plants, and the larger leaf area also allows for a higher photosynthetic rate and photosynthesis area, which make up for the organic matter consumed by the ectomycorrhizal fungi, ultimately resulting in the host plant outgrowing the uninoculated control. Sl group in this experiment showed 81.03% and 59.52% increase in fresh and dry weights, respectively, relative to the control. This is congruous with the findings of the study on the growth-promoting effects of different ectomycorrhizal fungi on \u003cem\u003eTilia amurensis\u003c/em\u003e seedlings, in which it was found that the different inoculation treatments increased the biomass of \u003cem\u003eT. amurensis\u003c/em\u003e seedlings. In the optimal treatment group, the biomass of the above-ground and below-ground portions of the \u003cem\u003eT. amurensis\u003c/em\u003e seedlings added by 38.9 and 31.1%, respectively, compared to the control group (Liu, et al. 2023).ECMF retains the gene for the restricted plant cell wall degrading enzyme PCWDEs, which can regulate the secretion of phytocellulose degrading enzymes by ECMF, resulting in a reduction in the cell wall cellulose content of the host when the fungus is colonized, which is not a complete degradation but mainly lies in symbiosis. \u003cem\u003eS. luteus\u003c/em\u003e inoculation increased \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e cellulose by 9.36% in this experiment, which is different from the above conclusion. This may be due to the secretion of cellulose degrading enzymes by ECMF mainly during the colonization phase of the fungus, and after the formation of mycorrhizal structures ECMF promotes enhanced growth potential of the host plant and increased cellulose synthesis. Plant symbiotic fungi can promote lignin deposition in host vascular tissues (Yadav et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e), in the present study lignin content of Sl was increased by 31.16% compared to CK group, as found in the previous study inoculated with \u003cem\u003eAmanita vaginata\u003c/em\u003e, \u003cem\u003eSuillus bovinus\u003c/em\u003e, the two ECMF-treated \u003cem\u003ePinus tabuliformis\u003c/em\u003e, the lignin content was elevated by 37.95% and 3.64%, respectively (Chu, 2017), which is consistent with the findings of this experiment.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. luteus\u003c/em\u003e promotes root growth of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e.Fungi can influence the root morphology of the host without forming mycorrhizae by secreting phytohormones, such as growth hormone (IAA) and ethylene (ET), to the outside world. For example, fungi can alter the dynamic equilibrium of plant endogenous IAA by secreting IAA, which inhibits the growth of primary roots and increases the branching of lateral roots, a result that facilitates mycorrhizal root formation by fungal colonization (Yu \u0026amp;Yuan, \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). The morphology of the root system was further affected by the formation of mycorrhizal structures, and in this study, a series of indexes, such as root length, root surface area, root projection area, root volume, number of root tips, and number of forks, were highly significantly increased in the treatment group. The results of the previous study using \u003cem\u003eS. luteus\u003c/em\u003e, \u003cem\u003eTrichoderma virens\u003c/em\u003e and two ECMF composite inoculation of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e proved that all the root growth indexes increased by more than 50.00%, except for the average root diameter, which increased by a small amount. (Qi \u0026amp; Song, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Some scholars inoculated \u003cem\u003ePinus massoniana\u003c/em\u003e seedlings with \u003cem\u003eSuillus placidus\u003c/em\u003e, \u003cem\u003eScleroderma citrinum\u003c/em\u003e, and the results proved that the 2 mycorrhizal fungi treatments significantly increased the indicators of root length, root tip number, and average diameter of seedlings under normal moisture (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eS. placidusk\u003c/em\u003e treatment under drought stress could increase root volume, number of root tips, and number of branches by 11.1%, 167%, and 55.8%; and \u003cem\u003eS. citrinum\u003c/em\u003e treatment significantly promoted the rest of the indexes except for the number of root tips (Li et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). This shows that ECMF inoculation can promote host root growth. However, in this experiment, the average diameter of roots after inoculation with \u003cem\u003eS. luteus\u003c/em\u003e was 16.95% lower than CK group, which may be due to the promotion of the finer lateral root branches and the inhibition of the growth of the thicker primary root by \u003cem\u003eS. luteus\u003c/em\u003e, which resulted in a highly significant increase in the number of roots, and a relative decrease in the average diameter of the root system. Secondly, after ECMF colonization, mycelial sheaths or harzian webs are formed mainly around the root tips, limiting the growth of absorbing roots, and ectomycorrhizal species rely mainly on epiphytic mycelium to increase the absorbing area and range of the root system, whereas thick root diameters increase the cost of root construction, and the increased thickness of the cortex affects the lateral transportation of nutrients by the root system, making the construction of thicker root diameters not necessary (Druebert et al. \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). Fine root diameters also lead to higher mycelial foraging precision in ectomycorrhizal tree species (Han et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). Root length, surface area and volume are important parameters to measure the extent of root distribution of a plant, the wider the root distribution of a plant, the larger the area that can be absorbed into the nutrient; Number of root tips and bifurcation is an essential parameter to measure the efficacy of root uptake.The inoculation of \u003cem\u003eS. luteus\u003c/em\u003e not only enlarged the area of nutrient uptake, but also increased the efficiency of uptake, which provided a good opportunity to increase the nutrient uptake efficiency for the organic matter in the seedlings of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings for organic matter formation and biomass accumulation.\u003c/p\u003e\n\u003cp\u003eThe mobility of phosphorus in soil is low, and most of the soil phosphorus exists in the form of inorganic PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e and organic matter that is easily immobilized by metal ions such as Ca\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e, or chelated by soil colloids. This greatly reduces the amount of effective phosphorus available to plants, and only 20% of soil phosphorus can be absorbed and utilized by plants (Yang et al., \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e). And the presence of ECMF will make this situation effectively alleviated. In this study, the effective phosphorus content of inter-mycorrhizal and inter-mycorrhizal soils was increased by 63% and 45%, respectively, compared with the rhizosphere soil not inoculated with \u003cem\u003eS. luteus\u003c/em\u003e, and the difference was significant after ANOVA test. When researchers conducted liquid culture of Entoloma clypeatum, they found that the effective phosphorus content of the treatment group under different refractory phosphorus sources increased by 4\u0026ndash;11 times compared with that of the control group (Hao et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e), which also verified the conclusion that ECMF can activate refractory phosphorus into effective forms.\u003c/p\u003e\n\u003cp\u003eIn this study, \u003cem\u003eS. luteus\u003c/em\u003e diminished soil pH from 6.48 (blank soil value) to 4.76 (intermycelial value) to 4.87 (intermycorrhizal value) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) significantly. The effect of soil pH decrease is mainly in the following aspects, firstly the acidic environment is favorable for the growth of \u003cem\u003eS. luteus\u003c/em\u003e, and the optimal growth pH of \u003cem\u003eS. luteus\u003c/em\u003e was found to be around 4.5 in the previous study (Liu, 2008), which shows 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 own survival. The release of protons enhances the solubilization of insoluble phosphates (Illmer et al., \u003cspan class=\"CitationRef\"\u003e1995\u003c/span\u003e), and provides the optimal pH for acid phosphatase to function, and the secretion of organic acids, such as citric acid and oxalic acid, is an important factor for the solubilization of insoluble phosphates furthermore the release of protons (Dai et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). For the solubilization of insoluble organic phosphorus, it mainly relies on phosphatase. Previous research found that the solubilization effect of insoluble phosphorus in soil was positively correlated with the activity of phosphatase in soil (Zhong, \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e). According to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e, it can be seen that soil effective phosphorus content showed highly significant positive relationship with acid phosphatase activity (r\u0026thinsp;=\u0026thinsp;0.84***)(r\u0026thinsp;=\u0026thinsp;0.84***) and highly negative relationship with soil pH (r=-0.95***), extremely significantly positively correlated with citric acid content (r\u0026thinsp;=\u0026thinsp;0.77**), and significantly positively correlated with oxalic acid content (r\u0026thinsp;=\u0026thinsp;0.60*). It can be seen that ECMF reduces soil pH and releases acid phosphatase by releasing protons and organic acids such as citric acid and oxalic acid, thus further activating insoluble phosphorus in the soil. Previous studies have also analyzed the relationship between pH, phosphatase, organic acids and the dissolution of insoluble phosphorus, such as (Liu et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e) showed that the titratable acidity and pH of the fermentation broths of four species of mycorrhizal fungi and their dissolved phosphorus amount showed a highly significant positive correlation (r\u0026thinsp;=\u0026thinsp;0.991**) and a highly significant negative correlation (r=-0.939**), respectively. were significantly positively correlated (r\u0026thinsp;=\u0026thinsp;0.998*). Some scholars used six ECMF strains cultured under various insoluble phosphorus sources, and found that acid phosphatase activity was significantly and positively correlated with phosphorus solubilization rate of the strains when calcium phytate and lecithin were used as the phosphorus sources (r\u0026thinsp;=\u0026thinsp;0.50\u0026ndash;0.95); the phosphorus solubilization rate was significantly and positively correlated with citric acid when aluminum phosphorus was used as the phosphorus source (r\u0026thinsp;=\u0026thinsp;0.55); and the solubilization rate was significantly and negatively correlated with the pH value (r=-0.939**) for all four types of insoluble phosphorus sources. negative correlation (r=-0.88~-0.70) (Jiang et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). It can be seen that ECMF lowers soil pH and releases acid phosphatase by releasing protons and organic acids, thus further activating insoluble phosphorus in soil.\u003c/p\u003e\n\u003cp\u003eThe process of activating insoluble phosphorus in soil does not only rely on the secretion of effective substances by ECMF. In the present study, the secretion of phosphatase and organic acid in the mycorrhizal zone was not simply the result of the superposition of root secretion and mycelial zone. This process may be related to the fact that the mycorrhizal interstice is a bacterial community, and it has been found that fructose secreted by mycorrhizal fungi can promote bacterial growth and act as a signaling molecule to stimulate the expression of bacterial phosphatase, phytase, and genes related to the secretion system, which in turn improves the bacterial ability to detoxify phosphorus (Mei et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). After a series of activation by ECMF, the effective phosphorus content of soil was significantly enhanced (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS.luteus\u003c/em\u003e promotes phosphorus uptake turnover in \u003cem\u003eP.sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings phosphorus uptake turnover. Many reports have indicated that the uptake and utilization of phosphorus by the host is significantly enhanced by the symbiosis of plant root systems with ECMF (Cao. et al., 2011). Quantitative analysis of this half of the root cross-section energy spectrum in Fig.\u0026nbsp;9 shows that a decreasing trend of phosphorus from the inter-root surface to the root center was formed in the control root system, whereas in the Sl group, the phosphorus content was relatively stable from the root surface to the root center. This phenomenon reflects that \u003cem\u003eS. luteus\u003c/em\u003e has the ability to promote \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e ' lateral absorption of phosphorus at the root surface.\u003c/p\u003e\n\u003cp\u003eIn this experiment, the phosphorus content of seedlings was quantitatively analyzed, and it can be seen that the treatment group inoculated with \u003cem\u003eS. luteus\u003c/em\u003e had their seedlings' root and leaf phosphorus content (Fig.\u0026nbsp;7) increased compared with the control group, and the semi-quantitative analysis in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e also shows that the inoculation of \u003cem\u003eS. luteus\u003c/em\u003e can make the Roi value of the root system increase very significantly.\u003cem\u003ePinus massoniana\u003c/em\u003e ectomycorrhizal fungi In this study, it was shown that the N and P contents of the leaves of \u003cem\u003eP. massoniana\u003c/em\u003e seedlings inoculated with \u003cem\u003eS. luteus\u003c/em\u003e were significantly higher than those of the control under two moisture conditions (Hao, \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e), indicating that \u003cem\u003eS. luteus\u003c/em\u003e inoculation increased the P content of plant leaves. Some scholars found that inoculation of ECMF can significantly increase the phosphorus content in plant roots, stems and leaves, in which the mixed inoculation of Calvatia uiacina and Cantharelles cibarius Fr. can increase the phosphorus content of all tissues of Rosa sterilis by about 1 times. This shows that inoculation of \u003cem\u003eS. luteus\u003c/em\u003e promotes the longitudinal turnover of phosphorus from roots to leaves in seedlings. The results showed that inoculation with ECMF promoted the phosphorus content of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings seedlings uptake and turnover of phosphorus in soil (Zhang et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe correlation results of this study indicate that \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings and \u003cem\u003eS. luteus\u003c/em\u003e form a mycorrhizal structure that increases the area of the plant for nutrient uptake by promoting root growth, improves physiological indices such as seedling height, diameter, fresh weight dry weight, promotes plant growth and improves the quality of wood by promoting the formation of cellulosic lignin. \u003cem\u003eS. luteus\u003c/em\u003e secretes organic acids, protons, and phosphatases to make \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings to activate inter-root soil phosphorus and increase the soil effective phosphorus content to facilitate the phosphorus turnover of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings for phosphorus uptake turnover. The results of this study revealed that in the process of nutrient uptake by plants, \u003cem\u003eS. luteus\u003c/em\u003e externally promotes soil phosphorus activation and internally improves the ability of plants to take up and turn over nutrients, which is conducive to \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings growth in poor soil.\u003c/p\u003e \u003cp\u003eIn summary, the formation of ECM can significantly promote the phosphorus uptake capacity of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings' ability to absorb phosphorus and improve the growth potential of seedlings. It provides a reliable theoretical basis for future poor soil afforestation and ecological management.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003eLiaoning Provincial Department of Education project(LJKZ0684)\u003c/p\u003e\n\u003cp\u003eFunding National Natural Science Foundation of China (31800542).\u003c/p\u003e\n\u003cp\u003eCompliance with Ethical Standards\u003c/p\u003e\n\u003cp\u003eConflict of interest The authors declare that they have no conflicts of interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBago B (2000) Putative sites for nutrient uptake in arbuscular mycorrhizal fungi. Plant Soil 226(2):263\u0026ndash;274\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBao SD (2000) Soil and agricultural chemistry analysis. 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China Academy of Forestry Sciences\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ectomycorrhizal fungi, Pinus sylvestris var. Mongolica, Growth promotion, Dephosphorization","lastPublishedDoi":"10.21203/rs.3.rs-3914697/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3914697/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eAims\u003c/strong\u003e: \u003cem\u003ePinus sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e is a major afforestation tree species in northern China. However, the soil in the natural growth area of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e often lacks nutrients. Applying ectomycorrhizal fungi (ECMF) to seedlings can effectively enhance their root system's absorption of phosphorus. However, how does mycorrhizal fungi activate insoluble phosphorus elements in the rhizosphere? The purpose of this study is to explain how ectomycorrhizal fungi activate insoluble phosphorus in the rhizosphere of seedlings into soluble phosphorus, which is supplied to seedlings for absorption.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods：\u003c/strong\u003e\u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica \u003c/em\u003eseedlings were inoculated with\u003cem\u003e Suillus luteus \u003c/em\u003eto study the efficacy of ECMF on the performance of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e seedlings. At the same time, the effects of \u003cem\u003eS. luteus \u003c/em\u003eon the activation of P in the interrhizosphere and the absorption of P in the rhizosphere of\u003cem\u003e P. sylvestris\u003c/em\u003evar.\u003cem\u003e Mongolica\u003c/em\u003e were studied by using a space-division grid device.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults：\u003c/strong\u003eThe outcome expressed that \u003cem\u003eS. luteus\u003c/em\u003e treatment could enhance the seedling growth. The content of cellulose and lignin increased by 9.36% and 31.16%; And the root growth was significantly improved. Inoculation with ECMF significantly reduced soil pH, and the release of acid phosphatase and organic acids led to a significant increase in soil active phosphorus content. Meanwhile, ECMF could increase P content in root and leaf of \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion：\u003c/strong\u003e\u003cem\u003eS. luteus \u003c/em\u003ecan activate soil phosphorus, promote the absorption and turnover of P by \u003cem\u003eP. sylvestris\u003c/em\u003e var. \u003cem\u003eMongolica\u003c/em\u003e, and improve growth potential.\u003c/p\u003e","manuscriptTitle":"Effects of Suillus luteus on growth promotion and root phosphorus activation and absorption of Pinus sylvestris var. Mongolica","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-13 05:07:32","doi":"10.21203/rs.3.rs-3914697/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"41b5ac35-b674-4c29-a6b5-eb69c24d168d","owner":[],"postedDate":"February 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-18T11:53:21+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-13 05:07:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3914697","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3914697","identity":"rs-3914697","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}