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Article

Effects of N-P-K Ratio in Root Nutrient Solutions on Ectomycorrhizal Formation and Seedling Growth of Pinus armandii Inoculated with Tuber indicum

by
Li Huang
1,†,
Rui Wang
1,†,
Fuqiang Yu
2,
Ruilong Liu
1,
Chenxin He
3,
Lanlan Huang
4,
Shimei Yang
1,2,
Dong Liu
2,* and
Shanping Wan
1,*
1
College of Resources and Environment, Yunnan Agricultural University, Kunming 650201, China
2
Germplasm Bank of Wild Species & Yunnan Key Laboratory for Fungal Diversity and Green Development & Yunnan International Joint Laboratory of Fungal Sustainable Utilization in South and Southeast Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
3
Meteorological Institute of Shaanxi Province, Xi’an 710016, China
4
College of Yunnan Rural Revitalizing Education, Yunnan Open University, Kunming 650221, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(7), 1749; https://doi.org/10.3390/agronomy15071749
Submission received: 23 June 2025 / Revised: 17 July 2025 / Accepted: 18 July 2025 / Published: 20 July 2025
(This article belongs to the Special Issue Innovations and Obstacles: Microbial Communities in the Journey of Soil Remediation)
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Abstract

Ectomycorrhizal symbiosis is a cornerstone of ecosystem health, facilitating nutrient uptake, stress tolerance, and biodiversity maintenance in trees. Optimizing Pinus armandii–Tuber indicum mycorrhizal synthesis enhances the ecological stability of coniferous forests while supporting high-value truffle cultivation. This study conducted a pot experiment to compare the effects of three root nutrient regulations—Aolu 318S (containing N-P2O5-K2O in a ratio of 15-9-11 (w/w%)), Aolu 328S (11-11-18), and Youguduo (19-19-19)—on the mycorrhizal synthesis of P. armandii–T. indicum. The results showed that root nutrient supplementation significantly improved the seedling crown, plant height, ground diameter, biomass dry weight, and mycorrhizal infection rate of both the control and mycorrhizal seedlings, with the slow-release fertilizers Aolu 318S and 328S outperforming the quick-release fertilizer Youguduo. The suitable substrate composition in this experiment was as follows: pH 6.53–6.86, organic matter content 43.25–43.49 g/kg, alkali-hydrolyzable nitrogen 89.25–90.3 mg/kg, available phosphorus 83.69–87.32 mg/kg, available potassium 361.5–364.65 mg/kg, exchangeable magnesium 1.17–1.57 mg/kg, and available iron 33.06–37.3 mg/kg. It is recommended to mix the Aolu 318S and 328S solid fertilizers evenly into the substrate, with a recommended dosage of 2 g per plant. These results shed light on the pivotal role of a precise N-P-K ratio regulation in fostering sustainable ectomycorrhizal symbiosis, offering a novel paradigm for integrating nutrient management with mycorrhizal biotechnology to enhance forest restoration efficiency in arid ecosystems.

1. Introduction

Truffles (Tuber) refer to a group of subterranean fungi with important economic and ecological values in the Tuberaceae, Pezizales, and Ascomycota [1,2]. Recent taxonomic and molecular phylogenetic studies of the Tuber have shown that at least 82 species exist in China [3]. As the value of truffles continues to rise, over-harvesting has led to the severe destruction of truffle habitats, causing a yearly decline in production and an increasingly serious threat to their existence [4]. In recent years, in order to protect and stabilize the natural resources and ecological environment, as well as to meet the market demand for edible ectomycorrhizal fungi, the artificial cultivation and resource conservation of ectomycorrhizal fungi have attracted much attention [5].
Truffles are among the world’s most valuable edible ectomycorrhizal fungi (EMF), capable of forming mutually beneficial mycorrhizal symbioses with dominant tree species in northern temperate forests, such as those in the genera Pinus (pines), Picea (spruces), Populus (poplars), Salix (willows), Corylus (hazels), and Quercus (oaks) [2]. EMF serve as a crucial interface between the soil matrix and host plants, facilitating bidirectional material exchange and signal transmission.
The formation of mycorrhizae not only makes the artificial cultivation of EMF possible but also enhances the ability of plants to absorb carbon, nitrogen, phosphorus, water, and mineral elements [6]. Specifically, in terms of improving resistance to abiotic stresses, EMF can expand the absorption range of host plant roots through their extensive mycelial networks, enabling more efficient uptake of soil moisture and nutrients under drought or nutrient-deficient conditions and alleviating stress [7]. For example, under drought stress, the hyphae of EMF can penetrate into tiny soil pores that root hairs cannot reach, absorbing and transporting water to the host plant while also regulating the expression of plant drought-responsive genes to reduce water loss [8]. In the case of heavy metal stress, EMF can sequester heavy metal ions in their mycelia through chelation and compartmentalization, preventing these toxic substances from entering the host plant’s vascular system, thus reducing damage [9]. Additionally, EMF can enhance the stability of plant cell membranes and the activity of antioxidant enzymes, helping plants mitigate oxidative damage caused by high temperatures, salinization, and other stresses [10,11,12,13,14]. Regarding carbon elimination and cycling, EMF play a vital role in the forest carbon cycle. Host plants transfer a portion of their photosynthetically fixed carbon to EMF, which is used for the growth and metabolism of the fungi. A significant portion of this carbon is allocated to the mycelial network in the soil, where some is decomposed into organic matter and stored in the soil, contributing to long-term carbon sequestration [15,16]. This process not only reduces the concentration of carbon dioxide in the atmosphere but also improves the soil’s organic carbon content, enhancing soil fertility and stability [17].
Due to the high profitability of truffles, their production has promoted the use of agricultural land in low-productivity areas through private forestation, as well as important associated ecosystem services such as carbon fixation and soil quality improvement [18]. Thus, EMF play a significant role in constructing forest ecological networks and maintaining the health and stability of ecosystems [19].
The soil matrix is a critical medium for the growth of ectomycorrhizal seedlings. To ensure the normal growth of both the seedlings and mycorrhizae, the soil must continuously and appropriately provide and balance essential factors for plant growth, such as water, nutrients, air, heat, and other necessary conditions [20]. Soil properties, including pH and nutrients (such as nitrogen, phosphorus, potassium, magnesium, calcium, and organic carbon), significantly influence the growth of EMF [2]. These soil environmental variables, in turn, interact with EMF, helping to regulate the carbon and nutrient balance at the interface between the soil matrix and the host plant [21,22].
Previous studies have found that EMF yields are higher in fertile soils compared to barren ones. The reasons for these differential results may lie in the effectiveness of soil background nutrients and the impact of exogenous nutrient additions, which influence the degree of nutrient limitation and alleviation in EMF fungi, thereby affecting the direction and extent of the mycelial response to nutrients. Throughout this extended life cycle, soil properties and climatic conditions are considered to be critical for the development of black truffle sporocarps [23,24]. Research indicates that phosphorus (P) is one of the key factors influencing truffle mycelial dynamics in natural forest plantations during black truffle production [25]. Meanwhile, the availability of nitrogen (N) significantly affects the growth characteristics (such as biomass, yield, density, and turnover rate) and functional traits of EMF mycelia [26,27]. Appropriate nitrogen supplementation increased EMF mycelial yield, biomass, and density by 79%, 39%, and 73%, respectively. Additionally, proper nitrogen application can enhance EMF phosphatase activity, thereby promoting phosphorus absorption in plants [28,29,30]. Recent evidence suggests that this relationship follows an optimum curve rather than a linear trend. In particular, for truffles, Barou et al. [25] revealed that phosphorus (P) availability shows a non-monotonic relationship with T. melanosporum mycelial biomass; it is beneficial up to 20 mg kg−1 but inhibitory at higher concentrations in plantation soils. Similarly, while nitrogen (N) availability significantly influences EMF’s growth characteristics (biomass, yield, and density) and functional traits [26,27], excessive N can disrupt the mutualistic balance, as demonstrated by the increases in mycelial parameters being contingent on maintaining appropriate C: N ratios [24].
Research has shown that inoculating EMF and the addition of a low amount of nitrogen (15 kg/(hm2·a)) exhibit a synergistic effect on the root morphology and nutrient content of P. sylvestris var. mongolica seedlings [31]. Through EMF inoculation and exponential fertilization, the growth indicators, nutrient accumulation (N and P), and mycorrhizal colonization rate of Scots pine seedlings were significantly higher than those under conventional fertilization [32]. In addition to using external additions to regulate the nutrient composition of the substrate [33], a reasonable substrate composition is also a key factor for the growth of mycorrhizal seedlings. Dupre et al. [34] studied the effects of different substrates, such as soil, vermiculite, and perlite, as well as factors such as soil N, P, and K content, on the mycorrhizal synthesis of T. melanosporum. The results showed that vermiculite as a growth substrate was the most conducive to the formation of truffle mycorrhizae, followed by perlite, while forest topsoil performed the worst. Chen et al. [35] used a mixed substrate of vermiculite, peat, and river sand in a certain volume ratio to inoculate T. melanosporum during the seedling stage of Castanopsis hystrix, achieving a mycorrhizal infection rate as high as 90% after 26 weeks, indicating that the mixed soil nutrients and structure are very suitable for the growth of T. melanosporum mycorrhizal seedlings. The growth and development of T. indicum mycorrhizae are also affected by soil nutrients and structure; sufficient nutrients and favorable soil environmental conditions increase the number of T. indicum mycorrhizae [36].
In addition, vegetation type is a fundamental factor influencing soil fungal communities and controlling the ecological functions of mycorrhizal systems [37,38,39]. Vegetation type can alter the physical and chemical properties of the soil (such as nutrient availability and pH) due to differences in growth rates, litter, and root exudates, thereby affecting the formation and growth of mycorrhizal fungi [40,41]. Therefore, the type of host plant can lead to significant differences in mycelial biomass due to its utilization of available soil nutrients. Research has shown that nitrogen application can increase mycelial biomass in P. armandii forests by 39%; specifically, nitrogen application can significantly increase the relative abundance of truffle mycelia in these forests [26]. Moreover, the strong selectivity and specificity of truffles for host tree species are evident even at the seedling stage, where the root morphology of the host plant can influence the appearance of the formed mycorrhizae. Thus, selecting suitable symbiotic plants for inoculation is not only a prerequisite for cultivating mycorrhizal seedlings but also a key factor in the success of truffle cultivation [42,43].
Pinus armandii, one of China’s native pine species, plays a significant role in ecological protection and regional socio-economic development. Proper fertilization has a marked effect on improving seedling productivity and expanding the spread of the root system. In particular, the use of nitrogen (N) and combinations of nitrogen with other plant nutrients can effectively shorten the harvesting period of trees and enhance the yield and quality of timber [44,45,46,47]. It has been pointed out that appropriate fertilization methods, substrate combinations, and fertilizer ratios can significantly promote the growth of container seedlings of P. armandii [48,49,50]. Pinus armandii is an ideal host for the synthesis of truffle mycorrhizae [51]. For truffle mycorrhizal seedlings, proper fertilization can promote the growth of both P. armandii and mycelia, thereby increasing the mycorrhizal colonization rate, which is crucial for the subsequent growth of truffles and the improvement of their yield.
In the field of mycorrhizology, research on the effects of fertilization on ectomycorrhizae is far less extensive than on other types of mycorrhizae. In particular, studies on the impact of fertilization on specific economically valuable mycorrhizal fungi and host combinations are rarely reported. While previous studies have examined ectomycorrhizal relationships [10,23] and fertilization effects on tree growth [34,46], few have systematically investigated how specific N-P-K ratios in root nutrients affect the economically important T. indicum–P. armandii symbiosis. Notably, no studies have compared slow-release versus quick-release fertilizers for this specific host–fungus combination. Thus, in this study, we hypothesized that slow-release fertilizers (Aolu 318S and 328S) with balanced N-P-K ratios would promote superior ectomycorrhizal formation and seedling growth compared to a quick-release fertilizer (Youguduo) due to sustained nutrient availability that matches fungal and plant requirements. An optimal substrate nutrient range exists that simultaneously maximizes both T. indicum mycorrhization and P. armandii growth. The interaction between specific N-P-K ratios and mycorrhization would create synergistic effects on plant performance beyond additive individual effects. Therefore, this study employed a pot experiment to comparatively analyze the effects of three types of compound fertilizers, Aolu 318S, Aolu 328S, and Yuguoduo, on the mycorrhizal synthesis of T. indicum with P. armandii and the growth of mycorrhizal seedlings. By analyzing changes in the seedling crown, plant height, ground diameter, aboveground and belowground biomass, substrate nutrient content, and the number of mycorrhizae of the seedlings, this study aims to explore the optimal nutrient substrate ratio and fertilization effect, providing valuable insights for the artificial cultivation of truffles, mycorrhizology research, and the efficient cultivation of robust P. armandii seedlings.

2. Materials and Methods

2.1. Sterile Seedling Cultivation and Inoculation

The experimental design used a mixed substrate with a peat/vermiculite/perlite volume ratio of 2:2:1. The moisture content was adjusted to 30%, and the substrate was sterilized at 121–126 °C. The pH was adjusted to 7.5 using calcium carbonate and magnesium carbonate. Mature ascomata of T. indicum were used as the inoculum, with an inoculation concentration of 5 × 106 spores/mL and an inoculation amount of 0.5 g per seedling. The inoculation time was 5 March 2023. Pinus armandii was fertilized on 5 June 2023 after inoculation. The seedlings used for inoculation were cultivated from germinated seeds of P. armandii. The spore suspension was inoculated onto the seedling roots in two stages, followed by covering with the substrate. Mycorrhizal formation was examined at 90 days (5 September 2023), 180 days (5 December 2023), and 270 days (5 March 2024) after fertilization. Each time, 5 seedlings were randomly selected from each of the 8 treatment groups for inspection. All seedlings were cultivated in an outdoor environment with good ventilation and sufficient sunlight and managed under conventional practices with alternating dry and wet cultivation conditions throughout the growth period.

2.2. Experimental Design for the Fertilization and Cultivation of Mycorrhizal Seedlings

This experiment used three types of compound fertilizers (Table 1) and designed 8 experimental treatments: CK (control), T (inoculation with T. indicum), 318S (application of Aolu 318S alone), 328S (application of Aolu 328S alone), P19 (application of Yuguoduo alone), T318S (inoculation with T. indicum + Aolu 318S), T328S (inoculation with T. indicum + Aolu 328S), and TP19 (inoculation with T. indicum + Yuguoduo). Fertilization was carried out three months after inoculation. Aolu 318S and Aolu 328S were evenly mixed into the substrate as solid fertilizers, with an application rate of 2 g per pot; Yuguoduo was diluted 600 times with water and applied to the roots at a rate of 0.66 g per pot. Each treatment group contained 15 seedlings, with a total of 120 seedlings across the 8 treatment groups. Mycorrhizal inspections were conducted at 90 days, 180 days, and 270 days after fertilization, with 5 seedlings randomly selected from each of the 8 treatment groups for inspection each time.

2.3. Methods for the Inspection, Identification, and Statistical Analysis of Mycorrhizae

Mycorrhizal inspection is conducted by observing morphological changes in plant roots or examining root tissues under a microscope to determine whether mycorrhizae have formed. For mycorrhizal identification, molecular-level identification is performed on the formed mycorrhizae. Before extracting DNA from mycorrhizal root tips using the modified CTAB method [52], the root tips must first be cleaned by rinsing gently to remove any attached substrate particles, then be soaking the roots repeatedly in a beaker filled with water to wash away impurities. After DNA extraction, PCR amplification and sequencing are carried out. The sequencing chromatograms are proofread and assembled using the sequence assembly software SeqMan v7.0. After removing low-quality sequences and primer regions, the sequences are uploaded to NCBI for NCBI Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 1 July 2025)) to confirm that the colonized mycorrhizae are the target mycorrhizae.
After completing mycorrhizal verification, statistical analysis is conducted using a stereomicroscope and a root scanner. First, root parameters are obtained by scanning with the root scanner. Then, the roots are observed under the stereomicroscope to count the mycorrhizal infection rate. The calculation formula is: mycorrhizal infection rate (%) = number of mycorrhizae/number of nutrient roots × 100%. Experimental data were analyzed using Microsoft Excel 2010 and SPSS 20.0.

2.4. Measurement of the Seedling Crown, Plant Height, Ground Diameter, and Aboveground and Belowground Biomass of Mycorrhizal Seedlings

For morphological index measurements, the dry weight of the seedlings in each treatment container was determined using an analytical balance. The ground diameter was measured with a vernier caliper, and the plant height and seedling crown were measured with a standard measuring tape. The determination of the dry weight of P. armandii seedlings requires drying treatment, with the specific procedure as follows. First, the seedlings are blanched at 105 °C for 30 min to quickly terminate cellular physiological activities and prevent substance decomposition. Then, they are placed in an environment at 80 °C to dry to constant weight. Among these steps, continuous drying at a low temperature of 80 °C can not only effectively remove moisture but also reduce the loss of volatile components in the seedlings. “Drying to constant weight” means that the weight difference between two weighings (with an interval of more than 2 h) does not exceed 0.001 g, which ensures the complete removal of moisture and guarantees the stability and accuracy of the determination results.

2.5. Measurement Indicators and Analytical Methods for Nutrient Substrates

The measurement methods for pH, organic matter, alkali-hydrolyzable nitrogen, available phosphorus, available potassium, exchangeable calcium and magnesium, and available iron in the nutrient substrate refer to those outlined in “Soil Agrochemical Analysis”, edited by Bao Shidan [53].

2.6. Statistical Analysis

To explore the significant differences in variables among different treatments, one-way analysis of variance (ANOVA) was conducted at a 95% confidence level (p < 0.05), followed by Tukey’s honest significant difference test (Tukey’s HSD). When the data showed a normal distribution and homogeneous variance, Tukey’s HSD test was used; if these assumptions were not met, the Kruskal–Wallis H non-parametric test was adopted instead.

3. Results

3.1. Effects of Different Treatments on the Seedling Crown, Plant Height, and Ground Diameter of T. indicum–P. armandii Seedlings

At the same growth stage, the growth of the seedling crown, plant height, and ground diameter of P. armandii varied under different fertilization treatments (Figure 1A–C, p < 0.05). Among the non-inoculated treatments, P. armandii seedlings treated with Aolu 318S and 328S had significantly higher seedling crowns, plant heights, and ground diameters compared to those treated with Yuguoduo and the control group (p < 0.05), while no significant difference was found between Aolu 318S and 328S (p > 0.05). The difference between Yuguoduo and the control group was not significant (p > 0.05).
Similarly, among the inoculated treatments, seedlings treated with Aolu 318S and 328S also exhibited significantly higher seedling crowns, heights, and ground diameters compared to those treated with Yuguoduo and the group inoculated with T. indicum alone (p < 0.05), with no significant difference between Yuguoduo and the group inoculated with T. indicum alone (p > 0.05).

3.2. Effects of Different Treatments on the Aboveground and Belowground Biomass Dry Weight of T. indicum–P. armandii Seedlings

At different growth stages, the aboveground and belowground biomass dry weights of P. armandii seedlings showed significant differences under different treatments during the same time period. On day 90, the aboveground and belowground biomass dry weights of the T328S treatment were higher than those of the T318S treatment, but the difference was not significant. However, both T318S and T328S treatments showed significant differences in biomass compared to other treatments. On days 180 and 270, the aboveground and belowground biomass dry weights of the T328S treatment were significantly higher than those of other treatments (Table 2). Throughout the entire growth period, inoculation combined with fertilization significantly increased the aboveground and belowground biomass dry weights of P. armandii seedlings. The effectiveness of fertilization on the P. armandii seedlings inoculated with T. indicum was as follows: Aolu 328S > Aolu 318S > Yuguoduo.

3.3. Effects of Different Fertilization Treatments on the Mycorrhizal Infection Rate of T. indicum–P. armandii Seedlings

The mycorrhizal infection rate showed significant differences under different treatments during the same growth period. In the T treatment, the mycorrhizal infection rate remained relatively stable between 40% and 50% across the three growth stages, with no significant changes. In the three treatments with both inoculation and fertilization, the mycorrhizal infection rate continuously increased throughout the growth period, with the rates in the T318S and T328S treatments being significantly higher than in the TP19 treatment. However, there was no significant difference in the mycorrhizal infection rate between the T318S and T328S treatments. Overall, the T318S and T328S treatments performed better than the TP19 and T treatments, indicating that the fertilizer effect of Osmocote 318S and 328S was superior to that of Yuguoduo. This suggests that fertilization is more beneficial than no fertilization in enhancing the mycorrhizal infection rate of P. armandii inoculated with T. indicum (Figure 2).
Under a stereomicroscope, the ectomycorrhizae formed by T. indicum and P. armandii displayed various morphological appearances (Figure 3). The ectomycorrhizal system was mostly bifurcated or irregularly pinnate, sometimes appearing coralloid or intermediate between these types. The young mycorrhizae were light yellow in color with numerous white emanating hyphae. As the mycorrhizae aged, their color deepened to dark brown. These are consistent with well-documented T. indicum–P. armandii morphotypes in peer-reviewed studies [49].

3.4. Nutrient Status Changes in the Substrate and Establishing the Optimal Nutrient Substrate Ratio

The changes in substrate pH under different treatments at different growth stages ranged between 6.18 and 7.63 (Figure 4A). According to the Chinese soil pH classification standards, the substrate pH values under different treatments and at different growth stages are within the weakly alkaline range. During the same growth period, there were significant differences in the organic matter content of the substrate among different treatments. Throughout the entire growth period, the organic matter content in each treatment showed a decreasing trend. However, the organic matter content in the T318S, T328S, and T treatments was slightly higher than in the CK, 318S, 328S, and P19 treatments. After 90 days of fertilization, the organic matter content in the T328S treatment reached 46.34 g/kg. In the T318S treatment, it reached 45.20 g/kg, and in the T treatment, it reached 45.93 g/kg. These three treatments were at relatively higher levels compared to the other treatments (Figure 4B).
During the same growth period, there were significant differences in the alkali-hydrolyzable nitrogen content of the substrate among different treatments. In the CK and T treatments, the alkali-hydrolyzable nitrogen content initially increased and then decreased, while in the other treatments, it showed a decreasing trend with no significant changes. The alkali-hydrolyzable nitrogen content in the substrate of all treatments was at a deficient level. After 90 and 180 days of fertilization, the alkali-hydrolyzable nitrogen content in the TF318S treatment was the highest among all treatments, whereas the CK treatment had the lowest content. From 180 to 270 days after fertilization, the alkali-hydrolyzable nitrogen content in the substrate showed no significant changes in treatments other than CK and T (Figure 4C).
The changes in available phosphorus in the substrate under different treatments during various growth stages showed an overall trend of first increasing and then decreasing. This may be related to the decomposition of applied fertilizers into the substrate and the subsequent consumption of a large amount of phosphorus in the later stages of growth. Among them, the available phosphorus content in the substrate for the TF318S and TF328S treatments was significantly higher than in other treatments, while the other treatments showed no significant changes throughout the growth period (Figure 4D).
The trend in available potassium in the substrate under different treatments during various growth stages, except for the CK treatment, was an increase followed by no significant changes. The available potassium content in the substrate of the 328S treatment showed a slight upward trend in the later stages. The available potassium content in the CK treatment was lower compared to other treatments. The maximum available potassium content in the substrate was 423.56 mg/kg, and the minimum was 236.49 mg/kg, indicating an extremely rich level (Figure 4E).
The changes in exchangeable magnesium content in the substrate across all treatments during different growth stages showed an initial increase followed by a decrease. In the T318S and T328S treatments, the exchangeable magnesium content in the substrate was significantly higher than in the other treatments (Figure 4F).
For available iron, the content in the substrate of the T328S, T318S, TP19, and P19 treatments first increased and then decreased. In the T treatment, the available iron content in the substrate continued to increase. Meanwhile, in the 318S and 328S treatments, the available iron content in the substrate remained relatively stable (Figure 4G).

4. Discussion

This study conducted a statistical analysis of data on seedling crown width, plant height, ground diameter, biomass, and mycorrhizal infection rate following fertilization, combined with changes in substrate nutrient content, to evaluate the effects of various treatments on seedling growth and mycorrhizal infection. The results indicate that pH has a significant impact on mycorrhizal formation, with the highest number of mycorrhizae observed at a pH of 6.53 to 6.86. This pH range is more conducive to the synthesis of T. indicum mycorrhizae. Research has shown that various truffle species also grow in slightly alkaline soils, and the pH of the soil in T. indicum truffle beds gradually shifts from slightly acidic to slightly alkaline during the maturation stage, indicating that it can regulate the pH of its growing environment [54,55,56,57]. Furthermore, T. indicum can form mycorrhizae under slightly acidic conditions, suggesting that the pH range for P. armandii inoculated with T. indicum to form mycorrhizae is relatively broad. However, excessively acidic or alkaline conditions can inhibit mycorrhizal formation, making neutral to slightly alkaline substrates the most suitable for growth. Within the tested pH range (6.18–7.63), mycorrhizal formation showed no significant correlation with pH variation across treatments (p > 0.05), suggesting that the observed differences in colonization rates were primarily driven by fertilizer types rather than pH effects. This aligns with field observations showing T. indicum’s broad ecological adaptability to soils ranging from pH 4.78 to 8.5 [58,59]. The homogeneity of pH across treatments suggests it was a permissive rather than limiting factor in this study.
Our results demonstrate that slow-release fertilizers (Aolu 318S and 328S) significantly enhanced seedling growth, with T328S increasing aboveground biomass by 30.7% (3.19 g vs. 2.44 g in the control) and mycorrhizal infection rates by 2.0-fold (50% vs. 24% in T-only) at 270 days (Table 2 and Figure 2). These improvements align with the sustained nutrient release of slow-release formulations, which matched the prolonged nutrient demands of both P. armandii and T. indicum. In contrast, quick-release Youguduo (19-19-19 NPK) led to transient nutrient availability, resulting in 12% lower biomass and 20% lower infection rates compared to slow-release treatments, likely due to leaching losses (Figure 4D,E) and microbial immobilization [25].
In this study’s statistical analysis, it was found that fertilization provided abundant nutrients for the mycelium and mycorrhizae, and mycorrhizal treatment effectively increased the organic matter content of the substrate, promoting the absorption and utilization of nitrogen, phosphorus, and potassium by P. armandii seedlings. This is because P. armandii–T. indicum mycorrhizal seedlings have a strong ability to convert unavailable nitrogen into available nitrogen during growth, and they can quickly activate insoluble phosphorus in the substrate, thereby promoting the growth of P. armandii seedlings and T. indicum–P. armandii mycorrhizal seedlings, with the latter showing significant growth advantages [56,59,60]. Research has shown that soil nitrogen nutrition plays a crucial role in the growth and development of truffles during their maturation period, and phosphorus may regulate truffle growth by affecting the fungal and bacterial communities in the soil [61,62,63], although the exact mechanism requires further study. In fact, ectomycorrhizae and host plants have a mutually beneficial symbiotic relationship. Kang [64] indicates that truffle inoculation can increase the nitrate nitrogen content in the rhizosphere soil of Quercus, reduce the available phosphorus content, enhance the activity of Quercus roots, and promote the colonization of denitrifying bacteria in the rhizosphere soil, thus affecting the soil’s physical and chemical properties.
The superior performance of T318S/T328S was correlated with elevated levels of substrate organic matter (46.34 g/kg versus 43.25 g/kg in the control) and available phosphorus (87.32 mg/kg versus 83.69 mg/kg in the control), which directly facilitated the expansion of fungal hyphae and the development of host roots (Figure 4B–D). The 11% higher phosphorus content in T328S likely stimulated mycelial growth, given that Tuber spp. upregulate phosphatase activity under phosphorus-limiting conditions [19]. Similarly, the increased concentration of exchangeable magnesium (1.57 mg/kg versus 1.17 mg/kg in the control) may have promoted enzymatic processes in both plant and fungal metabolism, considering magnesium’s essential role in ATP synthesis [59]. Notably, the transient peak of iron in T328S (37.3 mg/kg at 90 days, Figure 4G) might enhance the stress responses of fungi, as iron functions as a cofactor for multiple antioxidant enzymes in fungal cells [65].
Nitrogen, phosphorus, and potassium are essential nutrients for plant growth and development, and within a certain range, they can promote the growth of both aboveground parts and root systems [66,67]. This experiment showed that moderate levels of available phosphorus, quick-acting potassium, exchangeable magnesium, and available iron promote the formation of T. indicum mycorrhizae. Chen’s research on artificial truffle cultivation techniques pointed out that an excessive organic matter content in the substrate may inhibit mycorrhizal formation, while organic nitrogen sources such as aspartic acid and moderate levels of phosphorus significantly promote mycorrhizal synthesis [29]. The growth-promoting effects of different metal ions on truffle mycelium follow the order of Ca2+ > Na+ > K+ > Mg2+ [68,69,70,71]. The experimental results indicate significant differences between fertilization treatments. The quick-release fertilizer Youguduo may lead to nutrient loss before the mycelium and mycorrhizae have fully absorbed them. The fertilizer efficiency differences produced by applying different fertilizers may be potentially affected by various biotic and abiotic factors. For quick-release fertilizers such as Youguduo (19-19-19) in this research, rapid nutrient dissolution likely increased initial leaching risks, particularly for mobile N and K, as evidenced by the sharper decline in available P and K in P19/TP19 treatments compared to slow-release formulations (Aolu 318S/328S). Similar patterns were reported in truffle agroforestry systems, where irrigation accelerated nutrient leaching in sandy substrates [18]. Microbial immobilization may have further reduced available N, as ectomycorrhizal fungi (EMF) and associated bacteria compete for inorganic N [7]. The higher organic matter in T318S/T328S treatments (Figure 4B) suggests fungal-mediated nutrient retention, aligning with findings that EMF hyphae reduce leaching by binding nutrients [70]. Considering that there is no significant difference in substrate pH values during different stages of Youguoduo treatment, the possibility of non-biological nutrient loss caused by this is relatively small. These also explain the superior performance of slow-release fertilizers in maintaining the long-term nutritional supply of P. armandii and T. indicum symbiosis.
The experimental results indicate significant differences between fertilization treatments. The quick-release fertilizer Youguduo may lead to nutrient loss before the mycelium and mycorrhizae have fully absorbed them. Therefore, it is recommended to use the slow-release fertilizers Aolu 318S and Aolu 328S, which are more suitable for mycelium germination and promote fruiting body growth.
Under the context of climate change, the distribution range of P. armandii is gradually shrinking. The most concerning issue is that the suitable habitats for P. armandii in the Wushan region, which connects the Daba Mountains and the Yunnan–Guizhou Plateau, will gradually disappear, leading to the disruption of the current ecological corridor [72]. Fertilization and mycorrhization of seedlings play crucial roles in promoting the growth of P. armandii, enhancing stress resistance, improving soil structure, increasing nutrient utilization, and maintaining the health and development of forest ecosystems [73,74,75]. Therefore, protecting and restoring these critical habitats is of utmost importance. To address the ecological challenges posed by the shrinking distribution of P. armandii, it is essential to research and promote biotechnological measures based on ectomycorrhizal fungi. Ectomycorrhizal fungi not only enhance the stress resistance of P. armandii but also improve the soil’s physical and chemical properties, promote nutrient cycling, and enhance plants’ adaptability to environmental changes [10,76,77]. Moreover, by adopting appropriate fertilization strategies, particularly the use of slow-release fertilizers, the long-term healthy growth of seedlings can be supported, nutrient loss can be reduced, and soil fertility can be increased. This approach will help maintain and restore P. armandii populations while ensuring the stability and biodiversity of the entire forest ecosystem. Future research should further explore the optimal combination of mycorrhization treatments and fertilization strategies under different environmental conditions to provide scientific support for the conservation and sustainable development of P. armandii.

5. Conclusions

Under conditions of inoculation with Tuber indicum ascocarp inoculum at a spore concentration of 5 × 106 spores/mL, the optimal fertilization regime for Pinus armandii–T. indicum mycorrhizal synthesis and seedling development was determined as follows: Aolu 318S and 328S exhibited optimal efficacy at 2 g per plant, applied three months post-inoculation by uniform incorporation into the substrate. For Youguduo, the optimal dosage was 0.66 g per plant, administered three months after inoculation via the root application of a 600-fold water dilution. Comparative analysis revealed Aolu 318S to be the most effective formulation for promoting mycorrhizal formation and seedling growth. These findings provide robust scientific evidence and practical guidelines for cultivating P. armandii–T. indicum mycorrhizal seedlings, demonstrating that optimized fertilizer type, dosage, and application method can significantly enhance mycorrhizal establishment and seedling quality, thereby maximizing yields in truffle artificial cultivation. The superior performance of slow-release Aolu 318S over quick-release Youguduo offers critical insights for fertilizer selection and fertilization strategy development, enabling the optimization of tree-fungal symbiosis under diverse cultivation scenarios.

Author Contributions

L.H. (Li Huang), visualization, methodology, software, and formal analysis; R.W., writing—review and editing and data curation; R.L., writing—review and editing; S.Y., methodology, conceptualization, and supervision; F.Y., resources, supervision, and project administration; C.H., data curation and visualization; L.H. (Lanlan Huang), methodology and resources; D.L., supervision, validation, and writing—review and editing; S.W., validation, funding acquisition, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the Young Talents Special Project of the Xing Dian Talent Support Program of Yunnan Province (XDYC-QNRC-2023-0415), the National Natural Science Foundation of China (No. 32060008), and the Yunnan Technology Innovation Program (202205AD160036).

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effects of different treatments on the seedling crown (A), plant height (B), and ground diameter (C) of P. armandii at different growth stages. Note: CK: control treatment; T: inoculation with T. indicum only; 318S: application of Aolu 318S only; 328S: application of Aolu 328S only; P19: application of Youguduo only; T318S: combined inoculation with T. indicum and application of Aolu 318S; T328S: combined inoculation with T. indicum and application of Aolu 328S; TP19: combined inoculation with T. indicum and application of Youguduo. Data represent the mean of five replicates. Different letters indicate significant differences at the 0.05 level, while the same letter indicates no significant difference.
Figure 1. Effects of different treatments on the seedling crown (A), plant height (B), and ground diameter (C) of P. armandii at different growth stages. Note: CK: control treatment; T: inoculation with T. indicum only; 318S: application of Aolu 318S only; 328S: application of Aolu 328S only; P19: application of Youguduo only; T318S: combined inoculation with T. indicum and application of Aolu 318S; T328S: combined inoculation with T. indicum and application of Aolu 328S; TP19: combined inoculation with T. indicum and application of Youguduo. Data represent the mean of five replicates. Different letters indicate significant differences at the 0.05 level, while the same letter indicates no significant difference.
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Figure 2. Effects of different treatments on the mycorrhizal infection rate of P. armandii at different growth stages. Note: CK: control treatment; T: inoculation with T. indicum only; 318S: application of Aolu 318S only; 328S: application of Aolu 328S only; P19: application of Youguduo only; T318S: combined inoculation with T. indicum and application of Aolu 318S; T328S: combined inoculation with T. indicum and application of Aolu 328S; TP19: combined inoculation with T. indicum and application of Youguduo. Data represent the mean of five replicates. Different letters indicate significant differences at the 0.05 level, while the same letter indicates no significant difference.
Figure 2. Effects of different treatments on the mycorrhizal infection rate of P. armandii at different growth stages. Note: CK: control treatment; T: inoculation with T. indicum only; 318S: application of Aolu 318S only; 328S: application of Aolu 328S only; P19: application of Youguduo only; T318S: combined inoculation with T. indicum and application of Aolu 318S; T328S: combined inoculation with T. indicum and application of Aolu 328S; TP19: combined inoculation with T. indicum and application of Youguduo. Data represent the mean of five replicates. Different letters indicate significant differences at the 0.05 level, while the same letter indicates no significant difference.
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Figure 3. Morphological appearance of ectomycorrhizae formed by T. indicum and P. armandii. (A) bifurcated mycorrhiza; (B) coral-like mycorrhiza; (C) simple unbranched mycorrhiza.
Figure 3. Morphological appearance of ectomycorrhizae formed by T. indicum and P. armandii. (A) bifurcated mycorrhiza; (B) coral-like mycorrhiza; (C) simple unbranched mycorrhiza.
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Figure 4. Changes in substrate pH (A), organic matter (B), alkali-hydrolyzable nitrogen (C), available phosphorus (D), available potassium (E), exchangeable magnesium (F), and available iron (G) under different treatments at different growth stages. Note: CK: control treatment; T: inoculation with T. indicum only; 318S: application of Aolu 318S only; 328S: application of Aolu 328S only; P19: application of Youguduo only; T318S: combined inoculation with T. indicum and application of Aolu 318S; T328S: combined inoculation with T. indicum and application of Aolu 328S; TP19: combined inoculation with T. indicum and application of Youguduo. Data represent the mean of five replicates. Different letters indicate significant differences at the 0.05 level, while the same letter indicates no significant difference.
Figure 4. Changes in substrate pH (A), organic matter (B), alkali-hydrolyzable nitrogen (C), available phosphorus (D), available potassium (E), exchangeable magnesium (F), and available iron (G) under different treatments at different growth stages. Note: CK: control treatment; T: inoculation with T. indicum only; 318S: application of Aolu 318S only; 328S: application of Aolu 328S only; P19: application of Youguduo only; T318S: combined inoculation with T. indicum and application of Aolu 318S; T328S: combined inoculation with T. indicum and application of Aolu 328S; TP19: combined inoculation with T. indicum and application of Youguduo. Data represent the mean of five replicates. Different letters indicate significant differences at the 0.05 level, while the same letter indicates no significant difference.
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Table 1. Application methods, fertility duration, and nutrient content of the three fertilizers.
Table 1. Application methods, fertility duration, and nutrient content of the three fertilizers.
FertilizerFertilizer ApplicationFertile PeriodNPK Content and RatioTrace Element
Aolu 318Sroots9 months15-9-11 + TEFe0.45, Mg0.06, Cu0.05, Zn0.015, B0.03
Aolu 328Sroots9 months11-11-18 + TEFe0.25, Mg0.03, Cu0.055, Zn0.01, B0.01
Youguduoroots1 week19-19-19 + TEFe0.05, B0.12, Zn0.06, Mo0.015
Note: Data are expressed as the mean ± standard deviation, and the significance level is set at 0.05.
Table 2. Aboveground and belowground biomass dry weights (g) of P. armandii seedlings under different treatments at different growth stages.
Table 2. Aboveground and belowground biomass dry weights (g) of P. armandii seedlings under different treatments at different growth stages.
TreatmentsAboveground Dry WeightBelowground Dry Weight
90 Day180 Day270 Day90 Day180 Day270 Day
CK2.25 ± 0.04 d2.41 ± 0.06 d2.44 ± 0.04 d2.14 ± 0.03 d2.27 ± 0.08 d2.33 ± 0.07 e
T2.29 ± 0.08 cd2.42 ± 0.05 d2.46 ± 0.08 d2.17 ± 0.04 d2.30 ± 0.07 d2.35 ± 0.08 de
318S2.35 ± 0.08 bc2.49 ± 0.14 cd2.67 ± 0.06 c2.30 ± 0.12 c2.37 ± 0.07 cd2.44 ± 0.08 cd
328S2.39 ± 0.06 bc2.64 ± 0.05 b2.70 ± 0.03 c2.36 ± 0.08 bc2.43 ± 0.07 c2.47 ± 0.08 c
P192.30 ± 0.07 cd2.47 ± 0.08 cd2.49 ± 0.04 d2.20 ± 0.04 d2.31 ± 0.07 d2.34 ± 0.06 de
TF318S2.41 ± 0.05 ab2.71 ± 0.13 ab3.06 ± 0.17 b2.40 ± 0.04 ab2.67 ± 0.06 b2.71 ± 0.06 b
TF328S2.49 ± 0.08 a2.84 ± 0.15 a3.19 ± 0.12 a2.46 ± 0.04 a2.82 ± 0.14 a2.86 ± 0.09 a
TFP192.32 ± 0.08 cd2.55 ± 0.06 bc2.68 ± 0.08 c2.21 ± 0.04 d2.34 ± 0.05 cd2.38 ± 0.06 cde
Note: Data are expressed as the mean ± standard deviation, and the significance level is set at 0.05. Different letters (e.g., a, b, c, d, e) indicate significant differences between treatments at the 0.05 level based on ANOVA; treatments sharing the same letter are not significantly different from each other.
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MDPI and ACS Style

Huang, L.; Wang, R.; Yu, F.; Liu, R.; He, C.; Huang, L.; Yang, S.; Liu, D.; Wan, S. Effects of N-P-K Ratio in Root Nutrient Solutions on Ectomycorrhizal Formation and Seedling Growth of Pinus armandii Inoculated with Tuber indicum. Agronomy 2025, 15, 1749. https://doi.org/10.3390/agronomy15071749

AMA Style

Huang L, Wang R, Yu F, Liu R, He C, Huang L, Yang S, Liu D, Wan S. Effects of N-P-K Ratio in Root Nutrient Solutions on Ectomycorrhizal Formation and Seedling Growth of Pinus armandii Inoculated with Tuber indicum. Agronomy. 2025; 15(7):1749. https://doi.org/10.3390/agronomy15071749

Chicago/Turabian Style

Huang, Li, Rui Wang, Fuqiang Yu, Ruilong Liu, Chenxin He, Lanlan Huang, Shimei Yang, Dong Liu, and Shanping Wan. 2025. "Effects of N-P-K Ratio in Root Nutrient Solutions on Ectomycorrhizal Formation and Seedling Growth of Pinus armandii Inoculated with Tuber indicum" Agronomy 15, no. 7: 1749. https://doi.org/10.3390/agronomy15071749

APA Style

Huang, L., Wang, R., Yu, F., Liu, R., He, C., Huang, L., Yang, S., Liu, D., & Wan, S. (2025). Effects of N-P-K Ratio in Root Nutrient Solutions on Ectomycorrhizal Formation and Seedling Growth of Pinus armandii Inoculated with Tuber indicum. Agronomy, 15(7), 1749. https://doi.org/10.3390/agronomy15071749

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