The pot sample were three-month-old asexual group-cultivated seedlings of Eucalyptus urophylla × Eucalyptus grandis DH3229 from Gaoyaojiayao forestry development company. The inoculated AM fungi was Rhizophagus irregularis DAOM 197198, from the mycorrhizal biology team, College of Forestry and Landscape Architecture, South China Agricultural University. The conditions for the germination of R. irregularis were as follows: spores were washed with sterile water several times until the inhibitors were cleaned and stored in the refrigerator at 4°C. The spores were stored in a 12-well cell culture plate containing sterile water and placed in a dark incubator at 25°C to induce the spores to produce germination tubes. After 7 days of cultivation, the germinated spores were collected in centrifuge tubes and used for plant inoculation tests. The host plant was Zea mays and the resulting mycorrhizal agent was mixture of AM fungal spores, mycelium, root segments and soil. Spores were collected by wet sieving and sucrose centrifugation (Yamamura et al., 2003). Seedling roots were inoculated with R. irregularis, pipetting 1 mL of an aqueous solution containing approximately 400 spores to the vicinity of the root system. Each non-mycorrhizal seedling received the same amount of sterilized autoclaved inoculum (15 min in an autoclave at 121°C). One month after inoculating AM fungi, the root symbiosis of the seedlings was observed under a microscope to confirm successful inoculation, and then samples were collected.

In this study, there were 28 treatments in total, including with or without AM fungi inoculation, three organic N sources (glycine, L-glutamine and peptone) and four levels of N and P fertilization, with no application of organic N fertilizer as control. Four biological replicates were set up for each treatment. The plant growth conditions were: photoperiod of 16 h at 24°C during the day with a light intensity of 100-200 W m^-2 and 8 h at 19°C at night. Watering with Low (30 µM L^-1 NaH₂PO₄) Long Ashton (mLA) nutrient solution (Hewitt, 1966) during the previous month of mycorrhizal colonization. After mycorrhizal symbiosis, the improved LA nutrient solution with different levels of organic N, N and P was used for watering, with 50 mL of water per Eucalyptus seedling every three days. N concentration of 0.25 mM NaNO₃ was added to the base of organic N (glycine, L-glutamine, and peptone). Two level of organic N concentrations were set- in the study, 0.25 mM and 2.5 mM. Two level of P concentrations were set, 0.03 mM and 0.3 mM. N concentration of 1 mM NaNO₃ was added as control treatments with no organic N fertilization. The specific treatment of applying organic N and P concentration is shown in the table below ( Table 1 ).

The diameter and height of the seedlings were measured monthly from December 2023 to January 2024 using straightedge and vernier calipers. Eucalyptus seedling roots and leaves were harvested and weighed freshly, killed at 105°C for 15 min, then dried at 70°C until constant weight and weighed to calculate the biomass. Root length, diameter, surface area and root volume of seedlings were determined using a root scanner (WinRHIZO, China). The aboveground and underground part of each biological replicate was randomly divided into 5 groups for the determination of chlorophyll concentration, photosynthetic gas exchange parameters, enzyme activity, total nitrogen (TN) contents, total phosphorus (TP) contents and gene levels, respectively.

Gas exchange parameters of Eucalyptus leaves were determined with Li-6400 portable photosynthesis meter (LI-COR, USA), mainly including net photosynthetic rate, stomatal conductance, transpiration rate and intercellular CO₂ concentration. The measurements were made on the top 3 unfolded leaves of Eucalyptus seedlings between 08:00 to 12:00 am in the morning on a sunny day. Three measurement points were randomly selected for each leaf (Zhang et al., 2018).

For the determination of photosynthetic pigments, the ethanol extraction method was used. 0.2 g leaves were taken into a 50 mL centrifuge tube, and 25 mL of 95% ethanol was added sequentially to the tube and sealed, and then extracted for 24-36 h at room temperature in the dark, and then the extracts were diluted twice, and then colorimetrically compared with the UV-visible spectrophotometer at wavelengths of 665 and 649 nm. The concentrations of chlorophyll a, b and the total chlorophyll were calculated according to the OD values at each wavelength (Lichtenthaler and Wellburn, 1983).

Dry roots were fully ground and homogenized for nutrient analysis. The total nitrogen (TN) content was measured from 0.2 g of dried roots powder by the Kjeldahl method (Schuman et al., 1973). Nitrogen use efficiency (NUE) was calculated as the dry weights of the seedlings divided by the TN contents of seedlings (Shi et al., 2017).

To measure the total phosphorus (TP) contents, 0.4 g of dried roots powder was digested with HNO₃. The specimens were determined by inductively coupled plasma mass spectrometry (ICP-MS) after digestion and the internal standard method was used for determination of TP contents (Hansen et al., 2013). The glutamate synthase (GOGAT) activity of Eucalyptus in the leaves was determined using the Ionization assay (Singh and Srivastava, 1986). The GOGAT activity was expressed as the reduction of NADH in 0.1 g of sample after ten minutes’ reaction. The nitrate reductase (NR) activity of Eucalyptus in the leaves was determined using the ex vivo assay (Srivastava, 1980). The NR activity was expressed as the amount of nitrite produced by 0.15 g of sample after ten minutes’ reaction. The glutamine synthetase (GS) activity of Eucalyptus in the leaves was determined using the GS activity assay (Magalhaes and Huber, 1991). The GS activity was expressed as the amount of γ-glutamyl isohydroxycyclohexanoic acid produced by 0.05 g of sample.

Real-time fluorescence quantitative PCR (qRT-PCR) was used to investigate the relative expression of N transport-related genes in the root of Eucalyptus (Zhao et al., 2019). The samples were taken in liquid nitrogen and transferred to -80°C refrigerator for storage. RNA was extracted using E.Z.N.A.™ Plant RNA Protocol II kit (Guangzhou Omega Biotek, Ltd). HiScript^® III RT SuperMix for qPCR (+gDNA wiper) kit (Nanjing Vazyme Biotech Co., Ltd) was used to reverse transcription of the first strand cDNA. Furthermore, gene expression levels were determined in a 96-well Real time PCR system instrument (BioRed, Hercules, CA, USA), using the SGExcel FastSYBR Master Mix kit (Shanghai Sangon Biotech Co., Ltd.), with a reaction mixture of 10 µL SYBR qPCR Master Mix, 0.5 µL each of forward and reverse primers, 2 µL cDNA, and 7 µL ddH₂O. Nine primers were selected to be used in the qRT-PCR, with EgUBI3 applied to be the endogenous reference (Huang et al., 2014). Each PCR protocol was conducted with 3 biological replicates. Supplementary Table 1 presents all the primers used in the qRT-PCR.

In the current work, SPSS 27.0 (SPSS Inc., Chicago, IL, USA) was adopted for statistical analysis of the result data. The result data were analyzed based on one-way ANOVA (posthoc comparisons using Duncan’s test, P < 0.05) and two-way ANOVA (the variation sources were AM fungi inoculation, N application, P application, organic N and their association). T-test was analyzed between NM and AM treatments. After performing the homogeneity analysis of variance, the data were subjected to ANOVA. Pearson’s correlation coefficient was utilized to evaluate the correlation between different growth and physiological characters. Origin 8.0 software (Systat Software Inc., San Jose, CA, USA) was employed to construct figures.

The G2P2 treatment showed the most notable increase in Eucalyptus growth among various treatments. Eucalyptus seedlings treated with G2P2 had heights, ground diameters, and crown widths that were 3.23, 0.82, and 3.70 times higher than N0P1 treatment, respectively. Compared to non-mycorrhizal Eucalyptus seedlings, mycorrhizal Eucalyptus seedlings under both N1P1 and N2P1 treatments showed a substantial increase in plant height and crown width with glycine and peptone treatments ( Figures 1B, D ). The results indicated that glycine outperformed NM treatments among the three organic N treatments in terms of promoting mycorrhizal Eucalyptus growth ( Figure 1 ). Specifically, the height and crown width of mycorrhizal Eucalyptus seedlings treated with G1P1 were elevated by 60.72% and 41.56%, compared to NM treatments ( Figures 1B, C ). This implies that at the same N level, mycorrhizal Eucalyptus has a considerable growth advantage in low P conditions. However, the growth of Eucalyptus seedlings did not significantly increase after high N treatments at the same P level, suggesting that P is a more important factor restricting Eucalyptus seedling growth ( Figure 1 ).

The dry weight and fresh weight of mycorrhizal Eucalyptus seedlings treated with high organic N were found to be considerably higher than NM treatments. In particular, the dry weight of mycorrhizal Eucalyptus seedlings treated with G2P2 was increased by 58.79% compared to NM treatment ( Figure 1F ). Among the three organic N sources, glycine treatment led to a considerable increase in seedling biomass. There were substantial increases in seedling biomass under N2P2 treatments, the dry weights and fresh weights of Eucalyptus under G2P2 treatment were 13.39 and 12.93 times, respectively, higher than those under N0P1 treatment ( Figures 1E, F ). This suggests that Eucalyptus seedling biomass was more effectively promoted by higher levels of N and P. In terms of increasing the biomass of Eucalyptus seedlings, the G2P2 treatment performed the best overall.

Analysis of Eucalyptus seedlings root growth revealed significant increases in root length under glycine treatments ( Figure 2A ). Root length under G2P2 treatment was 5.38 times higher than that of N0P1 treatment. The root surface area was increased by 7.60 times under P2P2 treatment compared to N0P1 treatment ( Figure 2B ). Concurrently, the root average diameter and volume were significantly increased under L-glutamine treatments, compared to control treatment ( Figures 2C, D ). The average root volume of mycorrhizal Eucalyptus was elevated by 6.07 times compared to non-mycorrhizal Eucalyptus under G1P1 treatment. However, non-mycorrhizal Eucalyptus seedlings exhibited superior root growth, compared to mycorrhizal Eucalyptus seedlings under N2P2 treatment. Furthermore, compared to NM treatments, glycine treatment was more successful in lengthening the mycorrhizal Eucalyptus roots. Under high P treatments, root growth significantly increased in comparison to treatments at the same N level ( Figure 2 ). The root length of G2P2 treatment was increased by 0.84 times compared to G2P1 treatment, indicating that P plays a significant role in the root growth of Eucalyptus seedlings. There was an overall increase in root growth under P2P2 treatment. Compared to N0P1 treatment, the root length, surface area, average diameter and volume of Eucalyptus seedlings were increased by 3.23, 7.60, 3.38 and 5.76 times, accordingly. Overall, P2P2 treatment was the best in enhancing the Eucalyptus root growth.

There was no discernible increase in the net photosynthetic rate with the three organic N treatments. However, the net photosynthetic rate was increased by 0.38 and 0.40 times, respectively, under P1P2 and P2P1 treatment compared to N0P1 treatment ( Figure 3A ). Under the P1P2 treatment, net photosynthetic rate of mycorrhizal Eucalyptus seedlings was elevated by 113.10%, compared to NM treatment. Both the glycine and peptone treatments resulted in a considerable rise in the intercellular CO₂ concentration ( Figure 3B ). Under glycine treatment, stomatal conductance and transpiration rate increased significantly. Compared to N0P1 treatment, the stomatal conductance and transpiration rate under G2P2 treatment were increased by 1.92 and 3.33 times, respectively ( Figures 3C, D ). Furthermore, Eucalyptus net photosynthesis rate, intercellular CO₂ concentration, stomatal conductance, and transpiration rate were all increased under high P treatments in comparison to treatments at the same N level, suggesting that P is also a crucial component in boosting Eucalyptus seedling photosynthesis. The photosynthesis of Eucalyptus seedlings under the G2P2 treatment were increased notably. Overall, these results showed that glycine was superior in encouraging photosynthesis among the three organic N treatments.

Chlorophyll concentrations in Eucalyptus seedlings were shown to increase following P2P2 treatment over various treatments ( Figures 3E–G ). The concentrations of chlorophyll a, chlorophyll b and total chlorophyll under P2P2 treatment were 1.12, 0.69 and 1.75 times, accordingly, higher than those of N0P1 treatment. Also, the chlorophyll concentrations under treatments with high N and P increased significantly, revealing that high level of N and P increased the Eucalyptus seedlings’ chlorophyll concentrations. The findings demonstrated that mycorrhizal Eucalyptus seedlings treated with glycine had a higher chlorophyll concentration than non-mycorrhizal Eucalyptus seedlings. However, these results contrasted under G2P2 treatment, implying that higher level of N and P restricted the increase of mycorrhizal Eucalyptus seedlings’ chlorophyll concentrations.

Under different treatments, the activities of NR and GOGAT were significantly elevated under the P2P2 treatment. Compared to N0P1 treatment, NR and GOGAT activities under P2P2 treatment in Eucalyptus seedlings were increased by 0.81 and 3.82 times, respectively ( Figures 4A, B ). GS activity in mycorrhizal Eucalyptus was increased significantly, particularly under L1P1 treatment, where it was 1.75 times higher than NM treatment ( Figure 4C ). The activities of NR, GOGAT and GS in mycorrhizal Eucalyptus seedlings were elevated under G1P1 and G1P2 treatments compared to NM treatment ( Figure 4 ). High P treatments did not effectively promote the GS activity in Eucalyptus. Concurrently, activity of NR and GOGAT under G1P1 treatment was 0.25 and 2.27 times, respectively, higher than NM treatments, indicating that high N and P levels did not promote the activities of NR and GOGAT. The results demonstrated that among the three organic N treatments, glycine treatment was more effective in promoting N metabolism in mycorrhizal Eucalyptus compared to non-mycorrhizal Eucalyptus ( Figure 4 ).

The TN content in Eucalyptus seedlings was significantly elevated under G2P1 treatment. The TN content in mycorrhizal Eucalyptus seedlings under N2P1 treatment was increased by 0.47 times compared to NM treatment ( Figure 4D ). Additionally, compared to N0P1 treatment, the TN content in Eucalyptus was 0.67 times higher than that of G2P1 treatment. While high P treatments were less effective, high N fertilization significantly raised the TN contents. Concurrently, the TP content in mycorrhizal Eucalyptus seedlings treated with G1P2 was elevated by 93.13%, compared to non-mycorrhizal Eucalyptus ( Figure 4E ). Additionally, high N level had no discernible effect on the TP contents. Under various treatments, the N2P2 treatments was the most effective in promoting the NUE of mycorrhizal Eucalyptus. The mycorrhizal Eucalyptus showed notable benefits in boosting NUE when exposed to high levels of organic N fertilization, particularly glycine treatments. NUE of mycorrhizal Eucalyptus under G2P2 treatment was elevated by 24.03%, compared to non-mycorrhizal Eucalyptus. The results indicated that among the three organic N treatments, glycine treatment was more effective in increasing the TN contents, TP contents and NUE of mycorrhizal Eucalyptus compared to non-mycorrhizal Eucalyptus ( Figure 4 ).

Under different treatments, the relative expression levels of genes associated with N transport in mycorrhizal Eucalyptus significantly increased under G2P2 treatment ( Figure 5 ). The AM treatment markedly elevated the relative expression levels of EgAAP3, EgProt2, EgNPF4.5, EgNPF6.3, EgNPF8.1 and EgAMT2.1, compared to NM treatments under three organic N treatments. The relative expression of genes related to organic N transport (EgAAP3 and EgProt2), nitrate N transport (EgNPF4.5, EgNPF6.3 and EgNPF8.1) in mycorrhizal Eucalyptus were significantly up-regulated under high P treatments compared to treatments at the same organic N level. This suggests that P may be a key factor regulating nitrate N transport and amino acid transport in Eucalyptus ( Figure 5 ). Under G2P2 and L2P2 treatments, mycorrhizal Eucalyptus showed considerably greater relative expression levels of EgAMT2.1 than under NM treatments, whereas under L1P1 and L2P1 treatments, mycorrhizal Eucalyptus showed significantly lower relative expression levels of EgAMT3.1 than under NM treatments. This might indicate that plants use distinct ammonium ion transporter proteins to control ammonium ion absorption under various N supply situations.

Significant positive relationships between seedling growth features and photosynthetic characteristics were found based on the correlation plot among several Eucalyptus growth and physiological characters ( Figure 6 ). This suggests that photosynthesis is a major factor influencing seedling growth. Chlorophyll concentration and seedling growth characteristics showed highly significant positive associations with NUE (P < 0.001). Additionally, TC was shown to have highly significant positive associations (P < 0.001) with both NR and GOGAT activities. In summary, GOGAT activity, photosynthesis, and NUE are important variables affecting Eucalyptus growth. As presented in Supplementary Table 2 , the impacts of AM fungi, N application and P application, organic N and the interactions of the previous four significantly (P < 0.01) elevated the levels of evaluated growth and photosynthesis indexes.

AM fungi play a vital role in enhancing plant growth under nutrient-limited conditions. Our study revealed that AM treatments exhibited significantly greater biomass accumulation, compared to NM treatments, with low P fertilization under the same N level ( Figure 1 ). This aligns with previous observations that AM fungi symbiosis improves P acquisition efficiency in P deficient soil by extending the root absorption zone, thereby compensating for reduced root hair development (Qian et al., 2024). Notably, this growth advantage diminished under N2P2 treatments, where NM treatments outperformed their AM treatments, likely due to suppressed AM fungi colonization when P availability exceeds plant requirements ( Figure 2 ).

The AM fungi symbiosis further modulated seedlings’ organic N preference by enhancing NUE. Among three organic N sources, glycine emerged as the most effective in promoting mycorrhizal seedlings growth, contrasting with limited benefits observed in NM treatments. This finding corroborates evidence that forest-dominant plants, regardless of mycorrhizal type, preferentially acquire low-molecular-weight organic N compounds like glycine (Näsholm et al., 1998; Naz et al., 2024). Glycine’s efficiency in promoting Eucalyptus growth may be due to its small molecular weight and simple structure, making it easily absorbed by plants directly. The molecular simplicity of glycine likely facilitates direct uptake through AM-mediated pathways, bypassing energy-intensive mineralization processes required for complex N forms.

A synergistic mechanism between AM fungi and organic N was evident through coordinated nutrient interactions. While N fertilization independently promoted root proliferation, AM fungi inoculation amplified this effect by improving P co-utilization efficiency (Chen et al., 2020). The symbiosis induced physiological modifications in root architecture and exudation patterns, creating feedback loops that enhanced both N and P acquisition (Mazumder et al., 2025). This synergy was particularly pronounced under moderate fertilization regimes, where AM fungi optimized the balance between N assimilation and P mobilization. Our results demonstrate that AM-mediated nutrient coordination, rather than isolated nutrient applications, drives sustainable Eucalyptus growth, which is a critical insight for developing precision fertilization strategies in forest management.

In our study, treatments with high N and P significantly increased the seedlings chlorophyll concentration ( Figure 3 ), which corresponds with the previous finding that leaf chlorophyll content is positively correlated with N supply level. Increasing N supply typically elevates leaf chlorophyll content (Wu et al., 2017b; Chen et al., 2024). At the same time, AM fungi significantly improved photosynthetic efficiency of Eucalyptus seedlings under P-limited conditions. While high P treatments universally boosted net photosynthesis, stomatal conductance, and transpiration rate ( Figure 3 ), AM treatments exhibited superior photosynthetic performance compared to NM treatments under low P availability. This aligns with P’s critical role as a substrate, and deficiency or low level of P inhibit leaf photosynthetic rates (Yu et al., 2022). Notably, AM fungi symbiosis optimized N partitioning under low N conditions, redirecting N resources from structural components to photosynthetic machinery, thereby amplifying light energy capture and carbon assimilation (Wu et al., 2017a). This reallocation mechanism underscores AM fungi’s ability to mitigate nutrient limitations by prioritizing metabolic processes essential for growth under low fertilization (Sun J, et al., 2024).

The symbiosis between AM fungi and Eucalyptus selectively enhanced organic N utilization, with glycine emerging as the most effective N source for mycorrhizal seedlings. Under glycine treatments, AM treatments showed markedly elevated chlorophyll concentrations compared to NM treatments ( Figure 3 ), a response linked to enhanced NUE and upregulated activities of N metabolism enzymes (NR, GOGAT) ( Figure 6 ). These findings corroborate that AM fungi facilitate direct assimilation of low-molecular-weight organic N compounds, bypassing energy-intensive mineralization pathways (Näsholm et al., 1998; Naz et al., 2024). By increasing chlorophyll biosynthesis, AM fungi amplified capacity on harvesting light, creating a positive feedback loop where improved N metabolism reinforced photosynthetic output. This preferential utilization of glycine highlights AM fungi’s role in fine-tuning N source selection to maximize host plant productivity.

Several studies have demonstrated that AM fungi can facilitate plant uptake of soil nutrients such as N and P, increasing plant biomass, N concentration, P concentration, N uptake of whole plant (Che et al., 2022; Wu Y, et al., 2024). Our findings observed that high P treatments behaved better in increasing mycorrhizal Eucalyptus’s TP contents. This effect is most likely mediated by improved P acquisition in mycorrhizal plants compared to non-mycorrhizal plants under low P conditions. Outsourcing P acquisition to mycorrhiza may limit the value of P loss reduction by plants (Wang D, et al., 2025). AM fungi-organic N synergy drove coordinated improvements in N and P homeostasis. AM treatments exhibited simultaneous increases in TN and TP contents ( Figure 4 ), reflecting synergistic interactions exist between N and P uptake. While AM fungi primarily enhanced P uptake via extraradical hyphae that bypass rhizosphere depletion zones (Smith et al., 2011; Bhupenchandra et al., 2024), organic N fertilization indirectly boosted P availability by stimulating soil phosphatase activity, accelerating organic P mineralization (Gou et al., 2024). This dual mechanism direct AM-mediated P scavenging and N-driven P mobilization, creating a synergistic nutrient loop. High P treatments amplified this interaction, as AM fungi colonization under sufficient P conditions optimized N-P stoichiometry, further elevating chlorophyll synthesis and photosynthetic rates. Such interdependence underscores that AM fungi do not merely supplement nutrient uptake but actively rewire host plant metabolism to exploit nutrient co-limitation, transforming discrete nutrient inputs into multiplicative growth benefits.

AM fungi significantly upregulated key N metabolic enzymes in Eucalyptus leaves, even under N-deficient conditions. Mycorrhizal seedlings exhibited elevated activities of NR, GOGAT, and GS, compared to NM treatments ( Figure 4 ), consistent with AM fungi’s role in enhancing N uptake and assimilation. The GS-GOGAT cycle, which is critical for converting ammonium into glutamine and glutamate, was particularly amplified, thus providing precursors for amino acids, nucleic acids, and chlorophyll synthesis. This aligns with studies showing AM fungi colonization upregulates genes encoding nitrate transporters and N metabolism enzymes, enabling efficient N recycling under low nutrient conditions (Wang et al., 2020; Chen et al., 2023; Wu XL, et al., 2024). Notably, this enzymatic enhancement occurred independently of fertilization levels, suggesting AM fungi constitutively prime N assimilation pathways to mitigate nutrient scarcity.

The symbiosis selectively optimized organic N utilization by prioritizing ammonium assimilation via the GS-GOGAT pathway (Fortunato et al., 2023; Vaishnav et al., 2025). While NM treatments relied heavily on soil nitrate, mycorrhizal Eucalyptus exhibited a metabolic shift toward glutamine synthesis, a hallmark of organic N preference. This reprogramming explains why mycorrhizal seedlings maintained higher GS activity across all treatments ( Figure 4 ), as GS catalyzes the ATP-dependent fixation of ammonium, which is the key step in assimilating organic N breakdown products (Kojima et al., 2021). By bypassing energetically costly nitrate reduction, AM fungi-enabled ammonium assimilation conserved cellular resources, redirecting energy toward chlorophyll biosynthesis and photosynthetic output. Such metabolic flexibility underscores AM fungi’s ability to tailor N source utilization to environmental availability, favoring organic N forms like glycine under nutrient-limited condition.

The symbiosis between AM fungi and Eucalyptus created a nutrient acquisition feedback loop which N assimilation efficiency reinforced P utilization. Elevated GS activity in mycorrhizal roots increased demand for P-rich ATP, driving hyphal P scavenging via the AM pathway. Paradoxically, high N-P co-fertilization disrupted this synergy, excessive P suppressed AM fungi colonization, reducing NR and GOGAT activities despite nutrient abundance ( Figure 4 ). This aligns with the trade-off balance model, where AM fungi benefits diminish when soil P exceeds host demand, destabilizing the symbiosis (Smith et al., 2011; Bennett and Groten, 2022). Crucially, organic N inputs sustained the synergy by stimulating phosphatase-mediated P mineralization, whereas synthetic fertilization disrupted it (Manzoor et al., 2022). These findings reveal that AM fungi-mediated nutrient synergy thrives under moderate, organically balanced fertilization but collapses under inorganic nutrient overload, highlighting the importance of AM fungi-driven stoichiometric regulation in sustainable nutrient management.

AM fungi significantly upregulated key N transporter genes in Eucalyptus roots under nutrient-limited conditions. Mycorrhizal seedlings exhibited elevated expression of nitrate transporters (EgNPF4.5, EgNPF6.3, EgNPF8.1) and organic N transporters (EgAAP3, EgProt2), compared to NM treatments ( Figure 5 ), mirroring results observed in AM fungi-colonized maize and sorghum (Xie et al., 2022). This transcriptional reprogramming enables efficient N scavenging in low-fertility soils by activating dual acquisition pathways: direct nitrate uptake via NPF transporters and organic N assimilation through transport mediated by AAP3 and Prot2. Notably, P limitation amplified this response, as P starvation signals cross-regulate N transporter expression, positioning AM fungi as critical enhancers of N capture under co-limiting conditions (Zhao et al., 2021).

The symbiosis shifted Eucalyptus N acquisition strategy toward organic sources, evidenced by upregulation of EgAAP3 and EgProt2. While nitrate transporters (EgNPF4.5, EgNPF6.3, EgNPF8.1) were also induced, their activity likely supports secondary nitrate uptake from AM-released N pools in the peri-arbuscular space (Wang et al., 2020). Contrastingly, the downregulation of ammonium transporter EgAMT3.1 in mycorrhizal roots suggests a metabolic trade-off, AM fungi suppress energetically costly ammonium uptake pathways while prioritizing organic N and nitrate assimilation. This aligns with findings in Lycium barbarum, where AM fungi colonization favors nitrate over ammonium uptake, highlighting a conserved mycorrhizal strategy to optimize N source utilization based on symbiotic efficiency (Gong et al., 2023). The coordinated and specific expression of ammonium and nitrate transporters in mycorrhizae-colonized cortical cells suggests the crucial importance of fungal N transfer in plants (Rui et al., 2022).

AM fungi orchestrated a nutrient feedback loop which P availability fine-tuned N transporter expression. High P treatments amplified EgNPFs and EgAAP3 expression ( Figure 5 ), indicating that P sufficiency liberates carbon resources for N transporter synthesis, while P scarcity prioritizes organic N uptake to balance stoichiometric demands. This synergy mirrors the dual role of AM fungi hyphae: (1) delivering P via the AM pathway, which reduces plant investment in root P transporters, and (2) priming N transporters to exploit organic N mineralization driven by phosphatase activity (Wang S, et al., 2025). However, excessive P disrupted the balance, downregulating AM fungi-specific ammonium transporters and demonstrating that optimal AM fungi-driven nutrient synergy occurs under moderate P levels where N-P co-regulation remains intact (Calabrese et al., 2016).