# Interactive effects of soil phosphorus and nitrogen availability on mycorrhiza-mediated nutrition in wheat

Margot Trinquier, Eric Lecloux, Bruno Patrick, Virginie Gasciolli, Claire Jouany, Christophe Roux, Benoit Lefebvre, Agnès Ardanuy

► To cite this version:

Margot Trinquier, Eric Lecloux, Bruno Patrick, Virginie Gasciolli, Claire Jouany, et al., Interactive effects of soil phosphorus and nitrogen availability on mycorrhiza-mediated nutrition in wheat. Plant and Soil, 2026, 10.1007/s11104-025-08267-9. http://dx.doi.org/10.1007/s11104-025-08267-9

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# Interactive effects of soil phosphorus and nitrogen availability on mycorrhiza-mediated nutrition in wheat

Margot Trinquier* · Eric Lecloux · Bruno Patrick · Virginie Gasciolli · Claire Jouany · Christophe Roux · Benoit Lefebvre · Agnès Ardanuy

Received: 25 September 2025 / Accepted: 28 December 2025
© The Author(s) 2026

## Abstract

*Background and aims* Arbuscular mycorrhizal fungi (AMF) play an essential role in plant nutrition in both natural and agro-ecosystems. However, how soil nutrient availability simultaneously regulates AMF diversity and contribution to plant nutrition, requires more attention. We hypothesised that the interaction between the availability of key soil macronutrients phosphorus (P) and nitrogen (N) regulates AMF contribution to wheat nutrition and that nutrient availability will simultaneously influence AMF community composition.

*Methods* We tested this using a unique long-term P fertilisation trial, sampling wheat roots across two growing seasons. Expression of wheat mycorrhizal

nutrient transporters was quantified by RT-qPCR and AMF communities were characterised by ITS2 metabarcoding. Complementary experiments under controlled conditions examined how the interaction between P and N regulates arbuscular mycorrhizal function in plant nutrition.

*Results* Field-grown wheat showed campaign-specific effects of P fertilisation on AMF colonisation and nutrient transporter expression, which coincided with shifts in plant N status. Controlled experiments confirmed that colonisation depends on the limitation of either P or N, but that the regulation of peri-arbuscular phosphate, ammonium and nitrate transporters depended on the limiting nutrient. AMF communities also responded to soil P availability, with the genus

Responsible Editor: Xinhua He.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s11104-025-08267-9.

M. Trinquier · V. Gasciolli · B. Lefebvre (✉) ·

A. Ardanuy (✉)

Univ Toulouse, CNRS, INRAE, LIPME, 24 Chemin de Borde Rouge, Auzeville, 31326 Castanet Tolosan cedex, 52627 Toulouse, CS, France

e-mail: benoit.lefebvre@inrae.fr

A. Ardanuy

e-mail: agnes.ardanuy@inrae.fr

M. Trinquier · C. Roux

Univ Toulouse, CNRS, Toulouse INP, LRSV, Toulouse, France

E. Lecloux · C. Jouany

Univ Toulouse, INRAE, Toulouse INP, ENSFEA, AGIR, Toulouse, France

B. Patrick

Univ Toulouse, INRAE, Unité Expérimentale Grandes Cultures d'Auzeville, Toulouse, France
*Funneliformis* consistently dominating under high P conditions.

*Conclusions* Our findings demonstrate that P and N availability jointly shape root AMF communities and regulate their nutritive function in wheat. The combination of community profiling and mycorrhizal molecular markers provides a valuable approach for understanding the AMF contribution to plant nutrition across agroecosystems, and therefore can be used for optimising agroecological practices.

**Keywords** Arbuscular mycorrhiza · Peri-arbuscular nutrient transporters · Marker genes · ITS2 metabarcoding · Durum wheat · Long-term field trial · Fungal communities

Introduction

Nutrient availability in soil is a critical factor in plant productivity, influencing not only plant physiology but also plant interactions with beneficial soil microorganisms, such as arbuscular mycorrhizal fungi (AMF) (Smith and Smith 2011). AMF associate with roots of the majority of land plants, including most crops, and provide a variety of services to host plants in exchange for carbon, such as the delivery of nutrients acquired through their vast hyphal networks, stress tolerance and pathogen resistance (Brundrett 2002; Pozo and Azcón-Aguilar 2007; Bennett and Groten 2022).

This symbiosis has been shown to enhance plant nutrient acquisition under conditions of nutrient limitation, with AMF playing a crucial role in the uptake and delivery of phosphorus (P), nitrogen (N), potassium (K), sulphur, and micronutrients, such as iron, copper, manganese, and zinc (Hodge and Fitter 2010; Wipf et al. 2019). Mycorrhizal plants can acquire nutrients through two distinct pathways: (1) a direct pathway, in which nutrients are acquired through the root epidermis and root hairs, and (2) a mycorrhizal pathway, in which nutrients are acquired through the AMF extraradical mycelium and transferred to the root cortical cells containing AMF arbuscules, typical branched structures involved in plant-AMF nutrient exchanges (Smith et al. 2003). The relative contribution of each pathway to plant nutrition has been

shown to be dependent on various biotic and abiotic factors, including soil nutrient availability, pH, soil texture, plant genotype, and the identity of AMF taxa (Smith et al. 2004; Cavagnaro et al. 2005; Feddermann et al. 2010; Smith et al. 2011).

Soil nutrient enrichment is expected to reduce the cost-benefit balance of the symbiosis in terms of nutrients acquired by a plant via the mycorrhizal pathway vs the carbon allocated to the AMF (Johnson 2010; Olsson et al. 2010; Bennett and Groten 2022; Bunn et al. 2024). Therefore, intensive mineral fertilisation practices in agriculture can disrupt AMF colonisation and nutrient exchange and potentially lead to reduced AMF biomass and diversity in soils (Treseder and Allen 2002; Oehl et al. 2004; Egerton-Warburton et al. 2007; Oehl et al. 2010; Verbruggen et al. 2010; Edlinger et al. 2022). This reduction in diversity in soils potentially compromise the functional potential of AMF communities, as less diverse fungal communities may be less able to provide a full range of ecosystem functions and consequently less benefits to host plants (Powell and Rillig 2018). In a long-term experiment, repeated N, P, and K application has been reported to increase AMF alpha diversity in wheat roots (Ma et al. 2021), while it was shown that both AMF richness and phylogenetic diversity in sorghum roots were reduced when increasing short-term P fertilisation (Frew et al. 2023). These findings highlight the complexity of AMF responses to fertilisation and underscore the role of both environmental and host filtering in the assembly of functionally distinct communities.

In addition to the well-known effects of fertilisation on root colonisation levels and root/soil AMF community structure and composition, it is essential to understand how fertilisation influences the plant mycorrhiza-mediated nutrition. One promising approach is to track the expression level of genes coding for arbuscular mycorrhiza (AM)-specific nutrient transporters as a way to approximate nutritional benefit in ecologically realistic conditions. These transporters specifically located on the peri-arbuscular membrane, mediate plant nutrient uptake through the mycorrhizal pathway (Gamper et al. 2010; Wipf et al. 2019). By characterising the expression of these genes, it is possible to compare the activation of the mycorrhizal pathway for specific nutrient acquisition at a given time across contrasting fertilisation
treatments, AMF communities and environmen-
tal conditions (Feddermann et al. 2010; Wipf et al.
2019).

The aim of this study was to investigate the effect
of long-term P fertilisation on AMF communities and
mycorrhiza-mediated nutrition in wheat. We hypoth-
esised that (i) increasing soil P availability would
reduce AMF diversity and shift fungal community
composition towards species adapted to high P envi-
ronments; (ii) the contribution of mycorrhiza to wheat
nutrition would decline under high P conditions,
as the plant carbon cost of maintaining AM would
exceed the benefits of nutrient acquisition. To test
these hypotheses, we combined the characterisation
of fungal communities and wheat mycorrhiza-medi-
ated nutrition in a 50-year P fertilisation trial during
two distinct years on wheat. Differences observed
between the two field campaigns led us to hypothe-
sise that plant N status modulates AMF functioning
across P availability gradients. To explore this further,
we conducted controlled experiments to examine the
interaction between N and P availability on AM sym-
biosis. By combining AMF community profiling with
analysis of selected AM-specific nutrient transporter
expression in wheat roots, we aimed to link wheat
plant investment in the mycorrhiza-mediated nutri-
tion with shifts in root-associated AMF communities
under contrasting soil nutrient availability.

**Methods and materials**

Long-term P fertilisation field trial and root sampling

A long-standing phosphorus (P) fertilisation field trial (Colomb et al. 2007) is maintained since 1968 at the experimental unit “Crop Phenotyping and AgroEcology Experimental Facility” (https://doi.org/10.15454/1.5483266728434124E12) in Auzeville-Tolosane, South-Western France (43.31487; 1.30185). The trial is located on deep alluvial silty clay soil. The experimental design follows a split-plot design consisting of six randomised blocks of P treatment plots, each measuring 6 m × 15 m, with a 1 m buffer zone to prevent P dispersion. Three P treatments were evaluated: (1) No P, with no P fertilisation since 1968; (2) Low P, where P fertilisation balances annual P export from crop

harvest; and (3) High P, where P fertilisation was four-
fold (1968–1993), then threefold (1994–present) that
of Low P, gradually increasing soil P availability. Since
1994, the P input was supplied as Triple Superphos-
phate adjusted to 11 and 33 kg P ha⁻¹ yr⁻¹ for Low P
and High P treatments, respectively. In 1968, the mean
P Olsen was 6.2 mg/kg in the topsoil (0–30 cm depth)
and in 2021, it has decreased to 5.3 mg/kg in the No P
treatment, while it has increased to 10.0 and 48.0 mg/
kg in Low P and High P treatments, respectively. Soil
characteristics for each plot are described in Table S1.

Durum wheat (*Triticum turgidum* L. subsp. *durum* var. RGT Voilur) was cultivated over two seasons, from November to June/July in 2018–2019 and 2021–2022, at a density of 300 seeds m⁻². Soil preparation involved superficial tillage of the topsoil layer a month prior to sowing. Nitrogen (N) fertilisation, using 33.5% ammonium nitrate, was applied to achieve a total of 174 kg/ha N in 2019 and 160 kg/ha N in 2020, through 3 applications. Note that the 2019 sampling (May 2019) was done after the third ammonium nitrate application, while the 2022 sampling (April 2022) was done before the third application. The crop management practices for each campaign, sampling dates and meteorological data are summarised in Table S2 and Table S3. Root samples were collected just before the heading stage, during the stem extension phase (5 samples per plot in 2019, and 3 per plot in 2022). Each sample consisted of a pool of at least three plant root systems to a depth of 15 cm. Root samples were hand-washed with tap water to remove adhering soil, using 180 µm sieves to collect fine root fragments and immediately frozen in liquid N and stored at –80 °C for subsequent DNA and RNA extraction. In May 2022, a second sampling was conducted to evaluate AMF colonisation and collect soil for the controlled condition *experiment 2*. The roots N content (%) was analysed at the plot level (by pooling the plot samples) using an Elementar Analyser (Vario ElCube).

Experiments in controlled conditions

Experiments were carried out in a climatic chamber
at 25 °C and a photoperiod of 16:8 light:dark. Durum
wheat seeds (var. RGT Voilur) were rehydrated in
deionised water for an hour and were then germinated
on petri dishes containing gelose in the dark at 4 °C for 1 week followed by four days at 28 °C.

## Experiment 1 - testing specificity and efficiency of primers targeting the wheat AM-marker genes

Pregerminated seedlings were placed in 50 mL pierced falcon tubes filled with attapulgite (Oil Dri UK) and watered with a 0.5x modified Long Ashton nutrition solution (containing 7.5 µM $NaH_2PO_4$). Half of the tubes were inoculated (Myc+) with 200 spores of *Rhizophagus irregularis* DAOM 197198 (Agronutrition) and the other half (Myc-) were left as uninoculated controls. After 5 weeks, root systems were cleaned and immediately frozen with liquid N and stored at -80 °C. Root systems were pooled in groups of three for subsequent RNA extraction.

## Experiment 2 - complementary assays to long-term P fertilisation field experiments

To test whether the N:P interaction influences mycorrhiza-mediated nutrition in wheat, a factorial experiment was conducted using field trial soil from the No P and High P treatments in combination with an N fertilisation treatment. Briefly soils obtained from all the No P and High P plots were pooled according to treatment, homogenised and placed into 350 ml pots. Sterile pre-germinated wheat seedlings were transplanted into No P and High P soil pots and cultivated under two nutrient treatments: (1) control (watered with deionised water), and (2) N addition (watered with 2000 µM $KNO_3$) for 10 weeks ($n=7-9$ pots per treatment). The pots were watered at a frequency that followed the plant need, until the soil was saturated. At the end of the experiment, root systems were washed and split in two for AMF colonisation assessment (immediately placed in KOH 10%) and for RNA extraction (immediately frozen with liquid N and stored at -80 °C).

## Experiment 3 - evaluation of P and N interaction on mycorrhizal colonisation

Pre-germinated seedlings were planted in 50 mL Falcon containers filled with attapulgite (Oil Dri UK), and either inoculated with 200 spores of *Rhizophagus irregularis* DAOM 197198 (Agronutrition) per plant or left as non-mycorrhizal controls. Plants were grown under six distinct nutrient treatments with contrasting P ($KH_2PO_4$) and N ($KNO_3$) content: (A) 900 µM P, 1000 µM N, (B) 300 µM P, 2000 µM N, (C) 900 µM P, 2000 µM N, (D) 2400 µM P, 2000 µM N, 900 µM P, 5000 µM N, and (F) 2400 µM P, 5000 µM N. Details on the composition of the nutritive solution are provided in Table S5. Plants were harvested at 5 weeks and aboveground biomass was weighed and roots were separated for AMF colonisation assessment.

## Mycorrhizal colonisation

Roots were stained for 2 min at 95 °C in a 5% ink-vinegar solution after a 10% KOH treatment for 10 min at 95 °C. Roots were sectioned into 1 cm fragments, mixed and spread across gridded petri dishes. AMF colonisation was assessed by the “gridline intersect” method under a binocular microscope (Leica S6E, 40x) (Giovannetti and Mosse 1980). The presence or absence of mycorrhizal structures was noted at each intersection between a root fragment and a grid line. Mycorrhizal structures, including arbuscules, internal hyphae and vesicles were recorded for a minimum of 100 intersections per sample.

## DNA and RNA extraction

Frozen root samples were first manually ground in liquid N using a mortar and pestle. DNA extraction was performed with 100 mg of root powder using ZymoBIOMICS 96 DNA Kit (Zymo Research) with 50 µl elution buffer volume. Before extraction, samples were additionally grinded for 10 minutes using a mixer mill Retsch MM 400. For RNA extraction, 100 mg of root powder were transferred in 2 ml tubes containing one metal bead (4 mm)/tube, and further grinded twice for 1 minute in a mixer mill Retsch MM 400. Total RNA was extracted using the NucleoSpin® RNA Plus Kit (Macherey Nagel). The quality and integrity of a subset of the RNA samples were assessed using Agilent RNA 6000 Nano Chips (Agilent Technologies), while RNA quantity was measured using a Nanodrop spectrophotometer.
Analysis of AM marker gene expression

We identified by Blast search the orthologs in wheat
of genes coding for the well-conserved ammonium
transporter (SymAMT), nitrate transporter (SymNPF),
phosphate transporter (SymPT), energising proton
pump ATPase (SymATPase) involved in the mycor-
rhiza-mediated nutrition (Harrison et al. 2002; Wang
et al. 2014; Breuillin-Sessoms et al. 2015; Wang et al.
2020) and of a gene (AM3) coding for a secreted
LysM protein (Yu et al. 2023; Tian et al. 2024) used
as an early AMF colonisation marker gene (Gutjahr
et al. 2008). These marker genes were used as proxies
for symbiotic nutrient transport and the establishment
of the AM. Primers were designed, when possible, on
conserved regions in the homoeologous (A, B and D)
copies in various wheat genomes. The primers used
for amplification of the AM marker genes in wheat
and the reference genes (GAPDH, TUB, TEF) are
listed in Table S6.

Complementary DNA (cDNA) synthesis was then
performed through reverse transcription with the 5X
PrimeScript RT Master Mix (Takara), involving an
incubation step at 37 °C for 15 minutes, followed by
5 seconds at 85 °C. RT-qPCR was conducted at an
annealing temperature of 60 °C using Takyon No
ROX SYBR 2X MasterMix blue dTTP (Eurogentec)
on a CFX Maestro (Bio-Rad). Gene expression levels
were then normalised relative to three wheat house-
keeping genes with technical triplicates performed for
each biological replicate using CFX Maestro software.

Sequencing of root-associated fungal communities

ITS2 rRNA amplicon libraries were generated
through nested PCR: first, using rcAMDGR (5'-ATG
ATTAATAGGGATAGTTGGG-3') (Sato et al. 2005)
& ngsITS4 (5'-TCCTSCGCTTATTGATATGC-3')
(White et al. 1990) chosen to exclude wheat ITS2
rRNA, and secondly using ITS 86F (5'-GTGA+ATC
ATCGAATCTTTGAA-3') (Turenne et al. 1999) &
ngsITS4, which generate an amplicon size compat-
ible with Illumina MiSeq. For both PCR, GoTaq® G2
DNA polymerase (Promega) was employed within the
following mixes, PCR 1 (25 µl reaction volume) on
1 µl of DNA extraction: 1X GoTaq® Reaction Buffer,
0.08 mM dNTPs, 0.32 µM of both primers, 0.04 U/
µl of polymerase, and 2% DMSO; PCR 2 (60 µl reac-
tion volume) on 1 µl of PCR 1 product: 1X GoTaq®

Reaction Buffer, 0.2 mM dNTPs, 0.4 µM of both primers, 0.02 U/µl of polymerase, and 2.5 mM of MgCl2. Nuclease-Free Water for Molecular Biology (Sigma-Aldrich) was used for each PCR mix. PCR were performed with initial denaturation at 95 °C for 5 min and final elongation at 72 °C for 10 min. PCR 1 entailed 40 cycles at 95 °C for 5 sec, 62 °C for 30 sec, and 72 °C for 1 min. PCR 2 entailed 35 cycles at 95 °C for 30 sec, 55 °C for 30 sec, and 72 °C for 1 min. The resulting amplicon (300–500 bp) was visualised via electrophoresis on a 2% agarose gel. The subsequent library creation followed the Illumina two-step PCR protocol and underwent normalisation using SequalPrep plates (Thermofischer). Paired-end sequencing was performed at the Bio-Environment platform (University of Perpignan Via Domitia, Perpignan, France) on a MiSeq system (Illumina) using v2 chemistry with a 2×250 bp read length, following the manufacturer's protocol.

Bioinformatics

Raw reads were processed using the pipeline FROGS version 4.1 (Bernard et al. 2021) via the Galaxy platform hosted by INRAE Toulouse (https://vm-galaxy-prod.toulouse.inrae.fr/galaxy_main/). This involved a series of steps: merging reads, quality pre-filtering, dereplication, and trimming at 400 bp using VSEARCH version 2.17.0 (Rognes et al. 2016), flash version 1.2.11 (Magoč and Salzberg 2011), and cutadapt version 2.10 (Martin 2011). Next, clustering was performed through the SWARM algorithm (Mahé et al. 2014) and chimeras and singletons were eliminated. A minimum proportion filter of 0.005% of all sequences was applied to retain zero-radius Operational Taxonomic Units (zOTUs) (Escudié et al. 2018). An ITSx filter version 1.1.2 (Bengtsson-Palme et al. 2013) was employed to identify and extract ITS2. Taxonomic assignment was performed by blastn+ (Camacho et al. 2009) using the UNITE Fungi 8.3 database (Kõljalg et al. 2013). Clean sequences are deposited at SRA (bioproject ID PRJNA1291988 available at: http://www.ncbi.nlm.nih.gov/bioproject/1291988).

Data analysis

All statistical analyses were performed using R 4.1
using the packages detailed in each section.
*Effects of treatments on mycorrhizal functioning and mycorrhizal colonisation*

The effects of the experimental treatments on the expression of the AM marker genes (field & experiment 2), were analysed by ANOVA with a posterior Tukey test for multiple comparisons. ANOVA assumptions were evaluated using the R package ‘performance’ (Lüdecke et al. 2021). In the case a response variable violated assumptions of homogeneity of variances and/or residual normality, we performed a non-parametric Kruskal-Wallis test followed by a Dunn test for multiple comparisons with Benjamini-Hochberg correction (function ‘dunn. test’). For *experiment 3*, a linear model was fitted to assess the interaction of N and P availability on biomass and root colonisation by AMF. When significant, the interaction was visualised using the ‘interactions’ R package.

*Analyses of fungal communities in wheat roots*

**Datasets** Metabarcoding data of root-associated fungal communities were processed with the R ‘phyloseq’ package (McMurdie and Holmes 2013). We examined rarefaction curves for each sample to assess sequencing quality. Next, we merged biological replicates per plot in order to obtain the fungal communities for each experimental plot. Rarefaction curves generated for each plot can be found in Fig. S1. To evaluate changes of AMF communities (belonging to the *Glomeromycota* phylum) with P fertilisation treatments, we investigated community composition in terms of proportion of different taxa, indicator species analysis, alpha diversity, and we examined community structure with ordination and PERMANOVA analyses. Due to the issues associated with using rarefied or relative abundance data for community structure analyses of compositional data (McMurdie and Holmes 2014; Gloor et al. 2017), different approaches or data transformations were applied for each analysis (specified below).

**Community composition** zOTUs belonging to the *Glomeromycota* phylum were extracted from the fungal dataset and relative abundances at genus

level were calculated and plotted in compositional plots according to P treatment and sampling year. Unclassified and multi-affiliated taxa at genus level were merged into an unidentified genus group. To explore the contribution of specific zOTUs to community composition in each P treatment, we plotted rank abundance curves (R package ‘BiodiversityR’, Kindt 2023) and the 20 most abundant zOTUs (*Glomeromycota* phylum, CLR normalised) into a heatmap (R package ‘microbiomeutilities’, Shetty and Lahti 2025).

**Indicator species analysis** To explore preferential zOTU associations to P treatments, we used indicator species analysis (De Cáceres and Legendre 2009), which calculates an association index (IndVal) between taxa and treatment, and then tests for its significance with a permutation test (R package ‘indicspecies’). IndVal is a product of two components: A which corresponds to an estimate of the probability that a sample belongs to a treatment (specificity), and B which is an estimate of the probability finding a taxon in a treatment (fidelity). These components give additional information on why a taxon can be used as an indicator.

**Alpha diversity** Alpha diversity of AMF communities (Richness, Shannon index, Gini-Simpson index, Pielou’s evenness and dominance index) was estimated per P treatment (R package ‘microbiome’, Lahti and Shetty 2012). To calculate alpha diversity indices, raw data were rarefied to equal number of fungal reads (minimal sample reads: 20,704 reads in 2019 and 34,957 in 2022). Within a year, variation in alpha diversity indices across P treatments was evaluated using Kruskal-Wallis tests followed by a Dunn test for multiple comparisons with Benjamini-Hochberg correction.

**Beta diversity** A permutational multivariate analysis of variance (PERMANOVA, R package ‘vegan’, Oksanen et al. 2022) was then performed on Aitchison distances (CLR transformed AMF raw data) to test the effects of the P treatments and the experimental block on AMF community structure. Multiple pairwise comparisons were performed for significant PERMANOVA results and were corrected via the Bonferroni correction (R function ‘pairwise.adonis’).
All significant PERMANOVA results were further analysed with permutational multivariate analyses of dispersion (PERMDISP, R package 'vegan') to verify that all significant PERMANOVA results represented a difference in average community composition, rather than a difference in dispersion of communities among treatments. To visualise differences in AMF community composition, Aitchison distances were ordinated using principal components analysis (R package 'PCAtools', Blighe and Lun 2023).

## Results

Validation of the arbuscular mycorrhiza (AM) marker genes in controlled conditions

We tested the specificity of the primers and of the expression patterns of wheat genes orthologous to well-characterised symbiotic genes in other species (transporters SymAMT, SymNPF, SymPT; proton pump SymATPase; and the colonisation marker AM3) by measuring their expression in mycorrhizal and non-mycorrhizal roots of wheat grown under controlled conditions (experiment 1). All selected symbiotic genes tested were only detected in plants inoculated with R. irregularis (Fig. 1).

Nutrient transporter expression and N availability in field conditions

We then measured the expression of the nutrient transporters and of the colonisation marker gene in the roots of wheat grown in field plots that received various levels of long-term P fertilisation (No P, Low P and High P). Patterns of relative gene expression according to P treatments differed between years (Fig. 2). While in 2022 there was a pattern of decreased relative gene expression of AM marker genes as P fertilisation increased, this was not observed in 2019. In contrast, the expression of the symbiotic ammonium transporter (SymAMT) increased with increased P availability in 2019. We hypothesised that the effect of the years could be related to variability in the level of N availability at the sampling timepoint. To test it, we measured wheat root N content (%N), as a proxy of N availability, on the same root material used for RT-qPCR. Root N content differed between years with N (%) 1.5-fold greater in 2022 than in 2019 (Fig. 3). In 2019, %N content differed between P treatments with high P roots presenting lower N than the No P treatment. No significant differences in root %N were detected among P treatments in 2022, although a similar trend was observed.

<figure><img src="image_2.png" /><figcaption>**Fig. 1** Selected AM marker genes were specifically expressed in mycorrhizal wheat roots. Relative expression of genes coding for ammonium transporter (SymAMT), nitrate transporter (SymNPF), phosphate transporter (SymPT), and proton pump (SymATPase) well-known to be localised in the peri-arbuscular membrane in other plant species, and of an AMF colonisation</figcaption></figure>

marker gene (AM3) measured by RT-qPCR in mycorrhizal (inoculated with R. irregularis, Myc+) and non-mycorrhizal (control, Myc-) roots of wheat grown on attapulgite under controlled conditions (n = 3)). Bars represent the mean, individual plant pools are shown as black dots
<figure><img src="image_3.png" /></figure>

**Fig. 2** P fertilisation inhibited AM in wheat roots sampled in 2022 but not in those sampled in 2019. Relative expression of AM marker genes in wheat roots measured by RT-qPCR sampled in a long-term P fertilisation trial (treatments No P, Low P or High P) in 2019 or 2022 (n = 3–6). Bars represent the mean, individual plant pools are shown as black dots. Different letters indicate significant differences in AM marker

gene expression among P treatments (*p* value <0.05, detailed in Table S7) assessed by ANOVA followed by post hoc Tukey test or by Kruskal-Wallis test followed by Dunn test with Benjamini-Hochberg correction when ANOVA assumptions were not met. Note the difference of scale among AM marker genes and years

<figure><img src="image_4.png" /></figure>

**Fig. 3** Root N content (%) in field conditions was greater in 2022 than in 2019 (n = 5 to 8). Bars represent the mean, individual plant pools are shown as black dots. Different letters indicate significant differences (*p* value <0.05, detailed in Table S8) assessed by ANOVA followed by post hoc Tukey test. Asterisk shows a difference (*p* value <0.05, detailed in Table S8) between sampling years for all P treatments combined using ANOVA

## Effects of N and P availability on the AM interaction

To confirm that interaction between N and P availability influences AM functioning in wheat, soils collected from the long-term P field trial were used in combination with a N fertilisation treatment in a controlled condition experiment (experiment 2). AMF colonisation, measured by the expression of the marker gene *AM3* (Fig. 4a) and by microscopy (Fig. 4b), as well as the mycorrhiza-mediated nutrition (expression of the AM-specific nutrient transporters/proton pump *SymAMT*, *SymNPF*, *SymPT* and *SymATPase*, Fig. 4a) responded to the interaction between N and P availability in a similar manner as in field conditions. No difference in AMF colonisation was observed in the experimental treatments in which one of the nutrients was limiting, (i) No P, limited availability of both P and N, (ii) No P+N, limited availability of P but sufficient N availability,
<figure><img src="image_5.png" /><figcaption>**Fig. 4** Expression of the mycorrhiza-mediated nutrition genes and AMF colonisation are modulated by soil N and P availability in wheat plants grown in controlled conditions. (a) Relative expression of AM marker genes was measured by RT-qPCR on wheat plants grown under controlled conditions on soils with distinct P availability (soils from long-term P fertilisation field trial No P or High P) and N fertilisation treatment (+N corresponds to fertilisation 2000 µM of nitrate) (n = 3). Bars represent the mean, individual plant pools are shown as black dots. Different letters indicate significant differences (*p* value <0.05, detailed in Table S9) in the AM marker gene expression among P treatments assessed by ANOVA followed by post hoc Tukey test or by non-parametric Kruskal-Wallis test followed by Dunn test with Bonferroni correction when ANOVA assumptions were not met. (b) AMF root length colonisation was measured using the grid intersect method. Bars represent the mean, individual plants are shown as black dots (n = 7–9). Different scales are used according to the structures. Different letters indicate significant differences (*p* value <0.05, detailed in Table S9) using Kruskal-Wallis test followed by post-hoc Dunn test with Bonferroni correction. Note that the level of AMF root colonisation may be overestimated when wheat is grown in field soil, due to the potential misidentification of intracellular structures formed by endophytic fungi</figcaption></figure>

and (iii) High P, limited N availability but sufficient P availability. In contrast, AMF colonisation was inhibited in the High P+N treatment, where both N and P were sufficient. Interestingly, *SymNPF* and *SymATPase* expressions were upregulated by High P treatment only when N was limiting, while *SymPT* was upregulated in all treatments where P was limiting. In sum, wheat plants showed increased AMF

colonisation in treatments with a limiting nutrient, yet N and P transporters showed contrasting expression regulation depending on the identity of the limiting macronutrient.

A second controlled experiment (*experiment 3*) was performed to study in more detail the interaction of N and P availability in the soil on root AMF colonisation. As in *experiment 2*, results show an increase of
AMF colonisation when one macronutrient was limiting (Fig. 5b; considering treatment F as no limitation, see Fig. S2 for details on AMF structures). A linear model further confirmed that the interaction between P and N availability explained mycorrhizal colonisation of wheat roots (overall model $F_{3,40} = 128.7 p < .001$ and $R^2_{adj} = 0.89$; $P \beta = -0.38$, $p < 0.001$; $N \beta = -0.77$, $p < 0.001$, interaction $\beta = -0.42$, $p < 0.001$; interaction effects are represented in Fig. S3). Biomass, in turn, was greatly determined by N availability (Fig. 5c, treatments A-C-E & D-F) but not by P availability (Fig. 5c, treatments B-C-D & E-F).

AMF communities in wheat roots in a long-term P fertilisation assay

In addition to investigating the effect of P treatments on AMF colonisation and mycorrhiza-mediated nutrition in field-grown wheat, we analysed its effect on root AMF communities by metabarcoding. We used an original combination of already published primers to avoid amplification of wheat ITS2. After bioinformatic processing, 654,338 and 820,217 sequences obtained by Illumina sequencing of an ITS2 amplicon were retained and clustered into 580 and 738 zOTUs for 2019 and 2022, respectively. A total of 573 and 722 zOTUs were identified as fungi using the UNITE

database (with zOTUs not classified as fungi representing 3.60 and 8.83% of the reads). From this set, 524 zOTUs (2019) and 643 zOTUs (2022) were assigned to a specific fungal phylum. The phylum *Glomeromycota*, i.e. AMF, comprised 79 zOTUs in 2019 and 148 zOTUs in 2022, representing 4.66% and 6.31% of the total reads, respectively, with similar proportions across P treatments (Fig. S4). Although beyond the scope of this study, other fungal phyla increased their relative abundance with increased P fertilisation. The relative abundance of the *Olpidiomycota* increased with P fertilisation in both years, while an increase in *Mortierellomycota* relative abundance with P fertilisation was observed only for 2022. Taxonomic affiliation and zOTU tables are provided in Tables S11-S14.

AMF fungal communities in wheat roots were dominated by zOTUs belonging to the family *Glomeraceae* for all treatments and years (Fig. 6). There was a trend of increased relative abundance of the genus *Funneliformis* with long-term P fertilisation for both campaigns, and a complementary trend of decreased proportion of zOTUs belonging to non-identified *Glomeraceae*, with greater differences in relative abundance of *Funneliformis* genus between P treatments for the year 2022. Similar patterns were observed when AMF composition was

<figure><img src="image_6.png" /><figcaption>**Fig. 5** Limiting N and P enhances wheat root AMF colonisation by *R. irregularis* in artificial soil. Wheat plants were grown under controlled conditions in attapulgite using a range of 6 nutritional treatments with a gradient in N and P concentration. Wheat was grown for 5 weeks after inoculation with 200 spores of *R. irregularis* DAOM197198. (a) Details of the 6 nutrient conditions tested in terms of N (nitrate) and P concentration. (b) AMF root length colonisation measured using</figcaption></figure>

the grid intersect method and (c) Above-ground dry biomass. Bars represent the mean, individual plants are shown as black dots ($n = 7–9$). Different letters indicate significant differences (*p* value <0.05, detailed in Table S10) assessed by ANOVA followed by post hoc Tukey test (b), and Kruskal-Wallis test followed by post hoc Dunn test with Benjamini-Hochberg correction (c)
<figure><img src="image_7.png" /><figcaption>**Fig. 6** Relative abundance of AMF genera in wheat roots from a long-term P fertilisation field trial. P fertilisation treatments: No P, Low P, and High P. Wheat was sampled in (a) 2019 and</figcaption></figure>

(b) 2022. Relative abundances of AMF were calculated based on Illumina sequencing of an ITS2 amplicon

evaluated at the zOTU level. The most dominant zOTUs in 2019 were affiliated as *Funneliformis mosseae* [zOTU_126, zOTU_132 & zOTU_91] in Low P and High P treatments with the most abundant cluster [zOTU_126] presenting 15% of the total number of reads, while for the No P treatment the most abundant zOTU [zOTU_94], also *Funneliformis mosseae*, represented 8% of the reads. In 2022, the *Funneliformis mosseae* [zOTU_99] dominated communities in all P treatments but with increasing relative abundance as P fertilisation increased (9% for No P, 16% for Low P and 24% for High P). Rank abundance curves showing AMF zOTU dominance and heatmaps showing the distribution of the most abundant AMF zOTUs among plots and treatments can be found in Fig. S6 and S7. The patterns of root-associated AMF communities alpha diversity indices at the zOTU level differed between years. In 2019 there were no differences in alpha diversity indices across P fertilisation treatments. In contrast, in 2022, alpha diversity indices Gini-Simpson and Pielou's evenness decreased as P availability increased (Fig. 7, differences between P fertilisation extremes). Community dominance was the greatest under the high P treatment, which was

on average 2-fold greater than the No P treatment (Fig. 7). In sum, in 2022, plants grown in High P treatment soils presented less diverse communities which were dominated by a smaller number of zOTUs in comparison with the No P treatment (Fig. S6 and S7).

The structure of AMF communities at the zOTU level differed between P treatments for both years (Fig. S7). Pairwise contrasts revealed differences among No P and High P treatment after adjusting for multiple comparisons but not according to experimental block (Table S15). Differences in multivariate dispersions among P treatments were detected for year 2019, with higher variation in community structure for the No P treatment in comparison to the P addition treatments (Fig. S7). The visualisation of community structure by PCA showed that P treatments separated by the PC1 axis with 27.5 and 29.9% of variation explained in 2019 and 2022, respectively (Fig. S7).

In addition, an indicator species analysis showed that a number of zOTUs belonging to the *Funneliformis* genus (mostly *F. mosseae*) were indicators of P fertilisation in 2019 (11 zOTUs) and 2022 (9 zOTUs) (Fig. 8). In contrast, zOTUs belonging to the
<figure><img src="image_8.png" /><figcaption>**Fig. 7** Alpha diversity metrics of AMF zOTUs in wheat roots from a long-term P fertilisation field trial. Wheat plants were sampled in (a) 2019 and (b) 2022. Differences among treatments were assessed by Kruskal-Wallis test followed by post hoc Dunn test with Benjamini-Hochberg correction. Asterisks show significant differences among treatments (*p* < 0.05, detailed in Table S18)</figcaption></figure>

*Glomus* genus were indicators of both No P and Low P treatments in 2022, while few *Septoglomus* and unidentified *Glomeraceae* genus zOTUs were associated with No P in both years. The identity indicator zOTUs per P treatment and associated IndVal values are available in Tables S16 and S17 for 2019 and 2022, respectively.

## Discussion

In this study we investigated the effects of long-term P fertilisation on root-associated AMF communities in wheat and their role in nutrient acquisition under field conditions. It should be noted that, in this field experimental system, the gradient of availability

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 8** Number of AMF zOTUs which are indicator taxa closely associated with either No P, No P & Low P, Low P & High P or High P treatments in wheat roots from the long-term P fertilisation field trial. Wheat plants were sampled in (a) 2019 and (b) 2022</figcaption></figure>

of P is strong, ranging from very limited (No P) to an excess (High P) of P for crop development. This is reflected in the wheat yields during the two campaigns (Table S4) and in the yields of other crops previously grown in this field trial (Nawara et al. 2017). We measured the expression of peri-arbuscular transporters/pump involved in nutrient uptake and characterised the AMF community in roots of wheat grown in plots with varying P availability over two distinct years. We hypothesised that (i) the mycorrhiza-mediated nutrition is modulated by nutrient availability, and that the expression of AM transporters will follow a similar pattern; and that (ii) changing soil P availability will change AMF diversity shifting community composition towards species that better tolerate or compete in each environment. The results show that the composition and structure of root-associated AMF communities differed according to long-term P treatment and that the strength of differences varied depending on the year of sampling. Likewise, the differences in AM transporters expression among treatments was dependent on the year. The distinct N content in the roots at the time of sampling led us to hypothesise that the differences observed between the

different years were likely to be the result of the interaction between the availability of P and that of the other key macronutrient, N. The interaction between P and N for AM colonisation and functioning is well acknowledged in the literature (Sylvia and Neal 1990; Johnson 2010; Javot et al. 2011; Bonneau et al. 2013; Nouri et al. 2014; Püschel et al. 2016; Corrêa et al. 2024) and was further supported by experiments under controlled conditions. Finally, we showed that the expression of selected wheat AM-marker genes involved in arbuscule formation and plant nutrient uptake can be used to explore the contribution of the AMF community to plant nutrient acquisition.

Interaction in N and P availability on mycorrhiza-mediated wheat nutrition

Our results suggest that the interaction between the availability of the two major macronutrients, N and P, can partly explain the between-year differences in AM functioning in wheat in the same long-term P fertilisation trial. Although similar N fertilisation was applied in 2019 and 2022, the wheat N nutrition status differed between the campaigns. This could be due
to interactions between N fertilisation and meteorology, or crop succession (Geng et al. 2022; Zhao et al. 2022) (sunflower and soybean were grown before wheat in 2019 and 2022, respectively), which can dramatically affect N availability in soil. Alternatively, this could also be due to higher wheat N demand in 2019. Indeed, yield was 40% greater in 2019 than in 2022 for all P treatments (Table S4). Furthermore, the crop nutritional requirements change throughout the season influencing the formation and maintenance of mycorrhizae and their functioning. The formation and degeneration of arbuscules is highly dynamic (Kobae and Hata 2010; Kobae et al. 2016; Gao et al. 2019), allowing an adjustment of the AM to changes in plant nutritional status (e.g. nutrient limitation/starvation) and the soil nutritional context. By measuring expression of genes encoding peri-arbuscular transporters/ pump, we aimed to obtain a snapshot into the nutritional functions provided by AMF to their host plant at a given point in the season.

While in 2022 we observed inhibition of the AMF colonisation following increased soil P availability, in line with previously published data on the same field trial (Tang et al. 2016), and consequently a decrease in the expression of the AM transporters/ pump, we did not observe such responses in 2019. Our results suggest that the wheat N status greatly modulated mycorrhiza-mediated nutrition under field conditions. In 2019, N limitation may have reduced the need for enhanced P uptake in wheat plants under limiting P availability, while increasing the need for N uptake under non-limiting P availability. This aligns with previous work on maize showing that the activation of the transcriptional response to P starvation is contingent on N availability (Schlüter et al. 2012; Torres-Rodríguez et al. 2021), with the N limitation response dominating the transcriptome when plants were subjected to joint N and P limitation.

The controlled condition experiment using soil from the field trial confirmed that the expression of peri-arbuscular transporters regulated in wheat roots depends on the identity of the limiting macronutrient for the plant. P limiting conditions, no matter the N fertilisation treatment, promoted the expression of the symbiotic phosphate transporter (*SymPT*), while in P sufficient and limiting N conditions the symbiotic nitrate transporter (*SymNPF*) was upregulated. These regulations are independent of the level of colonisation since neither

the expression of the *AM3* marker gene nor the microsopy quantification changed between N-limiting/P-sufficient and N-sufficient/P-limiting conditions, while expression of *SymPT*, *SymAMT* and *SymNPF* changed drastically between these conditions. Interestingly, we found that *SymAMT* was upregulated in N-limiting/P-sufficient conditions in the field in 2019, while *SymNPF* was upregulated in controlled conditions under the same conditions. This difference in transporter expression may be a consequence of distinct N forms used as fertiliser (nitrate in controlled conditions and ammonium nitrate in the field) and suggests that the regulation of peri-arbuscular N transporters is specific to the nature of N available in the soil. In addition to *SymPT*, *SymAMT* and *SymNPF*, other members of the ammonium, nitrate and phosphate multigenic families are differentially regulated during AM, although their tissue expression partners and their role in nutrient exchange between plants and AMF are poorly characterised. Further studies are required to understand their contribution to mycorrhiz-mediated nutrition and how they are regulated depending on N and P availability.

Our results also demonstrate that despite being commonly used as markers of AMF colonisation, the expression of peri-arbuscular transporters is not a reliable indicator of colonisation. Conversely, the expression of the *AM3* marker gene in roots followed the same trend as the level of AMF colonisation quantified by microscopy under both controlled conditions and in the field trial (Fig. 4 and S8), suggesting that the expression of the *AM3* marker gene is a reliable indicator of AMF colonisation. The expression of *AM3* at a low colonisation level (when nutrition may not be the main benefit of AM) remains to be tested. Globally, our results support the idea that the use of these AM marker genes and transporters can be used as a proxy of mycorrhizal functioning of the AM at a moment in time when comparing treatments with contrasting nutrient availability. Next steps will be to link the transporter expression with measures of plant nutrient acquisition via the mycorrhizal pathway by means of isotopic tracer experiments.

## Long term P fertilisation effects on root-associated AMF communities

Our hypothesis that a long-term increase of soil P would lead to a reduction in AMF diversity and a
shift in AMF community composition towards spe-
cies adapted to high-P environments in wheat roots
was partly validated. We found that P fertilisation
affects the composition and structure of the wheat-
associated AMF community but not its richness, as
already described for other crops in previous field
trials (Beauregard et al. 2010; Lang et al. 2018;
Dueñas et al. 2020). The majority of AMF zOTUs
in wheat roots belonged to the *Glomeraceae* fam-
ily in all P treatments, as expected given that they
predominate in arable soils and that using the ITS2
fragment for metabarcoding is known to underrepre-
sent AMF basal lineages (Berruti et al. 2017; Lek-
berg et al. 2018). *Glomeraceae* in general exhibit
ruderal life-history traits according to Grime's
C-S-R framework (fast growth rates and preferential
allocation of biomass to roots rather than to extra-
radical hyphae), which are associated with condi-
tions of high disturbance and low stress, and are thus
dominant in agricultural ecosystems (Oehl et al.
2003; Ujvári et al. 2023). Within the *Glomeraceae*,
we observed a consistent shift in the root AMF com-
munity composition for both sampling years, with
zOTUs of the *Funneliformis* genus gaining preva-
lence with increased P availability. The increased
prevalence of the *Funneliformis* genus with P fer-
tilisation has previously been described in roots
of maize (Lang et al. 2018) and wheat (Peng et al.
2024). A similar correlation had also been observed
between the relative abundance of the *Funneliformis*
genus in roots and soil P availability throughout the
wheat growth cycle (Bainard et al. 2014). Interest-
ingly, the authors showed that while all soil AMF
exhibited negative correlations as P levels increased,
certain *Funneliformis* OTUs in the roots exhib-
ited positive correlations. They proposed that these
OTUs may shift their growth strategy with P avail-
ability, potentially investing less in extraradical
hyphae and more in root structures. In a long-term
fertilisation trial (Williams et al. 2017) reported
that soil AMF communities remained unaffected in
terms of composition with increased soil P, yet AMF
reduced their investment in extraradical biomass and
attributed such change to either a change in growth
habits or decreased carbon allocation to the fungi.
Such changes in biomass allocation of certain OTUs
(e.g. *Funneliformis*) with fertilisation could explain
our results. Another possible explanation could be
that certain *Funneliformis* zOTUs are more efficient

at the acquisition of other nutrients (e.g. N) that
become limiting with increased P fertilisation or that
they provide other non-nutritional benefits to plants
(Frew et al. 2023). Several studies on isolated strains
have indeed shown functional variability between
species and genotypes, including for ability to pro-
vide N or P to their host, supporting a possible shift
in the AMF community associated with nutritional
functionality (Munkvold et al. 2004; Avio et al.
2006; Mensah et al. 2015).

It is well established that the host plays a cru-
cial role in determining their associated AMF com-
munity. Various studies have shown contrasting
patterns in AMF community composition in roots
and soil in both natural and agricultural ecosys-
tems (Qin et al. 2020), increasing the challenge in
comparing findings across studies on distinct plant
species and potentially distinct nutritional statuses
(Johnson 2010). Interestingly, the strongest differ-
ences in root-associated AMF community structure
and diversity among long-term P treatments were
observed for year 2022, when N was non-limiting to
wheat plants (based on %N in wheat roots). In 2022,
a small number of unidentified *Glomeraceae* zOTUs
were most abundant in No P and Low P treatments
compared to High P, and indicators of No P and Low
P treatments. These zOTUs might be more adapted
at P mobilisation than dominant *Funneliformis mos-
seae* zOTUS which are ubiquitous but dominate in
the High P treatment. In contrast, N limitation in soil
could increase the competition between plant and
fungus for N (Kuzyakov and Xu 2013; Püschel et al.
2016). However, the question remains whether the
most abundant zOTUs in our treatments predomi-
nate due to their lifestyle traits or due to host filter-
ing and preferential allocation for resources.

Conclusion

A major goal in mycorrhizal research is to understand
how AMF diversity and community composition
determine host nutrient acquisition. A critical way to
achieve that will be to establish a link between AMF
communities and AM nutritional functions (Gamper
et al. 2010; Powell and Rillig 2018). The present
work performed on wheat, a major crop, shows the
potential to achieve that by enriching soil with N and
P, and combining characterisation of the AMF community composition with the expression of AM functional marker genes.

**Acknowledgments** We thank Nadine Bouzari for technical help. This study was developed within the framework of the "Laboratoires d'Excellences (LABEX)" TULIP (ANR 10 LABX 41) and of the "Ecole Universitaire de Recherche (EUR)" TULIP GS (ANR 18 EURE 0019).

**Author contributions** MT, CJ, CR, and BL designed the experiments. CJ, PB and EL performed the field assays and analysis. VG and MT performed RT-qPCR. MT performed all the others experiments, MT and AA analysed the data. MT, BL and AA wrote the manuscript.

**Funding** Open access funding provided by Université de Toulouse. MT PhD was co-funded by the Université Fédérale Toulouse Midi-Pyrénées (UFTMiP) and the Région Occitanie.

## Declarations

**Competing interests** The authors declare no competing interest.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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