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. 2026 Jun 1;21:97. doi: 10.1186/s40793-026-00913-8

Exploring the role of ergothioneine and mycorrhizal fungi in shaping the wheat soil microbiome

Sri Sai Nandini Ravi 1,2,#, Alexandra Pipinos 1,2,#, Nick Insley 1,2, Charanpreet Kaur 1,2, Jinjun Kan 3, Wade Heller 4, Andrew H Smith 5, Gladis Zinati 5, John Richie 6, Harsh Bais 1,2,✉
PMCID: PMC13455302  PMID: 42226086

Abstract

Background

The association between plants and soil microbes is critical for both soil and plant health. Introducing beneficial microbial inoculants, such as arbuscular mycorrhizal fungi (AMF), can enhance the uptake of nutrients and compounds in plants including ergothioneine (ERGO), a well-known antioxidant and anti-inflammatory compound that humans must obtain through diet. However, little is known about how ERGO and inoculated AMF influence ERGO uptake in plants or affect soil microbiome composition, including bacteria, archaea, and fungi. This study investigates the interaction between soil-applied ERGO and AMF inoculation in plant–microbe symbioses to assess (1) whether these treatments increase ERGO content in the staple crop wheat (Triticum aestivum), and (2) how they alter soil microbiome structure.

Results

Combined treatment with ERGO and AMF significantly increased wheat (Triticum aestivum) biomass and ERGO accumulation in roots and shoots. This co-treatment also led to higher bacterial diversity in rhizosphere. PERMANOVA analysis confirmed that both AMF and AMF/ERGO treatments significantly influenced microbiome composition, particularly bacterial communities. Indicator species analysis showed enrichment of Thioalkalivibrio, Chlorobi bacterium, Planctomycetes (Planctomycete A-2) and Candida subhashi, Paecilomyces zollerniae under ERGO and AMF/ERGO treatments.

Conclusions

Overall, the data show that plants can readily take up ERGO from the soil, with or without AMF, and that both ERGO and AMF amendments reshape soil microbiome communities. These findings highlight the broader role of ERGO and AMF in connecting soil microbiome to plant and human nutrition, a link that warrants further investigation.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40793-026-00913-8.

Keywords: Ergothioneine, AMF (arbuscular mycorrhizal fungi), Wheat, Rhizosphere, Soil microbiome

Background

In recent years, the crude protein content of agricultural crops has declined due to several interacting factors, including plant breeding, climate change, reduction in soil organic matter, and changes in environmental regulations [1, 2]. An estimated 2.4–2.9% decrease in crude protein content in grain crops has occurred over the past two decades [1]. Meta-analyses further indicate that rising atmospheric CO2 levels contribute to decreased protein levels in staple crops by limiting nitrogen (N) uptake and inhibiting protein biosynthesis pathways [3]. In addition to lower total protein concentrations, the protein quality in plants is generally lower due to reduced levels of essential amino acids, specifically lysine and sulfur-containing amino acids [4]. Because plant protein synthesis is tightly linked to soil N availability [5], recent research has increasingly focused on microbiome-based approaches to improve nutrient acquisition and crop nutritional quality [6]. Plant-associated microbial communities can substantially influence nutrient assimilation, including N acquisition, even in non-leguminous crops [5]. However, several important questions remain unresolved, including which microbial taxa directly contribute to N assimilation, how microbial interactions within the rhizosphere regulate protein formation, and how microbial inoculants alter indigenous soil microbial communities. Among these inoculants, arbuscular mycorrhizal fungi (AMF) are of particular interest because they form close symbiotic associations with most terrestrial plants, expanding effective root surface area and facilitating phosphorus (P) uptake [7]. AMF have also been shown to contribute to the uptake and transport of organic nitrogen compounds, such as amino acids [8]. However, the degree to which mycorrhizal associations improve plant protein content, as well as the ways in which AMF amendments alter local soil microbiomes and nutrient cycling processes, is still not fully understood.

Recent research has shown that ergothioneine (ERGO), an uncommon amino acid identified for its nutraceutical qualities in several model systems, can be biosynthesized by soil microbiota (fungi and certain bacteria), including some species with edible mushrooms [9]. Charles Tanret made the initial discovery of ERGO in 1909 when he isolated the amino acid from rye grain that had been infected by the ergot fungus, Claviceps purpurea [10]. Because of its anti-inflammatory and antioxidant effects on human health, ERGO has been referred to as a “longevity vitamin” based on numerous studies showing its cytoprotective qualities [11, 12]. Because ERGO tends to accumulate in tissues vulnerable to oxidative stress, it has become an important focus in human health research, particularly in relation to liver, cardiovascular, and neurological disorders [13]. However, neither plants nor animals can biosynthesize ERGO, despite its occurrence as a natural antioxidant in both systems [10, 14]. In plants and animals, ERGO is acquired primarily through the consumption of ERGO-producing fungi or through uptake from the soil, respectively. In soils, ERGO is likely synthesized by fungi and bacteria. Fungi produce ERGO from the precursor amino acid histidine via a two-gene pathway involving Egt1 and Egt2 [15, 16], whereas bacterial biosynthesis requires a five-gene pathway, EgtA-E [17].

The presence of numerous bacterial and/or fungal species in soil may consequently contribute to ERGO accumulation or uptake in plants [18]. Supporting this idea, recent studies across different plant species have shown that crops grown with AMF exhibited elevated ERGO levels [9]. For example, root inoculation with specific AMF species led to increased ERGO levels in wheat, oats, and barley compared to non-inoculated crops [18]. While a specific transporter for ERGO has been identified in animals [19], the mechanism underlying ERGO transport and uptake in plants remain largely unknown. Further research is needed to clarify how plants, such as cereal wheat, absorb ERGO and how this process can be optimized to enhance its dietary availability for humans. At the same time, there is a lack of comprehensive understanding regarding the responses and adaptations of soil and plant microbiomes to ERGO supplementation and AMF inoculation, necessitating further investigation.

To improve our understanding of how the root and soil microbiome influence ERGO uptake in cereal wheat (Triticum aestivum) as well as how ERGO and AMF influence soil microbiome, we conducted a seed-to-seed experiment using the following soil treatments: AMF inoculation, ERGO supplementation, combined AMF/ERGO treatment, and untreated control plants. We hypothesized that association with AMF would stimulate ERGO uptake in wheat and that the presence of both ERGO and AMF in the soil may further enhance ERGO uptake. We also predicted that AMF associations, with or without ERGO, could influence the plant-associated and soil microbiomes and enrich microbial taxa potentially involved in ERGO uptake. To assess the changes in microbiome structure in response to the treatments, amplicon high-throughput sequencing for both bacteria/archaea and fungi was performed for the root, rhizosphere, and bulk soils from all treatment groups.

Methods

Chemicals

L-ergothioneine (L-ERGO, purity > 99.99%) was obtained from MedChemExpress® (Monmouth Junction, NJ, USA). L-( +)-ergothioneine (ERGO) standard was purchased from Toronto Research Chemicals (Toronto, ON, CA), and L-( +)-ergothioneine-d9 (ERGO-d9) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA) as an internal standard. The ERGO inoculum for dose–response experiments was prepared by dissolving L-ERGO in 30 mL of nanopore water and further diluting the solution to 0.125 mg/mL and 0.375 mg/mL concentrations. These two exogenous concentrations were used based on the natural titers of ERGO found in biological or soil systems.

AMF

A consortium of AMF comprising various species was established. This consortium included Claroideoglomus etunicatum, Claroideoglomus claroideum, Rhizophagus irregularis, Rhizophagus intraradices, and Funneliformis mosseae, and was cultivated from agricultural soil managed organically at the Rodale Institute in Kutztown, PA. To create the inoculum, field soil was diluted with a mixture of autoclaved sand, soil, vermiculite, and turface (Applied Industrial Materials, Corp., Deerfield, IL, 60,015) prepared in the proportions of 1:0.75:1:0.75 (v/v) and thoroughly mixed to ensure homogeneity. This mixture was placed into 66-mL containers, into which individual seedlings of Bahia grass (Paspalum notatum) were transplanted, as described by Douds et al. [20]. A mixed species inoculum representing a native AMF community was propagated by preparing a 1:10 dilution of agricultural field soil from organically managed vegetable plots at the Rodale Institute in Kutztown, PA, USA [9]. Before being used in the experiment, the roots were cut into 1-cm segments, and the spore density was determined to be approximately 100 spores/mL [9]. The “control treatment” was prepared by autoclaving the inoculum at 121 °C for 60 min to control for the additional nutrients present in the compost.

Plant materials and growth conditions

Spring wheat (Triticum aestivum L. cv. Surpass) seeds obtained from Johnny’s Selected Seeds (Winslow, ME, USA) were used for all experiments. The seeds were directly sown at 3.81 cm dept measured from the soil surface using a ruler in 1 L (8.89 × 8.89 × 12.7 cm) pots containing pre-moistened potting media (ProMix,Quebec, Canada) and topped with a thin layer of vermiculite. The plants were grown in a greenhouse at 23 °C with a 16-h photoperiod, and a Photosynthetic Photon Flux Density (PPFD) of 600–900 µmol/m2/s.

ERGO and/or AMF treatment of Triticum aestivum

This experiment aimed to evaluate plant growth and soil microbiome responses to various treatments involving AMF and ERGO, with results compared across different growth phases of spring wheat. Germinating spring wheat seedlings were replanted into 160 pots containing ProMix without mycorrhizae. Four different treatments were planned for the wheat mesocosm experiments: Control (Untreated plants); ERGO, AMF and ERGO + AMF treatments respectively. For AMF treatments, inoculum containing 6000 spores was counted with hemocytometer [9] and homogenized with 40-L ProMix BX (PremierTech Horticulture, Riviere-du-Loup, Quebec, CA) and used to fill each set of 40 pots/treatment (~ 150 spores/pot). Mock inoculum that consisted of autoclaved inoculum added at the same rate to pots for the non-AMF treatments. For ERGO dosing, plants were treated with 60 mL of solution containing 0.025 mg L-ergothioneine per pot at day 0. Control plants for the ERGO treatment received 60 mL of distilled water. A booster application of 0.025 mg ERGO per pot was given on day 40 of growth.

Thus, for days 40, 50, and 60 harvest, 13 plants per treatment were harvested, for a total of 52 plants per day of harvest, and an overall total of 152 plants for all 4 treatments and 3 harvest days. For plant growth, images were taken, and plant samples were collected on the 60th day. For microbiome analysis, the roots were harvested and both root and adhering rhizosphere soil as collected for microbiome analysis. Bulk soil was collected from pots where plant roots did not expand. The plant roots, rhizospheric soil, and bulk soil samples were sent to Stroud Water Research Center (Avondale, PA), for microbial community analyses (described separately below).

ERGO analysis and sample preparation procedure

For ERGO analysis, the roots, shoots, and grain of each plant were harvested and placed in an oven dryer at 60 °C for at least 24 h. For ERGO quantification, the dried samples were ground to a powder using a small sample grinder coupled with a size 40 mesh (0.4 mm). The finely ground samples were then sent to Penn State College of Medicine for analysis by High-Performance Liquid Chromatography (HPLC) as described in Beelman et al. [11]. ERGO and ERGO-d9 stock solutions (1 mg/mL) were prepared in methanol. Working standards were prepared with a series of dilutions of the standard stock solution in methanol/water (50/50), resulting in concentrations from 10 to 10,000 ng/mL. ERGO-d9 working solution was similarly prepared using methanol/water (50/50). The ERGO standard curve was prepared by spiking an internal standard working solution into 50% methanol to make the standard concentrations from 1 to 1000 ng/mL The standard curves were constructed by plotting the ratio of the peak area of ERGO to that of the internal standard against the corresponding concentration.

The grain, root, and shoot samples were surface sterilized and ground into powder before processing. After weighing the powders (around 25 mg), 10 μL of the internal standard working solution was spiked, and 990 μL of 50% methanol was used to extract ERGO from the powder. The mixture was vortexed for 20 min under room temperature, then centrifuged at 4 °C, 8765 × g for 10 min. After transferring the supernatant to another vial, the pellet was re-extracted by 1 mL of 50% methanol. An equal volume of each supernatant was combined and vortexed before being loaded onto an HPLC–MS/MS system for analysis.

Ultra-performance liquid chromatography HPLC–MS/MS analysis

ERGO was analyzed using a Sciex 6500 + QTrap mass spectrometer coupled with an ExionLC HPLC separation system. A 1.7 μm ACQUITY UPLC BEH C18 analytical column (2.1 × 100 mm, Waters, Ireland) was used with the following mobile phase: mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of acetonitrile. The Sciex 6500 + QTrap mass spectrometer was equipped with an electrospray ionization probe in positive mode. The ion spray voltage was 5500 V, the temperature was 500 °C, the ion source gas 1 was 20 L/h, and the ion source gas 2 was 20 L/h. The multiple reaction monitoring mode was used to analyze and quantify ERGO and ERGO-d9, with transitions of m/z at 230 > 127 for ERGO and m/z at 239 > 127 for ERGO-d9. All peaks were integrated and quantified by Sciex OS 1.5.

ERGO recovery analysis from tissues

ERGO and AMF/ERGO treatments received 0.050 mg ERGO (50,000 ng), applied as 0.025 mg at day 0 and day 40. Grain, root, and shoot tissue weights were recorded at the time of harvest. ERGO concentrations (ng/mg) were quantified using UPLC–MS/MS as described above. Recovery (%) for each tissue was calculated as:

graphic file with name d33e519.gif

Here, ERGO represents the total amount applied per pot (0.050 mg = 50,000 ng).

Total recovery (%) was calculated as:

graphic file with name d33e527.gif

DNA extraction and sequencing

Genomic DNA from root, rhizosphere, and bulk soil samples were extracted by using DNeasy PowerSoil Pro kits (Qiagen, Hilden, Germany) following the manufacturer’s instructions. DNA quantity and quality were measured using an ND-2000 Nanodrop spectrometer (Thermo Fisher Scientific, Waltham, USA). Amplicon high throughput sequencing was conducted to characterize detailed bacteria/archaea and fungi communities. For bacteria and archaea, the V4 variable region of the 16S rRNA genes was amplified using 515f (5′-GTGYCAGCMGCCGCGGTAA-3′; [21] and 806r (5′-GGACTA CNVGGGTWTCTAAT-3′; [22] following the Earth Microbiome Project protocol [23]. For fungi, the ITS2 region was amplified using ITS3-F (5’-GCATCGATGAAGAACGCAGC-3′) and ITS4-R (5’-TCCTCCGCTTATTGATATGC-3′ [24]. 16S rRNA genes were amplified with following thermocycling program: 5 min at 94 °C for initialization; 30 cycles of 30 s denaturation at 94 °C, 30 s annealing at 53 °C, and 30 s extension at 72 °C; followed by 8 min final elongation at 72 °C. Fungal ITS regions were amplified with 3 min at 95 °C for initialization; 33 cycles of 20 s denaturation at 95 °C, 20 s annealing at 56 °C, and 30 s extension at 72 °C; followed by 5 min final elongation at 72 °C. Sequencing libraries were prepared by using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Massachusetts, USA) following manufacturer’s recommendations. High-throughput sequencing was performed at Magigene (Magigene Biotechnology, Guangzhou, China) on an Illumina Nova6000 platform (paired-end 250-bp mode). The sequence data is submitted to NCBI with the reference Sequence Read Archive (SRA) PRJNA1279644.

Statistical analysis of plant traits

For statistical analyses of plant traits, a two-way analysis of variance (ANOVA) test was run to compare each treatment group with the control group. Dunnett multiple comparisons test was conducted for hypothesis testing. To analyze ERGO accumulation in root, shoot, and grain, a two-way ANOVA test was used to compare treatments groups to control samples amongst plant tissue type. Dunnett multiple comparisons test was also conducted for hypothesis testing. All tests show significant results at p < α = 0.05.

Microbiome diversity and Community Analysis

Raw Illumina sequences were processed with the QIIME 2 software package (version 2021.11) [26]. After demultiplexing, all raw sequence reads were carried out with quality control, denoising, filtering, merging, and chimera removal through q2-DADA2. Amplicon sequence variants (ASVs) were generated, and a Naïve Bayes classifier artifact2 was applied to assign the ASVs to taxa at 99% using the Silva classifier 132 [27] for 16S rRNA genes and UNITE version 8.2 (February 20, 2020) for ITS regions. Contaminant ASVs were identified using prevalence in the three negative controls and removed from downstream sequencing analyses using the ‘decontam’ R package [25]. The ASVs were normalized by rarefaction approach performed with Qiime 2 pipeline, with cutoffs at 52,000 sequences for bacteria/ archaea and 6,400 for fungi. Both alpha and beta diversity metrics were analyzed to assess microbial diversity across various treatments and sample types (root, rhizosphere, and soil). Alpha diversity was evaluated using Shannon diversity for each treatment within the sample types. A one-way ANOVA was performed on Shannon diversity indices, and post hoc comparisons were performed using Tukey’s Honest Significance Difference (HSD) test [28, 29]. To identify significant pairwise differences plyloseq, ggplot2, dplyr, multcompView, tidyr, and viridis in R Studio was used per the cited literature [30]. Beta diversity was examined through Bray–Curtis dissimilarity matrices using the ordinate function in phyloseq [30] derived from ASV abundance data to investigate variations in microbiome composition. Principal Coordinates Analysis (PCoA) was conducted on the Bray–Curtis matrix to visualize the segregation of microbial communities based on treatment and sample type. The statistical significance of differences in community composition was tested using permutational multivariate analysis of variance (PERMANOVA) with 999 permutations, using the adonis2() function from the vegan package, focusing on the effects of treatments and sample types on microbiome structure. Additionally, PERMANOVA was employed to identify significant changes between individual treatment pairs and sample type [31].

Indicator species analysis

Indicator Species analysis was performed using the multipatt() function from indicator species package in R with 16s and ITS ASVs to identify microbial taxa significantly abundant at different sample types based on the applied treatments [32]. Only species with p < 0.05 were retained. Relative abundance was calculated by normalizing ASV count per sample and average treatment groups. Bubble plots and summary tables are generated for 16s and ITS data [33]. Each bubble plot represented a significant species-treatment association, where the size of the bubble plot reflected the mean relative abundance, and the fill color denoted the significance (p-values). Indicator values were displayed to each species for added interpretability. In addition, a summary table of indicator species analysis for both 16S and ITS were generated with its treatment association, relative abundance and p-value.

Results

Treatment of ERGO and AMF increased plant biomass in Triticum aestivum

Wheat plants were harvested on day 60 and were analyzed for biomass for all treatment groups including controls (See Fig. 1). Inoculation with ERGO, AMF and the combination of ERGO and AMF showed increased biomass over the untreated plants (Fig. 1 and Supplementary Online Fig. 1). Both ERGO and AMF may play a critical role in enhancing biomass in wheat plants.

Fig. 1.

Fig. 1

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Biomass in spring wheat plants inoculated with ergothioneine (ERGO), mycorrhizal fungi (AM)F, and both AMF/ERGO). The overall biomass was calculated on day 60 post-harvest. Control samples are not treated with either mycorrhizal species or ergothioneine. Asterisks indicate statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) between treatments

Wheat treated with ERGO or AMF/ERGO showed a temporal pattern of ERGO accumulation

The ERGO levels in T aestivum root, shoot, and grain were shown in Fig. 2. There was a significant increase in the ERGO accumulation in roots and shoots treated with exogenous ERGO or with AMF/ERGO (See Fig. 2). However, in the grains, no significant differences in ERGO accumulation were observed. In T. aestivum plants treated with ERGO, the highest ERGO content was observed in the roots. Interestingly, the amount of ERGO detected in the grains of ERGO-treated plants was lower than that observed in the control group. In plants treated with AMF, ERGO levels were lower than those in the control group across roots, shoots, and grains. It’s interesting to note that roots, shoots, and grains contained different overall percentages of ERGO recovery under ERGO and AMF/ERGO treatments (Supplementary Online Table 1). Compared to lone ERGO treatments, a systematic temporal ERGO uptake from roots to shoots to grains was seen when plants were treated with AMF/ERGO in soil (Supplementary Online Table 1).

Fig. 2.

Fig. 2

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Ergothioneine content in wheat plants inoculated with ergothioneine (ERGO), mycorrhizal fungi (AMF), and both AMF/ERGO). Plants were harvested on day 60 and ergothioneine content was analyzed in roots, shoots and grains. Asterisks indicate statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) between treatments

Impact of ERGO and co-inoculation of ERGO and AMF on soil microbiome assembly

The Shannon diversity plots for the bacteria/archaea and fungi showed differences across the treatments and from different sampling types (Fig. 3, 4). With a p-value < 0.05, annotations of significance above each boxplot showed the differences in microbial diversity within the root, rhizosphere, and bulk soil (See Fig. 3, 4) across different treatments. The bacterial diversity was higher in the rhizosphere and bulk soil across the treatments compared to the root (Fig. 3). The combination treatment of AMF/ERGO showed higher bacterial diversity compared to other treatments in rhizosphere (Fig. 3). All sample types showed the highest diversity with treatment AMF/ERGO, indicating a synergistic effect in boosting microbial communities (Fig. 3). In contrast, the lowest diversity indices were obtained in untreated plants (Fig. 3), demonstrating that both ERGO supplementation and AMF treatment may modulate the rhizosphere microbiome assembly in the wheat plants. Contrary to the bacterial diversity indices, there are no significant differences between fungal diversity across sample types (roots, rhizosphere, and bulk soil) or treatments (Fig. 4).

Fig. 3.

Fig. 3

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Boxplot illustrates the Shannon diversity index of bacteria (16S ASVs) across different treatments and sample types (root, rhizosphere, and bulk soil). The data reveals variations in bacterial diversity levels based on treatment conditions, with distinct distributions evident for each sample type with a p-value < 0.05. The letters above denotes the statistical significance among treatment groups within each sample type, based on post-hoc Tukey HSD tests. Control samples are not treated with either mycorrhizal species or ergothioneine. The other treatment depicts: AMF/ERGO’ treatment represents wheat plants inoculated with both mycorrhizal species and ergothioneine. ‘ERGO’ treatment represents wheat plants inoculated with ergothioneine. ‘AMF’ treatment in represents wheat plants inoculated with mycorrhizal species

Fig. 4.

Fig. 4

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Boxplot illustrating the Shannon diversity index of bacteria (ITS ASVs) across different treatments and sample types (root, rhizosphere, and bulk soil). The data reveals variations in bacterial diversity levels based on treatment conditions, with distinct distributions evident for each sample type with a p-value < 0.05. The letters above denotes the statistical significance among treatment groups within each sample type, based on post-hoc Tukey HSD tests. Control samples are not treated with either mycorrhizal species or ergothioneine. The other treatment depicts: AMF/ERGO’ treatment represents wheat plants inoculated with both mycorrhizal species and ergothioneine. ‘ERGO’ treatment represents wheat plants inoculated with ergothioneine. ‘AMF’ treatment in represents wheat plants inoculated with mycorrhizal species

The impact of ERGO, AMF or both on assembly of bacterial and fungal communities (β-diversity) across different sampling types (root, rhizosphere, and bulk soil) were shown in Figs. 5 and 6. PERMANOVA analysis indicated that microbiomes were significantly influenced by AMF and AMF/ERGO co-inoculation (p = 0.001 for 16S and p = 0.002 for ITS) (Fig. 5). Treatment accounted for approximately 21.4% of the observed differences, while sample type (root, rhizosphere, and soil) explained 18.1% of bacterial data. Whereas in fungal data, around 7.3% of the observed differences were due to treatment and 20% difference in abundance levels was related sample types.

Fig. 5.

Fig. 5

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Bray–Curtis dissimilarity index and PCoA (Principal Coordinates Analysis) analysis of the 16S ASV data shows the relationships between the bacterial communities from several sample types (bulk soil, rhizosphere, and root) across different treatments with a p-value of 0.001. Every point on the plot represents a sample; various colors denote specific treatments, and various shapes indicate different sorts of samples. The spots’ spatial organization shows clustering patterns, indicating that sample type and treatment impact the composition of the microbial community. Control samples are not treated with either mycorrhizal species or ergothioneine. The other treatment depicts: AMF/ERGO’ treatment represents wheat plants inoculated with both mycorrhizal species and ergothioneine. ‘ERGO’ treatment represents wheat plants inoculated with ergothioneine. ‘AMF’ treatment in represents wheat plants inoculated with mycorrhizal species

Fig. 6.

Fig. 6

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Bray–Curtis dissimilarity index, the PCoA (Principal Coordinates Analysis) analysis of the ITS ASV data shows the relationships between the fungal communities from several sample types (bulk soil, rhizosphere, and root) across different treatments with a p-value of 0.002. Every point on the plot represents a sample; various colors denote treatments, and various shapes indicate different sorts of samples. The spots’ spatial organization shows clustering patterns, indicating that sample type and treatment impact the composition of the microbial community. Control samples are not treated with either mycorrhizal species or ergothioneine. The other treatment depicts: AMF/ERGO’ treatment represents wheat plants inoculated with both mycorrhizal species and ergothioneine. ‘ERGO’ treatment represents wheat plants inoculated with ergothioneine. ‘AMF’ treatment in represents wheat plants inoculated with mycorrhizal species

Significant changes in the composition of the bacterial communities were observed when ERGO and Compared to bulk soil, the clustering in the rhizosphere for ERGO and AMF was more significant compared to other treatments and sampling types (Fig. 5). In contrast, the PCoA analysis for fungal communities across the sampling zones and treatments showed a specific effect of the treatments and the location of microbiome sampling, suggesting that both sampling location and treatment does influence fungal community composition (Fig. 6). Although PERMANOVA detected a significant treatment effect (p = 0.001), the low effect size (R2 = 0.074) and overlapping clustering patterns indicated limited separation in the distribution of fungal communities across ERGO and AMF treatments (Fig. 6).

AMF and ERGO amendments on indicator species

The 16S indicator species plot highlighted bacterial species whose abundance was significantly influenced by treatment and sampling type, illustrating distinct microbiome shifts in the incubation (Supplementary Online Figs. 3 and 4; and Supplementary Online Tables 2 and 3). The indicator species analysis highlighted taxa with the highest indicator values, representing species strongly associated with specific treatments and sample types. For bacteria and archaea (16S), Planctomycete A-2 exhibited notably high indicator values and were relatively abundant under the combination treatment across all sample types, indicating a strong and consistent association (See Supplementary Online Figs. 3, 4; and Supplementary Online Tables 2, 3). Similarly, Thioalkalivibrio demonstrated high indicator strength in the ERGO treatment, as well as its relative abundance is moderately high across sample types. On the contrary, Holophaga sp. was shown moderately in relative abundance across all treatments, but its indicator value was moderately significant. Further, Chlorobi bacteria were exclusively detected in the combination treatment AMF/ERGO across root, rhizosphere, and bulk soil, marked by strong indicator values and consistently moderate relative abundance, underscoring their specificity to this treatment. Flavobacterium caeni displayed a moderate relative abundance and slightly high in indicator value under the AMF treatment in root samples as well as in combination-treated rhizosphere and soil samples. Chitinophaga pinensis was spotted only in ERGO treatment for rhizosphere and bulk soil samples with high indicator value and moderately high relative abundance. In addition, Acidobacteria bacterium showed moderate relative abundance across all treatments and all sample types. In summary, the rhizosphere and bulk soil exhibited higher species diversity and number of significant indicator taxa across all treatments, reflecting strong microbial community differentiation across plant-associated niches.

The ITS-based indicator species analysis identified a limited number of fungal taxa with strong treatment-specific associations. Metarhizium anisopliae exhibited notable specificity to the AMF treatment, demonstrated by high indicator values despite its low relative abundance, suggesting a strong but selective response. In contrast, Trichomonascus valeeneenianus was relatively abundant and showed moderate indicator values across both control and ERGO treatments in rhizosphere and bulk soil samples, indicating a consistent but weaker association with these conditions. The GS05 fungal group was more abundant under AMF treatment, though its association strength varied across different sample types. Notably, Candida subhashii was detected at relatively high abundance exclusively in the combined AMF/ERGO treatment within bulk soil samples, accompanied by moderately high indicator values, suggesting a strong and niche-specific association. Interestingly, Paecilomyces zollerniae was found in both root and rhizosphere samples under the ERGO treatment, as well as in control bulk soil. However, its relative abundance was higher in ERGO-treated root and rhizosphere samples compared to the control bulk soil, pointing to a niche-dependent indicator response (See Supplementary Online Figs. 2, 3 and Supplementary Online Tables 2, 3).

Discussion

In nature, minimal ERGO levels found in some plants suggest that AMF may act as both a reservoir of ERGO biosynthesis and a driver to facilitate ERGO uptake in the plants. Recent studies have shown that mycorrhizae, particularly AMFs, play a role in increasing ERGO content in grain, but it is currently unknown if that is due to uptake from the soil or biosynthesis by AMF [9]. These experiments, conducted under natural soil conditions, showed that the association of AMF with multiple crops led to the accumulation of ERGO in the grain [9]. It is argued that while mammalian systems do not synthesize ERGO, they can absorb it through known transporters [34]. Recent research has demonstrated that ERGO can be absorbed by invertebrate model systems, like Caenorhabditis elegans, which increases the worms’ lifespan and general health [35]. Although the exact mechanism by which plants absorb ergothioneine (ERGO) remains unknown, our findings suggest that AMF may play a role in facilitating its uptake. The presence and abundance of AMF in the soil imply that plant–fungal associations could be involved in mediating ERGO transport into plant tissues. However, introducing ERGO into soils containing AMF under natural conditions may influence the complexity of the native soil microbiome, potentially altering the diversity and dynamics of existing root–microbe interactions within the rhizosphere [36]. Since plants actively shape their associated soil microbial communities, any changes to the microbial structure can significantly affect plant development, phenotype, and overall health [37].

Our results showed that wheat plants supplemented with ERGO exhibited improved growth traits compared with untreated control, suggesting a beneficial role for ERGO in plant growth. This growth-promoting effect is consistent with observations in both invertebrate and mammalian model systems, where ERGO has been reported to enhance growth, extend lifespan, and provide additional health benefits [38]. The positive effects observed following direct ERGO supplementation also indicate that plants can absorb ERGO independently of AMF colonization, implying the presence of a receptor or transporter involved in ERGO uptake and translocation from roots to other plant tissues. Indeed, direct ERGO application to the wheat rhizosphere resulted in the temporal accumulation of ERGO in roots, shoots, and grains. ERGO concentrations in roots were higher in the ERGO and AMF/ERGO treatments than in the AMF-only treatment, suggesting that AMF associations may facilitate ERGO uptake, as reported previously [9]. At the same time, the uptake of ERGO following direct application of pure ERGO to the roots demonstrates that plants are capable of absorbing ERGO both in the presence and absence of AMFs, potentially through distinct uptake pathways. In contrast, ERGO accumulation in grains was greatest in the AMF/ERGO treatment compared with the individual AMF or ERGO treatments, suggesting that ERGO transport and grain fortification may involve mechanisms distinct from those governing uptake and accumulation in roots and shoots.

Amino acid composition in the rhizosphere is influenced by both the plant’s ability to secrete amino acids and microbial transformation of these compounds in the soil [39]. ERGO is an unusual amino acid known to be biosynthesized by certain fungal species and a few actinobacteria [40]. ERGO is a dietary thione/thiol and it improves lifespan and muscle density in both invertebrate and mammalian model systems [35], effects believed to be mediated, at least in part, through modulation of the host microbiome [41]. However, the role of ERGO, either synthesized by soil fungi/bacteria or by direct supplementation into the soil, has not been investigated. Microbial diversity in the rhizosphere plays a critical role in plant growth and health [36]. In our study, we observed that the diversity and structure of the wheat rhizosphere bacterial microbiome were more strongly affected by ERGO and AMF treatments than those associated with root tissues, suggesting that ERGO may play a role in modulating microbial communities in the rhizosphere (Fig. 3). Notably, both bacterial and fungal populations were enriched in the presence of combined ERGO and AMF treatments, pointing toward synergistic effects on the soil microbiome.

The microbial responses observed under ERGO treatment reveal diverse ecological and metabolic adaptations across bacterial and fungal taxa.​ Notabely, Thioalkalivibrio species exhibited a high indicator value in response to ERGO, which aligns with earlier findings [42], wherein the function of EgtC was characterized in the involment of ERGO biosynthesis. Although Thioalkalivibrio is a Gram-negative bacterial genus not typically associated with canonical ERGO biosynthetic pathways, the presence of egtC-like homologs in its genome suggests a potential role in ERGO-related metabolism. This indicates that certain non-canonical bacterial taxa may engage in ERGO transformation or utilization through alternative metabolic routes [42].​ Results from this study showed moderate abundance of Holophaga (a member of the phylum Acidobacteria) under all treatments. A previous study indicated that Holophaga foetida utilizes methoxylated aromatic compounds in the lignin of plant cell walls, through transmethylation processes, to produce volatile sulfur compounds [43]. This metabolic specialization may confer an advantage in ERGO-enriched environments, possibly due to structural similarities between ERGO or its derivatives and the substrates preferred by Holophaga.​ Furthermore, a Chlorobi-affiliated bacterium was predominantly present in the root, rhizosphere, and bulk soil compartments under combination treatments (AMF/ERGO) [44]. The study identified an oxygen-independent pathway for ERGO biosynthesis in Chlorobium limicola, mediated by the enzyme EanB [44]. This alternative pathway allows for ERGO production under anaerobic conditions, which are characteristic of the root microenvironment, suggesting that ERGO biosynthesis may occur even in low-oxygen niches within the rhizosphere. The high indicator value of this Chlorobi bacterium suggests a strong association with ERGO-enriched conditions, potentially reflecting its metabolic adaptation to utilize or produce ERGO in anoxic niches.

Paecilomyces zollerniae emerged as an indicator species under ERGO treatment in Indicator species analysis among fungal communities. While not directly studied for ERGO biosynthesis, other species within the Paecilomyces genus, such as Paecilomyces tenuipes, are known producers of ERGO in fruiting bodies [45]. ERGO levels in Paecilomyces tenuipes increase significantly when cultures are supplemented with methionine, a known precursor in ERGO biosynthesis. Although P. zollerniae and P. tenuipes differ taxonomically, they share potential for similar metabolic capabilities. Enriched P. zollerniae under ERGO treatment may reflect either a tolerance to or utilization of ERGO, highlighting possible unexplored biosynthetic potential within this genus. Collectively, these observations underscore the multifaceted roles of ERGO in soil microbial ecology, influencing community composition and metabolic functions across diverse taxa (Supplementary Online Figs. 2, 3; Supplementary Online Tables 2, 3). These findings demonstrate that ERGO, both independently and in combination with AMF, significantly influenced microbiome composition and diversity.

Conclusion

This study demonstrates that both ERGO and AMF, individually and in combination, enhance wheat growth and significantly influence soil microbial communities. ERGO uptake occurred across plant tissues, with its dynamics modulated by the presence of AMF, suggesting multiple transport pathways. Co-application of ERGO and AMF not only boosted biomass accumulation but also increased bacterial diversity and reshaped microbial community structures, particularly through synergistic effects. Indicator species and microbial network analyses revealed treatment-specific associations, indicating that AMF may facilitate beneficial microbial interactions while modulating EGT-utilizing taxa. Moreover, the findings suggest that plants possess an active mechanism for ERGO transport and mobilization. Given ERGO’s well-documented antioxidant properties in mammalian systems, it is plausible that ERGO contributes to enhanced antioxidant defenses in plants, thereby improving resilience against biotic and abiotic stressors. Overall, these findings highlight the potential of ERGO and AMF as complementary treatments to improve crop productivity and support soil microbiome function, offering a promising strategy for sustainable agriculture.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (476.3KB, pptx)
Supplementary Material 2 (14.8KB, docx)
Supplementary Material 3 (8.9KB, csv)
Supplementary Material 4 (6.8KB, csv)

Acknowledgements

The work was conducted with the grant received by authors from USDA FFAR (CA20-SS-0000000152).

Author contribution

SNR, AP, JK, and HPB wrote the initial draft of the manuscript. The work performed udder Figs. 1, 2, 3, 4, 5 and 6 and Supplementary Fig. 1, 2, 3 and Table 2, 3 were conducted by SNR, AP and NI. The data in Table 1 was conducted by CK. The Ergothioneine estimation shown in Fig. 2 was performed by JR. JK and SNR performed all the analysis of the microbiome data. WH, JK, AS, GZ and HPB worked on the edits of the manuscript for its submission. WH, JK, AS, GZ and HPB secured funding and managed the project to its completion.

Funding

Funding was provided by the U.S. Department of Agriculture.

Data availability

All the analyzed datasets including 16S, ITS ASVs, metadata and R scripts regarding this research are included in this article as its supplementary information and are deposited in a publicly available in the following github repository https://github.com/HPBaisLab/AMF_ERGO_MICROBIOME. The sequencing data for both 16S and ITS is submitted to NCBI with the reference Sequence Read Archive (SRA) PRJNA1279644.

Declarations

Competing interests

The authors declare that they have no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Sri Sai Nandini Ravi and Alexandra Pipinos have equally contributed to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (476.3KB, pptx)
Supplementary Material 2 (14.8KB, docx)
Supplementary Material 3 (8.9KB, csv)
Supplementary Material 4 (6.8KB, csv)

Data Availability Statement

All the analyzed datasets including 16S, ITS ASVs, metadata and R scripts regarding this research are included in this article as its supplementary information and are deposited in a publicly available in the following github repository https://github.com/HPBaisLab/AMF_ERGO_MICROBIOME. The sequencing data for both 16S and ITS is submitted to NCBI with the reference Sequence Read Archive (SRA) PRJNA1279644.

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