Structural analyzes suggest that MiSSP13 and MiSSP16.5 may act as proteases inhibitors during ectomycorrhiza establishment in Laccaria bicolor.

•The signaling process during mycorrhiza establishment involves intense molecular communication between symbionts. It has been suggested that a group of protein effectors, the so-called MiSSPs, plays a broader function in the symbiosis metabolism, however, many of these remain uncharacterized structurally and functionally. •Herein we used three-dimensional protein structure modeling methods, ligand analysis, and molecular docking to structurally characterize and describe two protein effectors, MiSSP13 and MiSSP16.5, with enhanced expression during the mycorrhizal process in Laccaria bicolor. •MiSSP13 and MiSSP16.5 show structural homology with the cysteine and aspartate protease inhibitor, cocaprin (CCP1). Through structural analysis, it was observed that MiSSP13 and MiSSP16.5 have an active site similar to that observed in CCP1. The protein-protein docking data showed that MiSSP13 and MiSSP16.5 interact with the papain and pepsin proteases at sites that are near to where CCP1 interacts with these same targets, suggesting a function as inhibitor of cysteine and aspartate proteases. The interaction of MiSSP13 with papain and MiSSP16.5 with pepsin was stronger than the interaction of CCP1 with these proteases, suggesting that the MiSSPs had a greater activity in inhibiting these classes of proteases. Based on the data supplied, a model is proposed for the function of MiSSPs 13 and 16.5 during the symbiosis establishment. Our findings, while derived from in silico analyses, enable us formulate intriguing hypothesis on the function of MiSSPs in ectomycorrhization, which will require experimental validation.

Assembly collapsing versus heterozygosity oversizing: detection of homokaryotic and heterokaryotic Laccaria trichodermophora strains by hybrid genome assembly.

Genome assembly and annotation using short-paired reads is challenging for eukaryotic organisms due to their large size, variable ploidy and large number of repetitive elements. However, the use of single-molecule long reads improves assembly quality (completeness and contiguity), but haplotype duplications still pose assembly challenges. To address the effect of read length on genome assembly quality, gene prediction and annotation, we compared genome assemblers and sequencing technologies with four strains of the ectomycorrhizal fungus Laccaria trichodermophora. By analysing the predicted repertoire of carbohydrate enzymes, we investigated the effects of assembly quality on functional inferences. Libraries were generated using three different sequencing platforms (Illumina Next-Seq, Mi-Seq and PacBio Sequel), and genomes were assembled using single and hybrid assemblies/libraries. Long reads or hybrid assemby resolved the collapsing of repeated regions, but the nuclear heterozygous versions remained unresolved. In dikaryotic fungi, each cell includes two nuclei and each nucleus has differences not only in allelic gene version but also in gene composition and synteny. These heterokaryotic cells produce fragmentation and size overestimation of the genome assembly of each nucleus. Hybrid assembly revealed a wider functional diversity of genomes. Here, several predicted oxidizing activities on glycosyl residues of oligosaccharides and several chitooligosaccharide acetylase activities would have passed unnoticed in short-read assemblies. Also, the size and fragmentation of the genome assembly, in combination with heterozygosity analysis, allowed us to distinguish homokaryotic and heterokaryotic strains isolated from L. trichodermophora fruit bodies.

Populus MYC2 orchestrates root transcriptional reprogramming of defence pathway to impair Laccaria bicolor ectomycorrhizal development.

The jasmonic acid (JA) signalling pathway plays an important role in the establishment of the ectomycorrhizal symbiosis. The Laccaria bicolor effector MiSSP7 stabilizes JA corepressor JAZ6, thereby inhibiting the activity of Populus MYC2 transcription factors. Although the role of MYC2 in orchestrating plant defences against pathogens is well established, its exact contribution to ECM symbiosis remains unclear. This information is crucial for understanding the balance between plant immunity and symbiotic relationships. Transgenic poplars overexpressing or silencing for the two paralogues of MYC2 transcription factor (MYC2s) were produced, and their ability to establish ectomycorrhiza was assessed. Transcriptomics and DNA affinity purification sequencing were performed. MYC2s overexpression led to a decrease in fungal colonization, whereas its silencing increased it. The enrichment of terpene synthase genes in the MYC2-regulated gene set suggests a complex interplay between the host monoterpenes and fungal growth. Several root monoterpenes have been identified as inhibitors of fungal growth and ECM symbiosis. Our results highlight the significance of poplar MYC2s and terpenes in mutualistic symbiosis by controlling root fungal colonization. We identified poplar genes which direct or indirect control by MYC2 is required for ECM establishment. These findings deepen our understanding of the molecular mechanisms underlying ECM symbiosis.

Three New Species of Laccaria (Hydnangiaceae) from India (Darjeeling Hills) Based on Molecular and Morphological Evidence.

Three new species of Laccaria infundibuliformis, L. pallidus, and L. darjeelingensis, collected from Darjeeling, India, are described based on morphological and molecular evidence. Laccaria infundibuliformis is characterized by its small infundibuliform basidiocarps, and echinulate basidiospores with spines up to 1.36 µm long. Laccaria pallidus is characterized by medium-sized greyish-red basidiocarps, and echinulate basidiospores with spines up to 1.9 µm long. Laccaria darjeelingensis is characterized by dull red basidiocarps, and echinulate basidiospores with spines up to 1.27 µm long. Altogether, the study shows that these three Laccaria species are previously unknown to science.

Laccaria murina and L. pumila from Pakistan, a study based on SEM and nrITS molecular marker.

Two species of the genus Laccaria, are described as new reports for Pakistan. Laccaria murina has been collected from a Himalayan moist temperate forest in Khanspur, KP, while L. pumila was found in the moist temperate forests of Kumrat Valley in Dir Upper, KP, and at higher altitudes of the Deosai plains of Gilgit-Baltistan, Pakistan. Phylogenetic analysis based on the nrITS region clustered the Pakistani species with L. murina sequences with other Asian collections and L. pumila from USA and Netherlands. SEM of basidiospores along with detailed micro-morphological data are provided. General distribution, habitat, ecology, and diagnostic features are also discussed. RESEARCH HIGHLIGHTS: It has new reports from Pakistan, studies based on light, scanning electron microscopy, and nrITS molecular markers. These species have been described with detailed micro-morphological and molecular phylogenetic analyses. General distribution, ecology, diagnostic features, and comparisons with closely related specimens have been provided. Graphical representation of DNA extraction and geographical locations of sampling sites are also illustrated (Figures 1 and 2). Very few members of this genus are already described from Pakistan.

Ectomycorrhizal fungi, two species of Laccaria, differentially block the migration and accumulation of cadmium and copper in Pinus densiflora.

The root tips of host plant species can establish ectomycorrhizae with their fungal partners, thereby altering the responses of the host plants to heavy metal (HM) toxicity. Here, two species of Laccaria, L. bicolor and L. japonica, were investigated in symbiosis with Pinus densiflora to study their potential for promotion of phytoremediation of HM-contaminated soils in pot experiments. The results showed that L. japonica had significantly higher dry biomass than L. bicolor in mycelia grown on modified Melin-Norkrans medium containing elevated levels of cadmium (Cd) or copper (Cu). Meanwhile, the accumulations of Cd or Cu in L. bicolor mycelia were much higher than that in L. japonica at the same level of Cd or Cu. Therefore, L. japonica displayed a stronger tolerance to HM toxicity than L. bicolor in situ. Compared with non-mycorrhizal P. densiflora seedlings, inoculation with two Laccaria species significantly increased the growth of P. densiflora seedlings in absence or presence of HM. The mantle of host roots blocked the uptake and migration of HM, which led to the decrease of Cd and Cu accumulation in the P. densiflora shoots and roots, except for the root Cd accumulation of L. bicolor-mycorrhizal plants when 25 mg kg-1 Cd exposure. Furthermore, HM distribution in mycelia showed Cd and Cu are mainly retained in the cell walls of mycelia. These results provide strong evidence that the two species of Laccaria in this system may have different strategies to assist host tree against HM toxicity.

DNA hypomethylation of the host tree impairs interaction with mutualistic ectomycorrhizal fungus.

Ectomycorrhizas are an intrinsic component of tree nutrition and responses to environmental variations. How epigenetic mechanisms might regulate these mutualistic interactions is unknown. By manipulating the level of expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1) and two demethylases DEMETER-LIKE (DML) in Populus tremula × Populus alba lines, we examined how host DNA methylation modulates multiple parameters of the responses to root colonization with the mutualistic fungus Laccaria bicolor. We compared the ectomycorrhizas formed between transgenic and wild-type (WT) trees and analyzed their methylomes and transcriptomes. The poplar lines displaying lower mycorrhiza formation rate corresponded to hypomethylated overexpressing DML or RNAi-ddm1 lines. We found 86 genes and 288 transposable elements (TEs) differentially methylated between WT and hypomethylated lines (common to both OX-dml and RNAi-ddm1) and 120 genes/1441 TEs in the fungal genome suggesting a host-induced remodeling of the fungal methylome. Hypomethylated poplar lines displayed 205 differentially expressed genes (cis and trans effects) in common with 17 being differentially methylated (cis). Our findings suggest a central role of host and fungal DNA methylation in the ability to form ectomycorrhizas including not only poplar genes involved in root initiation, ethylene and jasmonate-mediated pathways, and immune response but also terpenoid metabolism.

Laccaria bicolor pectin methylesterases are involved in ectomycorrhiza development with Populus tremula × Populus tremuloides.

The development of ectomycorrhizal (ECM) symbioses between soil fungi and tree roots requires modification of root cell walls. The pectin-mediated adhesion between adjacent root cells loosens to accommodate fungal hyphae in the Hartig net, facilitating nutrient exchange between partners. We investigated the role of fungal pectin modifying enzymes in Laccaria bicolor for ECM formation with Populus tremula × Populus tremuloides. We combine transcriptomics of cell-wall-related enzymes in both partners during ECM formation, immunolocalisation of pectin (Homogalacturonan, HG) epitopes in different methylesterification states, pectin methylesterase (PME) activity assays and functional analyses of transgenic L. bicolor to uncover pectin modification mechanisms and the requirement of fungal pectin methylesterases (LbPMEs) for ECM formation. Immunolocalisation identified remodelling of pectin towards de-esterified HG during ECM formation, which was accompanied by increased LbPME1 expression and PME activity. Overexpression or RNAi of the ECM-induced LbPME1 in transgenic L. bicolor lines led to reduced ECM formation. Hartig Nets formed with LbPME1 RNAi lines were shallower, whereas those formed with LbPME1 overexpressors were deeper. This suggests that LbPME1 plays a role in ECM formation potentially through HG de-esterification, which initiates loosening of adjacent root cells to facilitate Hartig net formation.

Mycorrhiza-induced mycocypins of Laccaria bicolor are potent protease inhibitors with nematotoxic and collembola antifeedant activity.

Fungivory of mycorrhizal hyphae has a significant impact on fungal fitness and, by extension, on nutrient transfer between fungi and host plants in natural ecosystems. Mycorrhizal fungi have therefore evolved an arsenal of chemical compounds that are hypothesized to protect the hyphal tissues from being eaten, such as the protease inhibitors mycocypins. The genome of the ectomycorrhizal fungus Laccaria bicolor has an unusually high number of mycocypin-encoding genes. We have characterized the evolution of this class of proteins, identified those induced by symbiosis with a host plant and characterized the biochemical properties of two upregulated L. bicolor mycocypins. More than half of L. bicolor mycocypin-encoding genes are differentially expressed during symbiosis or fruiting body formation. We show that two L. bicolor mycocypins that are strongly induced during symbiosis are cysteine protease inhibitors and exhibit similar but distinct localization in fungal tissues at different developmental stages and during interaction with a host plant. Moreover, we show that these L. bicolor mycocypins have toxic and feeding deterrent effect on nematodes and collembolans, respectively. Therefore, L. bicolor mycocypins may be part of a mechanism by which this species deters grazing by different members of the soil food web.

Estimation of the most suitable nitrogen concentration for sporocarp formation in Laccaria japonica colonizing Pinus densiflora seedlings through in vitro mycelial culture.

Many ectomycorrhizal (ECM) fungi produce commercially valuable edible sporocarps. However, the effects of nitrogen (N) application on ECM fungal sporocarp formation remain poorly understood. In this study, we investigated the effect of application of various N concentrations (0, 5, 25, 50, 100, and 200 mg/L) on the growth of Laccaria japonica mycelia in vitro for 1 month. The results showed that L. japonica mycelial biomass was highest in the 50 mg/L treatment and was significantly inhibited at N concentrations higher than 200 mg/L. Next, we investigated the effects of N application on mycorrhizal colonization and sporocarp formation in L. japonica colonizing Pinus densiflora seedlings in pots. The seedlings were watered with nutrient solutions containing 0, 5, 25, 50, or 100 mg N/L. The biomass, photosynthetic rate, and mycorrhizal colonization rates of the seedlings were measured at 45 days (first appearance of primordia), 65 days (sporocarp appearance on the substrate surface), and 4 months after seedlings were transplanted. The numbers of primordia and sporocarps were recorded during the experimental period. Total carbon (C) and N content were determined in seedlings at 4 months after transplantation, and in L. japonica sporocarps. Both mycelial growth and sporocarp production reached their maximum at an N application concentration of 50 mg/L, suggesting that the most suitable N concentration for ECM fungal sporocarp formation can easily be estimated in vitro during mycelial growth. This finding may help determine the most suitable N conditions for increasing edible ECM fungus sporocarp production in natural forests.

Distribution and regulatory roles of oxidized 5-methylcytosines in DNA and RNA of the basidiomycete fungi Laccaria bicolor and Coprinopsis cinerea.

The formation of three oxidative DNA 5-methylcytosine (5mC) modifications (oxi-mCs)-5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)-by the TET/JBP family of dioxygenases prompted intensive studies of their functional roles in mammalian cells. However, the functional interplay of these less abundant modified nucleotides in other eukaryotic lineages remains poorly understood. We carried out a systematic study of the content and distribution of oxi-mCs in the DNA and RNA of the basidiomycetes Laccaria bicolor and Coprinopsis cinerea, which are established models to study DNA methylation and developmental and symbiotic processes. Quantitative liquid chromatography-tandem mass spectrometry revealed persistent but uneven occurrences of 5hmC, 5fC and 5caC in the DNA and RNA of the two organisms, which could be upregulated by vitamin C. 5caC in RNA (5carC) was predominantly found in non-ribosomal RNA, which potentially includes non-coding, messenger and small RNA species. Genome-wide mapping of 5hmC and 5fC using the single CG analysis techniques hmTOP-seq and foTOP-seq pointed at involvement of oxi-mCs in the regulation of gene expression and silencing of transposable elements. The implicated diverse roles of 5mC and oxi-mCs in the two fungi highlight the epigenetic importance of the latter modifications, which are often neglected in standard whole-genome bisulfite analyses.

Identification and characterization of eight metallothionein genes involved in heavy metal tolerance from the ectomycorrhizal fungus Laccaria bicolor.

Metallothioneins (MTs) are small, cysteine-rich, heavy metal-binding proteins involved in metal homeostasis and detoxification. The increasing numbers of available genomic sequences of ectomycorrhizal (ECM) fungi enable deeper insights into the characteristics of MT genes in these fungi that form the most important symbiosis with the host trees in forest ecosystems. The aim of this study was to establish a comprehensive, genome-wide inventory of MT genes from the ECM fungus Laccaria bicolor. Eight MT genes in L. bicolor were cloned, and the expression patterns of their transcripts at various developmental stages based on expressed sequence tag (EST) counts were analyzed. The expression levels of four MTs were significantly increased during symbiosis stages. Quantitative real-time PCR (qRT-PCR) analysis revealed that transcripts of LbMT1 were dominant in free-living mycelia and strongly induced by excessive copper (Cu), cadmium (Cd), and hydrogen peroxide (H2O2). To determine whether these eight MTs functioned as metal chelators, we expressed them in the Cu- and Cd-sensitive yeast mutants, cup1∆ and yap1∆, respectively. All LbMT proteins provided similar levels of Cu(II) or Cd(II) tolerance, but did not affect by H2O2. Our findings provide novel data on the evolution and diversification of fungal MT gene duplicates, a valuable resource for understanding the vast array of biological processes in which these proteins are involved.

Study on the molecular mechanism of Laccaria bicolor helping Populus trichocarpa to resist the infection of Botryosphaeria dothidea.

AIMS: This study explored the specific molecular mechanism of Laccaria bicolor to help Populus trichocarpa resist infection by Botryosphaeria dothidea. METHODS AND RESULTS: Transcriptome technology was used to sequence P. trichocarpa under disease stress, and a total of 6379 differentially expressed genes (DEGs) were identified. A total of 536 new DEGs were induced by L. bicolor during the infection of B. dothidea. L. bicolor helps to prevent and alleviate the infection of B. dothidea by regulating related genes in the cell wall pathway, signal transduction pathway, disease-resistant protein synthesis pathway and antioxidant enzyme synthesis pathway of P. trichocarpa. CONCLUSION: The inoculation of L. bicolor can regulate the expression of genes in the cell wall pathway and enhance the physical defense capabilities of plants. Under disease stress conditions, L. bicolor can regulate signal transduction pathways, disease-resistant related pathways and reactive oxygen species (ROS) clearance pathways to help P. trichocarpa alleviate the disease. SIGNIFICANCE AND IMPACT OF THE STUDY: The research reveals the mechanism of L. bicolor inducing resistance to canker of P. trichocarpa from the molecular level and provides a theoretical basis for the practical application of mycorrhizal fungi to improve plant disease resistance.

The ectomycorrhizal basidiomycete Laccaria bicolor releases a GH28 polygalacturonase that plays a key role in symbiosis establishment.

In ectomycorrhiza, root penetration and colonization of the intercellular space by symbiotic hyphae is thought to rely on the mechanical force that results from hyphal tip growth, enhanced by the activity of secreted cell-wall-degrading enzymes. Here, we characterize the biochemical properties of the symbiosis-induced polygalacturonase LbGH28A from the ectomycorrhizal fungus Laccaria bicolor. The transcriptional regulation of LbGH28A was measured by quantitative PCR (qPCR). The biological relevance of LbGH28A was confirmed by generating RNA interference (RNAi)-silenced LbGH28A mutants. We localized the LbGH28A protein by immunofluorescence confocal and immunogold cytochemical microscopy in poplar ectomycorrhizal roots. Quantitative PCR confirmed the induced expression of LbGH28A during ectomycorrhiza formation. Laccaria bicolor RNAi mutants have a lower ability to establish ectomycorrhiza, confirming the key role of this enzyme in symbiosis. The purified recombinant LbGH28A has its highest activity towards pectin and polygalacturonic acid. In situ localization of LbGH28A indicates that this endopolygalacturonase is located in both fungal and plant cell walls at the symbiotic hyphal front. These findings suggest that the symbiosis-induced pectinase LbGH28A is involved in the Hartig net formation and is an important determinant for successful symbiotic colonization.

Nucleus-directed fluorescent reporter system for promoter studies in the ectomycorrhizal fungus Laccaria bicolor.

Currently ectomycorrhizal research suffers from a lack of molecular tools specifically adapted to study gene expression in fungal symbionts. Considering that, we designed pReNuK, a cloning vector for transcriptional promoter studies in the ectomycorrhizal basidiomycete Laccaria bicolor. The pReNuK vector offers the use of a nuclear localizing and chromatin incorporating histone H2B-mCherry fluorescent reporter protein and it is specifically optimized for efficient transgene expression in Laccaria. Moreover, pReNuK is designed to work in concert with Agrobacterium-mediated transformation under hygromycin B resistance selection. The functionality of the pReNuK reporter system was tested with the constitutive Laccaria glyceraldehyde 3-phosphate dehydrogenase gene promoter and further validated with the nitrogen source regulated nitrate reductase gene promoter. The expression of the nucleus-directed H2B-mCherry reporter is highly stable in time. Moreover, the transformation of Laccaria with pReNuK and the expression of the reporter do not have negative effects on the growth of the fungus. The pReNuK offers a novel tool for studying in vivo gene expression regulation in Laccaria, the leading fungal model for ectomycorrhizal research.

Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris.

l-amino acid oxidases (LAAOs) catalyze the oxidative deamination of l-amino acids to corresponding α-keto acids. Here, we describe the heterologous expression of four fungal LAAOs in Pichia pastoris. cgLAAO1 from Colletotrichum gloeosporioides and ncLAAO1 from Neurospora crassa were able to convert substrates not recognized by recombinant 9His-hcLAAO4 from the fungus Hebeloma cylindrosporum described earlier thereby broadening the substrate spectrum for potential applications. 9His-frLAAO1 from Fibroporia radiculosa and 9His-laLAAO2 from Laccaria amethystine were obtained only in low amounts. All four enzymes were N-glycosylated. We generated mutants of 9His-hcLAAO4 lacking N-glycosylation sites to further understand the effects of N-glycosylation. All four predicted N-glycosylation sites were glycosylated in 9His-hcLAAO4 expressed in P. pastoris. Enzymatic activity was similar for fully glycosylated 9His-hcLAAO4 and variants without one or all N-glycosylation sites after acid activation of all samples. However, activity without acid treatment was low in a variant without N-glycans. This was caused by the absence of a hypermannosylated N-glycan on asparagine residue N54. The lack of one or all of the other N-glycans was without effect. Our results demonstrate that adoption of a more active conformation requires a specific N-glycosylation during biosynthesis.

Identification and Differentiation of Wide Edible Mushrooms Based on Lipidomics Profiling Combined with Principal Component Analysis.

Mushroom, as a kind of higher fungus, is a precious homology resource of medicine and foods. In this study, total lipids were extracted from eight wild edible mushrooms and subsequently characterized by ultra-high-performance liquid chromatography-Quadrupole-Exactive Orbitrap mass spectrometry. 20 lipid classes and 173 molecular species were identified and quantified. Lipid molecules and their concentrations in Boletus speciosus, Boletus bainiugan, and Tricholoma matsutake exhibited significantly different behaviors compared with the remaining mushrooms. Hierarchical cluster analysis revealed that lipid profiles of B. bainiugan were most similar to B. speciosus followed by T. matsutake, Canthar-ellus cibarius, Sarcodon aspratu, Termitomyces eurrhizus, Laccaria laccata, and Thelephora ganbajun. In addition, several differential lipids can be considered as potential biomarkers to distinguish different mushroom species, for instance, lysophosphatidylethanolamine (16:1) and ceramide non-hydroxy fatty acid-dihydrosphingosine (d23:0-10:0). This study provided a new perspective to discriminate the mushroom species from the perspective of lipidomics.

Transcriptomic markers of fungal growth, respiration and carbon-use efficiency.

Fungal metabolic carbon acquisition and its subsequent partitioning between biomass production and respiration, i.e. the carbon-use efficiency (CUE), are central parameters in biogeochemical modeling. However, current available techniques for estimating these parameters are all associated with practical and theoretical shortcomings, making assessments unreliable. Gene expression analyses hold the prospect of phenotype prediction by indirect means, providing new opportunities to obtain information about metabolic priorities. We cultured four different fungal isolates (Chalara longipes, Laccaria bicolor, Serpula lacrymans and Trichoderma harzianum) in liquid media with contrasting nitrogen availability and measured growth rates and respiration to calculate CUE. By relating gene expression markers to measured carbon fluxes, we identified genes coding for 1,3-ß-glucan synthase and 2-oxoglutarate dehydrogenase as suitable markers for growth and respiration, respectively, capturing both intraspecific variation as well as within-strain variation dependent on growth medium. A transcript index based on these markers correlated significantly with differences in CUE between the fungal isolates. Our study paves the way for the use of these markers to assess differences in growth, respiration and CUE in natural fungal communities, using metatranscriptomic or the RT-qPCR approach.

Benefits provided by four ectomycorrhizal fungi to Pinus taeda under different external potassium availabilities.

Ectomycorrhizal fungi contribute to the nutrition of many woody plants, including those in the Pinaceae family. Loblolly pine (Pinus taeda L.), a native species of the Southeastern USA, can be colonized by multiple species of ectomycorrhizal fungi. The role of these symbionts in P. taeda potassium (K+) nutrition has not been previously investigated. Here, we assessed the contribution of four ectomycorrhizal fungi, Hebeloma cylindrosporum, Paxillus ammoniavirescens, Laccaria bicolor, and Suillus cothurnatus, in P. taeda K+ acquisition under different external K+ availabilities. Using a custom-made two-compartment system, P. taeda seedlings were inoculated with one of the four fungi, or kept non-colonized, and grown under K+-limited or -sufficient conditions for 8 weeks. Only the fungi had access to separate compartments in which rubidium, an analog tracer for K+, was supplied before harvest. Resulting effects of the fungi were recorded, including root colonization, biomass, and nutrient concentrations. We also analyzed the fungal performance in axenic conditions under varying supply of K+ and sodium. Our study revealed that these four ectomycorrhizal fungi are differentially affected by external K+ and sodium variations, that they are not able to provide similar benefits to the host P. taeda in our growing conditions, and that rubidium may be used with some limitations to estimate K+ transport from ectomycorrhizal fungi to colonized plants.