Water Relations, Gas Exchange, Chlorophyll Fluorescence and Electrolyte Leakage of Ectomycorrhizal Pinus halepensis Seedlings in Response to Multi-Heavy Metal Stresses (Pb, Zn, Cd)

The success of mine site restoration programs in arid and semi-arid areas poses a significant challenge and requires the use of high-quality seedlings capable of tolerating heavy metal stresses. The effect of ectomycorrhizal fungi on different physiological traits was investigated in Pinus halepensis seedlings grown in soil contaminated with heavy metals (Pb-Zn-Cd). Ectomycorrhizal (M) and non-ectomycorrhizal (NM) seedlings were subjected to heavy metals stress (C: contaminated, NC: control or non-contaminated) soils conditions for 12 months. Gas exchange, chlorophyll fluorescence, water relations parameters derived from pressure–volume curves and electrolyte leakage were evaluated at 4, 8 and 12 months. Ectomycorrhizal symbiosis promoted stronger resistance to heavy metals and improved gas exchange parameters and water-use efficiency compared to the non-ectomycorrhizal seedlings. The decrease in leaf osmotic potentials (Ψπ100: osmotic potential at saturation and Ψπ0: osmotic potential with loss of turgor) was higher for M-C seedling than NM-C ones, indicating that the ectomycorrhizal symbiosis promotes cellular osmotic adjustment and protects leaf membrane cell against leakage induced by Pb, Zn and Cd. Our results suggest that the use of ectomycorrhizal symbiosis is among the promising practices to improve the morphophysiological quality of seedlings produced in forest nurseries, their performance and their tolerance to multi-heavy metal stresses.

Morphoanatomical and phylogenetic characterization of the ectomycorrhiza between Laccaria squarrosa with Pinus pseudostrobus and its relevance for reforestation programs

Background: Pinus (Coniferophyta) and Laccaria (Basidiomycota) establish ectomycorrhizal symbioses in natural forests. However, their detailed morphoanatomical and phylogenetic characterization have received little attention. Accurate identification of native host symbionts is of paramount relevance to the production of mycorrhized seedlings for successful reforestation programs. Questions/Objective: We aimed to determine if L. squarrosa is able to establish ectomycorrhizal symbiosis with gymnosperms, thereby widening its host range and highlighting its relevance as a potential inoculant for pine seedlings. Currently, L. squarrosa is only known from its type collection associated with the angiosperm Fagus grandifolia var. mexicana. Studied species: The fungus L. squarrosa and Pinus pseudostrobus, a tree endemic to Mexico. Study site and dates: A Pinus-Quercus forest in Piedra Canteada, Nanacamilpa, Tlaxcala; 2018-2020. Methods: L. squarrosa basidiomata were identified and ectomycorrhizal roots were collected and morphoanatomically characterized. For molecular identification, DNA was extracted, PCR was performed targeting the nuclear ribosomal internal transcribed spacer region (nucrDNA ITS) for the mycobiont identification and the chloroplastic single-locus trnL region for the phytobiont. Results: In the phylogenetic analyses, our sequences from basidiomata and ectomycorrhizae clustered together with L.squarrosa with high values of supporting identity. Meanwhile, P. pseudostrobus was molecularly identified as the phytobiont. Conclusions: This is one of the few worldwide characterizations of Laccaria ectomycorrhiza under field conditions and contributes to the understanding of the ecology, distribution, and economic relevance of the symbiotic association. Our data suggest that L. squarrosa has potential for use as a native inoculant for P. pseudostrobus tree production. Translate stop Translate stop

Metabolomics and transcriptomics to decipher molecular mechanisms underlying ectomycorrhizal root colonization of an oak tree

Mycorrhizas are known to have a positive impact on plant growth and ability to resist major biotic and abiotic stresses. However, the metabolic alterations underlying mycorrhizal symbiosis are still understudied. By using metabolomics and transcriptomics approaches, cork oak roots colonized by the ectomycorrhizal fungus Pisolithus tinctorius were compared with non-colonized roots. Results show that compounds putatively corresponding to carbohydrates, organic acids, tannins, long-chain fatty acids and monoacylglycerols, were depleted in ectomycorrhizal cork oak colonized roots. Conversely, non-proteogenic amino acids, such as gamma-aminobutyric acid (GABA), and several putative defense-related compounds, including oxylipin-family compounds, terpenoids and B6 vitamers were induced in mycorrhizal roots. Transcriptomic analysis suggests the involvement of GABA in ectomycorrhizal symbiosis through increased synthesis and inhibition of degradation in mycorrhizal roots. Results from this global metabolomics analysis suggest decreases in root metabolites which are common components of exudates, and in compounds related to root external protective layers which could facilitate plant-fungal contact and enhance symbiosis. Root metabolic pathways involved in defense against stress were induced in ectomycorrhizal roots that could be involved in a plant mechanism to avoid uncontrolled growth of the fungal symbiont in the root apoplast. Several of the identified symbiosis-specific metabolites, such as GABA, may help to understand how ectomycorrhizal fungi such as P. tinctorius benefit their host plants.

Evolutionary priming and transition to the ectomycorrhizal habit in an iconic lineage of mushroom-forming fungi: is preadaptation a requirement?

The ectomycorrhizal symbiosis is an essential guild of many forested ecosystems and has a dynamic evolutionary history across kingdom Fungi, having independently evolved from diverse types of saprotrophic ancestors. In this study, we seek to identify genomic features of the transition to the ectomycorrhizal habit within the Russulaceae, one of the most diverse lineages of ectomycorrhizal fungi. We present comparative analyses of the pangenome and gene repertoires of 21 species across the order Russulales, including a closely related saprotrophic member of Russulaceae. The ectomycorrhizal Russulaceae is inferred to have originated around the Cretaceous-Paleogene extinction event (73.6-60.1 million years ago (MY)). The genomes of the ectomycorrhizal Russulaceae are characterized by a loss of genes for plant cell-wall degrading enzymes (PCWDEs), an expansion of genome size through increased transposable element (TE) content, a reduction in secondary metabolism clusters, and an association of genes coding for certain secreted proteins with TE “nests”. The saprotrophic sister group of the ectomycorrhizal Russulaceae, Gloeopeniophorella convolvens, possesses some of these aspects (e.g., loss of some PCWDE and protease orthologs, TE expansion, reduction in secondary metabolism clusters), resulting from an accelerated rate of gene evolution in the shared ancestor of Russulaceae that predates the evolution of the ectomycorrhizal habit. Genomes of Russulaceae possess a high degree of synteny, including a conserved set of terpene secondary metabolite gene clusters. We hypothesize that the evolution of the ectomycorrhizal habit requires premodification of the genome for plant root association followed by an accelerated rate of gene evolution within the secretome for host-defense circumvention and symbiosis establishment.

Draft Genome Sequences of the Black Truffles Tuber brumale Vittad. and Tuber indicum Cook & Massee

ABSTRACT Tuber brumale and Tuber indicum (Pezizomycetes) are two edible black truffles establishing ectomycorrhizal symbiosis with trees and shrubs. T. brumale is ubiquitous in Europe, and T. indicum is mainly found in China. Here, we present the draft genome sequences of T. brumale and T. indicum.

Fungal Shaker-like channels beyond cellular K+ homeostasis: A role in ectomycorrhizal symbiosis between Hebeloma cylindrosporum and Pinus pinaster

Potassium (K+) acquisition, translocation and cellular homeostasis are mediated by various membrane transport systems in all organisms. We identified and described an ion channel in the ectomycorrhizal fungus Hebeloma cylindrosporum (HcSKC) that harbors features of animal voltage-dependent Shaker-like K+ channels, and investigated its role in both free-living hyphae and symbiotic conditions. RNAi lines affected in the expression of HcSKC were produced and used for in vitro mycorrhizal assays with the maritime pine as host plant, under standard or low K+ conditions. The adaptation of H. cylindrosporum to the downregulation of HcSKC was analyzed by qRT-PCR analyses for other K+-related transport proteins: the transporters HcTrk1, HcTrk2, and HcHAK, and the ion channels HcTOK1, HcTOK2.1, and HcTOK2.2. Downregulated HcSKC transformants displayed greater K+ contents at standard K+ only. In such conditions, plants inoculated with these transgenic lines were impaired in K+ nutrition. Taken together, these results support the hypothesis that the reduced expression of HcSKC modifies the pool of fungal K+ available for the plant and/or affects its symbiotic transfer to the roots. Our study reveals that the maintenance of K+ transport in H. cylindrosporum, through the regulation of HcSKC expression, is required for the K+ nutrition of the host plant.

Phosphate availability and ectomycorrhizal symbiosis with Pinus sylvestris have independent effects on the Paxillus involutus transcriptome

Many plant species form symbioses with ectomycorrhizal fungi, which help them forage for limiting nutrients in the soil such as inorganic phosphate (Pi). The transcriptional responses to symbiosis and nutrient-limiting conditions in ectomycorrhizal fungal hyphae, however, are largely unknown. An artificial system was developed to study ectomycorrhizal basidiomycete Paxillus involutus growth in symbiosis with its host tree Pinus sylvestris at different Pi concentrations. RNA-seq analysis was performed on P. involutus hyphae growing under Pi-limiting conditions, either in symbiosis or alone. We show that Pi starvation and ectomycorrhizal symbiosis have an independent effect on the P. involutus transcriptome. Notably, low Pi availability induces expression of newly identified putative high-affinity Pi transporter genes, while reducing the expression of putative organic acid transporters. Additionally, low Pi availability induces a close transcriptional interplay between P and N metabolism. GTP-related signalling was found to have a positive effect in the maintenance of ectomycorrhizal symbiosis, whereas multiple putative cytochrome P450 genes were found to be downregulated, unlike arbuscular mycorrhizal fungi. We provide the first evidence of global transcriptional changes induced by low Pi availability and ectomycorrhizal symbiosis in the hyphae of P. involutus, revealing both similarities and differences with better-characterized arbuscular mycorrhizal fungi.

The communities of ectomycorrhizal fungal species associated with Betula pendula Roth and Pinus sylvestris L. growing in heavy-metal contaminated soils

Aims Pioneer tree species such as Betula pendula and Pinus sylvestris encroach soils contaminated with heavy metals (HMs). This is facilitated by ectomycorrhizal fungi colonizing tree roots. Thus, we evaluated the ectomycorrhizal fungal (EMF) communities of B. pendula and P. sylvestris growing in HM-contaminated soils compared to non-contaminated soils. We also studied the effect of HMs and soil properties on EMF communities and soil fungal biomass. Methods Roots of B. pendula and P. sylvestris were collected from three HM-contaminated sites and from two non-contaminated sites located in Poland. EMF species were identified using DNA barcoding. Soil fungal biomass was determined by soil ergosterol. Results B. pendula and P. sylvestris growing in HM-contaminated soils had similar EMF communities, where Scleroderma, Rhizopogon and Russula as well as ectomycorrhizae of the long-distance exploration type dominated. Among all of the examined soil factors studied, toxicity index (TITotal) was the most significant factor shaping the composition of EMF communities. Despite significant differences in the structure of the EMF communities of trees growing in HM-contaminated sites compared to control sites, no differences in overall diversity were observed. Conclusions Only well-adapted EMF species can survive toxic conditions and form ectomycorrhizal symbiosis with encroaching trees facilitating the forest succession on contaminated soils.

Tuber melanosporum shapes nirS-type denitrifying and ammonia-oxidizing bacterial communities in Carya illinoinensis ectomycorrhizosphere soils

Background NirS-type denitrifying bacteria and ammonia-oxidizing bacteria (AOB) play a key role in the soil nitrogen cycle, which may affect the growth and development of underground truffles. We aimed to investigate nirS-type denitrifying bacterial and AOB community structures in the rhizosphere soils of Carya illinoinensis seedlings inoculated with the black truffle (Tuber melanosporum) during the early symbiotic stage. Methods The C. illinoinensis seedlings inoculated with or without T. melanosporum were cultivated in a greenhouse for six months. Next-generation sequencing (NGS) technology was used to analyze nirS-type denitrifying bacterial and AOB community structures in the rhizosphere soils of these seedlings. Additionally, the soil properties were determined. Results The results indicated that the abundance and diversity of AOB were significantly reduced due to the inoculation of T. melanosporum, while these of nirS-type denitrifying bacteria increased significantly. Proteobacteria were the dominant bacterial groups, and Rhodanobacter, Pseudomonas, Nitrosospira and Nitrosomonas were the dominant classified bacterial genera in all the soil samples. Pseudomonas was the most abundant classified nirS-type denitrifying bacterial genus in ectomycorrhizosphere soils whose relative abundance could significantly increase after T. melanosporum inoculation. A large number of unclassified nirS-type denitrifying bacteria and AOB were observed. Moreover, T. melanosporum inoculation had little effect on the pH, total nitrogen (TN), nitrate-nitrogen (NO${}_{3}^{-}$-N) and ammonium-nitrogen (NH${}_{4}^{+}$-N) contents in ectomycorrhizosphere soils. Overall, our results showed that nirS-type denitrifying bacterial and AOB communities in C. illinoinensis rhizosphere soils were significantly affected by T. melanosporum on the initial stage of ectomycorrhizal symbiosis, without obvious variation of soil N contents.