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

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 qPCR. The biological relevance of LbGH28A was confirmed by generating RNAi-silenced LbGH28A mutants. We localized the LbGH28A protein by immunofluorescence confocal and immunogold cytochemical microscopy in poplar ectomycorrhizal roots. qPCR confirmed the induced expression of LbGH28A during ectomycorrhiza formation. L. 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. The present findings suggest that the symbiosis-induced pectinase LbGH28A is involved in the Hartig net formation and is an important determinant for successful symbiotic colonization.

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.

Amanita subjunquillea and its ectomycorrhizal association, reported as new for Pakistan

Amanita subjunquillea and its ectomycorrhizal association are reported for the first time from moist temperate Himalayan forests of Pakistan. The sample was studied based on morphological characters and nucleotide sequence analyses of the ITS region generated from basidiomata and ectomycorrhizal roots of Quercus floribunda. Our collection differs from the type in its dark orange pileus disc and pale yellow margins. Remaining morphological and molecular data are consistent with previously reported specimens. This represents the first report of A. subjunquillea from Pakistan.