In order to assess the actual role of ectomycorrhizae in ion uptake by the ectomycorrhizal root system, we used a microelectrode ion flux estimation methodology that provided access to local values of net fluxes. This made it possible to investigate the heterogeneity of ion fluxes along the different types of roots of Pinus pinaster associated or not with ectomycorrhizal species. We compared two fungi able to grow with nitrate in pure culture, Rhizopogon roseolus and Hebeloma cylindrosporum , the former having a positive effect on host tree shoot growth ( c. +30%) and the latter a negative effect ( c. − − − − 30%). In non-mycorrhizal plants (control), NO 3 -was taken up at higher rates by the short roots than by the long ones, whereas K + uptake occurred mainly in growing apices of long roots. In mycorrhizal plants, H. cylindrosporum did not modify K + uptake and even decreased NO 3 -uptake at the level of ectomycorrhizal short roots, whereas R. roseolus strongly increased K + and NO 3 -fluxes at the level of ectomycorrhizal short roots without any modification of the fluxes measured along the fungus-free long roots. The measurement of ion influxes at the surface of the ectomycorrhizal roots can provide a way to reveal actual effects of mycorrhizal association on ion transport in relation to mycorrhizal efficiency in natural conditions.

The genetic relationship among Tricholoma matsutake and T. nauseosum strains collected from various parts of the Northern Hemisphere was investigated using sequence analysis of the rDNA ITS region and PCR-RFLP analysis of the rDNA IGS-1 region. ITS sequence similarity between T. matsutake and T. nauseosum ranged between 98.1% and 100%. The strains of T. matsutake from coniferous forests and those from broad-leaved forests showed more than 99.8% similarity in their ITS sequences. Three distinct RFLP types were detected when IGS-1 regions were digested with Cfr13I. RFLP patterns showed no variability among the strains of T. nauseosum and those of T. matsutake from broad-leaved forests. This pattern corresponded to the dominant RFLP type in the Japanese population of T. matsutake. Thus, strains belonging to this RFLP type are widely distributed throughout East Asia and Europe and associated with many tree species of Pinaceae and Fagaceae. The result suggests that T. matsutake in coniferous and broad-leaved forests and T. nauseosum should be treated as the same species genetically.

There has been conflicting debate over many years regarding the trophic status of Matsutake. Here we examined the morphology, structure and ultrastructure of Pinus densiflora roots collected from a Tricholoma matsutake Shiro within a pure Japanese red pine stand. Molecular investigations (PCR-RFLP analyses) indicated that T. matsutake was the highly dominant fungus within both the Shiro and the colonized root tips, suggesting that reported root morphology modifications can be attributed to T. matsutake infection. The external morphology of Matsutakecolonized roots is consistent with previous descriptions. The presence of extraradical mycelium, mantle, and intracortical Hartig net hyphae indicates clearly that T. matsutake forms an ectomycorrhizal association with P. densiflora in naturally occurring Shiros. The elucidation, for the first time, of the Hartig net ultrastructure at the host-fungus interface provides further and convincing evidence of a conventional ectomycorrhizal association. The progressive blackening, observed from base to tip in dominant mycorrhizal types, due to increased deposition of polyphenol and subsequent necrosis, appears to be a result of infection. However, the presence of highly nucleated vascular tissue indicates the viability of the vascular cylinder in these roots bearing necrotic cortices. Such a preponderance of black necrotic cortical tissues among colonized roots may reflect some atypical behaviour of T. matsutake.

The mycorrhizal relationships between pines and two edible species of Lactarius sect. Dapetes were investigated by optimizing the experimental conditions of mycelial growth and of mycorrhizal colonization of pine seedlings. In vitro mycelial growth of Lactarius deliciosus and L. sanguifluus was improved on a buffered medium containing glucose, amino acids, and vitamins. Two methods of mycorrhization of pines with Lactarius deliciosus were tested. The mycorrhizal colonization was rapid and intense under non-aseptic conditions with a low nutrient supply and without exogenous glucose. A positive influence of mycorrhizal colonization on Pinus sylvestris growth was subsequently observed. Under axenic conditions and with a high nutrient supply, mycorrhization was stimulated at 10 g/L of exogenous glucose, irrespective of the phosphorus concentration. At high phosphorus level (1 mM) and 0.1, 1.0, or 10.0 g/L glucose, growth of Pinus sylvestris was reduced by inoculation. Stability and development of Pinus spp./Lactarius deliciosus symbioses were assayed in a climatic chamber using containers filled with a synthetic substrate. Over a 2-year culture period, the root systems of the pine seedlings were heavily colonized by Lactarius deliciosus. One year following inoculation, Lactarius deliciosus fruit-body primordia appeared associated with Pinus sylvestris seedlings. Six months later, two mature basidiomata were obtained. This is the first report of soilless fruit-body formation of this edible mushroom.

An inter-simple sequence repeat (ISSR)-suppression-PCR technique established to develop microsatellite markers of plant species was applied to an ectomycorrhizal fungus, Tricholoma matsutake. Six polymorphic SSR markers were developed. All six polymorphic SSR markers were single-locused and co-dominant. Alleles produced by these six single-locused markers ranged from two to nine per locus and the expected heterozygosities were calculated as values from 0.098 to 0.803. The results indicated that the ISSR-suppression-PCR technique was effective and applicable to the development of microsatellite markers from ectomycorrhizal fungi. Furthermore, the six microsatellite loci did not amplify DNA from any other ectomycorrhizal species investigated, except for Tricholoma nauseosum (Swedish matsutake) and Tricholoma fulvocastaneum, suggesting that population genetics and reproduction of T. matsutake could be investigated by the SSR markers developed in the present study.

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Saprotrophic growth of Tricholoma matsutake isolates was investigated over Pinus densiflora bark fragments either on soil or on agar media. Preferential colonization of pine bark fragments by hyphae, in glucose-deprived environments suggested that Matsutake was able to extract some nutrients to sustain its growth. This was confirmed in glucose-free liquid nutrient medium, where bark as sole carbon source significantly stimulated (up to two-fold) growth of T. matsutake isolates. The addition of surfactants (Tween 80 and Tween 40) in liquid medium further stimulated mycelium growth over pine bark by up to 55%. Such growth stimulation was associated with a sharp increase in protein and beta-glucosidase excretion by hyphae in culture filtrates. As T. matsutake has some saprotrophic ability, the initiation and extension of Matsutake Shiro in forest soil might require simultaneously nutrients derived from the host plant and from soil organic compounds. Data reported here may contribute to the formulation of new culture substrates adapted to the co-culture of T. matsutake and its host plant under controlled conditions.

Edible mycorrhizal fungi (EMF) include truffles (Périgord black truffle, Italian white truffle etc.) and other highly prized forest mushrooms (porcini, chanterelles, matsutake etc.). Most edible EMF truffle species belong to Ascomycota, while EMF mushrooms belong to Basidiomycota. They are abundant in boreal and temperate forests, where they live in ectomycorrhizal symbiosis with trees, colonizing their roots and transforming them into well-defined ectomycorrhizae, mixed organs where fungal and plant tissues are merged. EMF are, however, also present in dry and desert areas, e.g. the Mediterranean Basin, where the so-called 'desert truffles' engage in a polyvalent mycorrhizal relationship with Cistaceae Juss. of the genus Helianthemum Mill. (Morte, Gutiérrez, & Navarro Ródenas, 2020). Mycorrhizae enable both partners to live together and exchange mutual services (Smith & Read, 2008). The fungus supplies the tree with water and nutrients that its extensive mycelium, made of microscopic hyphae, pumps or mobilizes from the soil. In exchange, the tree, or shrub, reallocates photosynthates to its fungal symbiont, which in most cases, are otherwise inaccessible to EMF, given their inability to decompose carbohydrate polymers such as starch, cellulose or lignin. From a practical point of view, this symbiotic relationship leads to (1) fast-growing and resilient mycorrhizal host trees (