Description, identification, and growth of Tuber borchii Vittad. mycorrhized Pinus sylvestris L. seedlings on different lime contents.

Tuber borchii forms ectomycorrhiza with oaks, hazel, and pines, including Pinus sylvestris. However, its ectomycorrhiza morphotype with P. sylvestris was not comprehensively described so far, and molecular analyses are missing despite a high danger of misidentification of T. borchii ectomycorrhiza with other closely related and less valuable truffle species. We described for the first time the morphology and anatomy of T. borchii-P. sylvestris ectomycorrhiza using differential interference contrast technique and semi-thin sections in combination with molecular confirmation of identity. Color of ectomycorrhiza is reddish to dark brown, and morphotypes are unevenly but densely covered by warts-bearing pin-like cystidia. All layers of the hyphal mantle are pseudoparenchymatous with outer mantle layer formed of epidermoid cells. T. borchii ectomycorrhiza was identified by a molecular comparison with fruitbodies used for inoculation and its respective ectomycorrhizae. T. borchii has a wide ecological amplitude. To get a better insight in mycorrhization requirements, we investigated growth of P. sylvestris and its ectomycorrhiza infection rate with T. borchii in substrate with different lime content. The mycorrhization of P. sylvestris with T. borchii in the mycorrhization substrate and cultivation in greenhouse conditions was successful, with colonization of P. sylvestris varying between 36.5 and 48.1%. There was no significant correlation of mycorrhization to applied lime contents, and consequently to pH in substrate, while the increased levels of lime improved growth of the P. sylvestris seedlings.

Scleroderma areolatum ectomycorrhiza on Fagus sylvatica L.

Despite its broad host range and distribution and its potential applications in commercial plantation forests, comprehensive descriptions of Scleroderma ectomycorrhizae are available only for Scleroderma citrinum, Scleroderma bovista and Scleroderma sinnamariense. This study provides a morphological and anatomical description of tree nursery derived ectomycorrhizae of Scleroderma areolatum on Fagus sylvatica, grown for several years in a climatized room. Ectomycorrhizae of S. areolatum were silvery white with abundant rhizomorphs; all mantle layers were plectenchymatous, rhizomorphs of type E, with prominent emanating hyphae with thick cell wall. The distal ends of emanating hyphae of rhizomorphs were inflated and often merged with other emanating hyphae. All parts of the mycorrhiza were clampless. In hyphae of the outer mantle layer, rhizomorphs and emanating hyphae, oily droplets were observed that did not stain in sulfo-vanillin and disappeared in lactic acid after a few hours. Although the phylogenetic analysis positioned the newly described ectomycorrhiza together with Scleroderma verrucosum and Scleroderma cepa in a single clade with a taxon name SH005470.07FU, the ectomycorrhizae of these three species can be morphologically well separated based on rhizomorph type.

Molecular and morphological analyses confirm Rhizopogon verii as a widely distributed ectomycorrhizal false truffle in Europe, and its presence in South America.

The genus Rhizopogon includes species with hypogeous or subepigeus habit, forming ectomycorrhizae with naturally occurring or planted pines (Pinaceae). Species of the genus Rhizopogon can be distinguished easily from the other hypogeous basidiomycetes by their lacunose gleba without columella and their smooth elliptical spores; however, the limit between species is not always easy to establish. Rhizopogon luteolus, the type species of the genus, has been considered one of the species that are more abundant in Europe, as well as it has been cited in pine plantation of North and South America, different parts of Africa, Australia, and New Zealand. However, in this study, based on molecular analyses of the ITS nuclear ribosomal DNA (nrDNA) sequences (19 new sequences; 37 sequences from GenBank/UNITE, including those from type specimens), we prove that many GenBank sequences under R. luteolus were misidentified and correspond to Rhizopogon verii, a species described from Tunisia. Also, we confirm that basidiomes and ectomycorrhizae recently collected in Germany under Pinus sylvestris, as well as specimens from South of Brazil under Pinus taeda belong to R. verii. Thanks to the numerous ectomycorrhizal tips collected in Germany, a complete description of R. verii/P. sylvestris ectomycorrhiza is provided. Moreover, since in this paper the presence of R. verii in South America is here reported for the first time, a short description of basidiomes collected in Brazil, compared with collections located in different European herbaria, is included.

The ectomycorrhizal morphotype Pinirhiza sclerotia is formed by Acephala macrosclerotiorum sp. nov., a close relative of Phialocephala fortinii.

Relatively few ectomycorrhizal fungal species are known to form sclerotia. Usually, sclerotia are initiated at the extraradical mycelium. In this study, we present anatomical and ultrastructural evidence for the formation of sclerotia directly in the hyphal mantle of the mycorrhizal morphotype Pinirhiza sclerotia. A dark-pigmented fungal strain was isolated from Pinirhiza sclerotia and identified by molecular tools as Acephala macrosclerotiorum sp. nov., a close relative of Phialocephala fortinii s.l. As dark septate fungi are known to be mostly endophytic, resyntheses with Pinus sylvestris and A. macrosclerotiorum as well as Populus tremula x Populus tremuloides and A. macrosclerotiorum or P. fortinii s.l. were performed under axenic conditions. No mycorrhizas were found when hybrid aspen was inoculated with A. macrosclerotiorum or P. fortinii. However, A. macrosclerotiorum formed true ectomycorrhizas in vitro with P. sylvestris. Anatomical and ultrastructural features of this ectomycorrhiza are presented. The natural and synthesized ectomycorrhizal morphotypes were identical and characterized by a thin hyphal mantle that bore sclerotia in a later ontogenetic stage. The Hartig net was well-developed and grew up to the endodermis. To our knowledge, this is the first evidence at the anatomical and ultrastructural level that a close relative of P. fortinii s.l. forms true ectomycorrhizas with a coniferous host.

Nutrient amounts of ectomycorrhizae analysed by EDX using ESEM and ICP.

Energy-dispersive X-ray (EDX) analysis coupled with an environmental scanning electron microscope (ESEM) was tested as an alternative to the inductively coupled argon plasma (ICP) spectrometer method for nutrient analyses of ectomycorrhizae. The results of EDX-ESEM and ICP were compared for 12 ectomycorrhizal morphotypes collected in beech and Scots pine forests in northern Brandenburg. The amounts of Al, Ca, Mg and S analysed in the outer hyphal layers of the sheath with the EDX-ESEM technique correlated well with the amounts of these elements in the whole mycorrhiza as assessed by ICP. For the elements P and K, no such correlation existed, indicating an uneven distribution of these elements in the ectomycorrhiza. It is concluded that the EDX-ESEM technique could be a useful and reliable tool for the analysis of nutrient elements in ectomycorrhizae, especially for studies focussing on small-scale soil heterogeneity or on infrequent morphotypes.