Mycorrhizal fungi are mutualists that play crucial roles in nutrient acquisition in terrestrial ecosystems. Mycorrhizal symbioses arose repeatedly across multiple lineages of Mucoromycotina, Ascomycota, and Basidiomycota. Considerable variation exists in the capacity of mycorrhizal fungi to acquire carbon from soil organic matter. Here, we present a combined analysis of 135 fungal genomes from 73 saprotrophic, endophytic and pathogenic species, and 62 mycorrhizal species, including 29 new mycorrhizal genomes. This study samples ecologically dominant fungal guilds for which there were previously no symbiotic genomes available, including ectomycorrhizal Russulales, Thelephorales and Cantharellales. Our analyses show that transitions from saprotrophy to symbiosis involve (1) widespread losses of degrading enzymes acting on lignin and cellulose, (2) co-option of genes present in saprotrophic ancestors to fulfill new symbiotic functions, (3) diversification of novel, lineage-specific symbiosis-induced genes, (4) proliferation of transposable elements and (5) divergent genetic innovations underlying the convergent origins of the ectomycorrhizal guild.

Fungal and actinobacterial communities were analyzed together with soil chemistry and enzyme activities in order to profile the microbial diversity associated with the economically important mushroom Tricholoma matsutake. Samples of mycelium-soil aggregation (shiro) were collected from three experimental sites where sporocarps naturally formed. PCR was used to confirm the presence and absence of matsutake in soil samples. PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting and direct sequencing were used to identify fungi and actinobacteria in the mineral and organic soil layers separately. Soil enzyme activities and hemicellulotic carbohydrates were analyzed in a productive experimental site. Soil chemistry was investigated in both organic and mineral soil layers at all three experimental sites. Matsutake dominated in the shiro but also coexisted with a high diversity of fungi and actinobacteria. Tomentollopsis sp. in the organic layer above the shiro and Piloderma sp. in the shiro correlated positively with the presence of T. matsutake in all experimental sites. A Thermomonosporaceae bacterium and Nocardia sp. correlated positively with the presence of T. matsutake, and Streptomyces sp. was a common cohabitant in the shiro, although these operational taxonomic units (OTUs) did not occur at all sites. Significantly higher enzyme activity levels were detected in shiro soil. These enzymes are involved in the mobilization of carbon from organic matter decomposition. Matsutake was not associated with a particular soil chemistry compared to that of nearby sites where the fungus does not occur. The presence of a significant hemicellulose pool and the enzymes to degrade it indicates the potential for obtaining carbon from the soil rather than tree roots.

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.

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.

Raulí (Nothofagus nervosa) is a highly valuable and fast growing Chilean native tree species, therefore being of great interest for the establishment of plantations. The success of plantations depends upon several factors, among which nursery management can be decisive. This study evaluated the effects of slow release fertilization (FLC) in doses of 2.5, 5.0 and 7.5 kg m-3 , and of topping (with and without) on the survival and growth of raulí in a plantation with restoration purposes in the Valdivian Andes of Chile (650 m asl). Average survival was 82 % the first year, 70 % the second and 62 % the third (mortality due to frosts). The mean height growth for the period was 120 cm. However, the trend was that survival and growth were greater with the two higher fertilization doses, and to a lesser extent with topping. During the first three years of field evaluation we did not detect significant differences of FLC and top pruning on survival, height growth and diameter at breast height among the six treatments. However, the trend was that there were higher survival rates and growth rates with the two higher FLC doses, which also occurred with top pruning (better perforrmance with pruning), but to a lower extent. Results show that with the range of plant quality used, similar performance results can be obtained when outplanted in the field. RESUMEN Nothofagus nervosa (raulí) es una de las especies nativas chilenas de mayor valor y crecimiento y, por ello, en el último tiempo concita alto interés para el establecimiento de plantaciones. El éxito del establecimiento de plantaciones depende de diversos fac-tores, entre los cuales el manejo en vivero puede ser decisivo. El presente estudio evaluó los efectos de fertilización de liberación controlada (FLC en dosis de 2,5, 5,0 y 7,5 kg m-3) y de poda apical (con y sin) en vivero sobre la supervivencia y el crecimiento de N. nervosa en plantaciones con fines de restauración en Los Andes valdivianos de Chile (650 m s.n.m.). La supervivencia fue de 82 % en el primer año, 70 % en el segundo y 62 % en el tercero (mortalidad por heladas). El crecimiento promedio en altura durante el trienio estuvo alrededor de 120 cm. Durante los primeros tres años de evaluación en terreno no se observaron efectos significativos de las dosis de FLC y la poda sobre la supervivencia, crecimientos en altura y en diámetro a la altura del pecho. Sin embargo, la tendencia fue de mayor supervivencia y crecimiento con ambas dosis mayores de FLC, lo cual también ocurrió con poda apical, pero en menor magnitud. Los resultados demuestran que en el rango de calidad de planta obtenida en vivero se pueden alcanzar similares resultados en terreno. Palabras clave: restauración, estrés abiótico, mortalidad, crecimiento.

Tricholoma matsutake is an economically important ectomycorrhizal fungus of coniferous woodlands. Mycologists suspect that this fungus is also capable of saprotrophic feeding. In order to evaluate this hypothesis, enzyme and chemical assays were performed in the field and laboratory. From a natural population of T. matsutake in southern Finland, samples of soil-mycelium aggregate (shiro) were taken from sites of sporocarp formation and nearby control (PCR-negative) spots. Soil organic carbon and activity rates of hemicellulolytic enzymes were measured. The productivity of T. matsutake was related to the amount of utilizable organic carbon in the shiro, where the activity of xylosidase was significantly higher than in the control sample. In the laboratory, sterile pieces of bark from the roots of Scots pine were inoculated with T. matsutake and the activity rates of two hemicellulolytic enzymes (xylosidase and glucuronidase) were assayed. Furthermore, a liquid culture system showed how T. matsutake can utilize hemicellulose as its sole carbon source. Results linked and quantified the general relationship between enzymes secreted by T. matsutake and the degradation of hemicellulose. Our findings suggest that T. matsutake lives mainly as an ectomycorrhizal symbiont but can also feed as a saprotroph. A flexible trophic ecology confers T. matsutake with a clear advantage in a heterogeneous environment and during sporocarp formation.

This study aimed to test the ability of Tricholoma matsutake isolates to form mycorrhizas with aseptic seedlings of Pinus sylvestris L. and Picea abies (L.) Karst. Germinated seedlings of Scots pine and Norway spruce were separately inoculated with either isolates originating from Finland or Japan. Eight months after inoculation, the Finnish isolate had formed a sheath and Hartig net on both host species. Ectomycorrhizal Scots pine seedlings inoculated with the Finnish isolate showed the same shoot height and dry mass as the controls. Ectomycorrhizal Norway spruce seedlings inoculated with the Finnish isolate had similar shoot height but slightly less dry mass than the control seedlings. For both tree species, inoculation with the Finnish isolate resulted in reduced total nitrogen content per seedling, but carbon content was unaffected. Inoculation with the Japanese isolate resulted in an initial Hartig net-like structure in pine but not in spruce. No typical Hartig net was observed on either tree species. Furthermore, seedlings of both species inoculated with the Japanese isolate showed significantly reduced growth, dry mass, nitrogen, and carbon content per seedling and shoot height (in spruce) compared to the controls. This study documents and describes the in vitro ectomycorrhization between T. matsutake and Scots pine or Norway spruce and the variable mycorrhizal structures that matsutake isolates can form.

The root systems of 1 1-wk-old Pinus densiflora seedlings were inoculated with a hyphal suspension of Tricholoma matsutake and aseptically incubated for 4 wk in a forest soil without supplying exogenous carbohydrates. One week following inoculation, fungal hyphae had colonized the root surface and bound soil particles together establishing a rootsubstrate continuum. Fungal hyphae were visible within the main root cortex following clearing, bleaching and staining. In the ensuing days, fungal colonization was observed within elongating lateral roots in which Hartig net formation was confirmed 4 wk after inoculation. This is the first report of rapid ectomycorrhizal infection of P. densiflora seedlings by T. matsutake.