RESUMEN
Soils are the foundation of all terrestrial ecosystems1. However, unlike for plants and animals, a global assessment of hotspots for soil nature conservation is still lacking2. This hampers our ability to establish nature conservation priorities for the multiple dimensions that support the soil system: from soil biodiversity to ecosystem services. Here, to identify global hotspots for soil nature conservation, we performed a global field survey that includes observations of biodiversity (archaea, bacteria, fungi, protists and invertebrates) and functions (critical for six ecosystem services) in 615 composite samples of topsoil from a standardized survey in all continents. We found that each of the different ecological dimensions of soils-that is, species richness (alpha diversity, measured as amplicon sequence variants), community dissimilarity and ecosystem services-peaked in contrasting regions of the planet, and were associated with different environmental factors. Temperate ecosystems showed the highest species richness, whereas community dissimilarity peaked in the tropics, and colder high-latitudinal ecosystems were identified as hotspots of ecosystem services. These findings highlight the complexities that are involved in simultaneously protecting multiple ecological dimensions of soil. We further show that most of these hotspots are not adequately covered by protected areas (more than 70%), and are vulnerable in the context of several scenarios of global change. Our global estimation of priorities for soil nature conservation highlights the importance of accounting for the multidimensionality of soil biodiversity and ecosystem services to conserve soils for future generations.
Asunto(s)
Biodiversidad , Conservación de los Recursos Naturales , Mapeo Geográfico , Microbiología del Suelo , Suelo , Animales , Conservación de los Recursos Naturales/métodos , Suelo/parasitología , Invertebrados , ArchaeaRESUMEN
Plant-soil biodiversity interactions are fundamental for the functioning of terrestrial ecosystems. Yet, the existence of a set of globally distributed topsoil microbial and small invertebrate organisms consistently associated with land plants (i.e., their consistent soil-borne microbiome), together with the environmental preferences and functional capabilities of these organisms, remains unknown. We conducted a standardized field survey under 150 species of land plants, including 58 species of bryophytes and 92 of vascular plants, across 124 locations from all continents. We found that, despite the immense biodiversity of soil organisms, the land plants evaluated only shared a small fraction (less than 1%) of all microbial and invertebrate taxa that were present across contrasting climatic and soil conditions and vegetation types. These consistent taxa were dominated by generalist decomposers and phagotrophs and their presence was positively correlated with the abundance of functional genes linked to mineralization. Finally, we showed that crossing environmental thresholds in aridity (aridity index of 0.65, i.e., the transition from mesic to dry ecosystems), soil pH (5.5; i.e., the transition from acidic to strongly acidic soils), and carbon (less than 2%, the lower limit of fertile soils) can result in drastic disruptions in the associations between land plants and soil organisms, with potential implications for the delivery of soil ecosystem processes under ongoing global environmental change.
Asunto(s)
Embryophyta , Microbiota , Microbiología del Suelo , Biodiversidad , Suelo/químicaRESUMEN
Explaining the large-scale diversity of soil organisms that drive biogeochemical processes-and their responses to environmental change-is critical. However, identifying consistent drivers of belowground diversity and abundance for some soil organisms at large spatial scales remains problematic. Here we investigate a major guild, the ectomycorrhizal fungi, across European forests at a spatial scale and resolution that is-to our knowledge-unprecedented, to explore key biotic and abiotic predictors of ectomycorrhizal diversity and to identify dominant responses and thresholds for change across complex environmental gradients. We show the effect of 38 host, environment, climate and geographical variables on ectomycorrhizal diversity, and define thresholds of community change for key variables. We quantify host specificity and reveal plasticity in functional traits involved in soil foraging across gradients. We conclude that environmental and host factors explain most of the variation in ectomycorrhizal diversity, that the environmental thresholds used as major ecosystem assessment tools need adjustment and that the importance of belowground specificity and plasticity has previously been underappreciated.
Asunto(s)
Biodiversidad , Bosques , Hongos/clasificación , Hongos/fisiología , Interacciones Microbiota-Huesped , Micorrizas/fisiología , Microbiología del Suelo , Europa (Continente) , Hongos/aislamiento & purificación , Mapeo GeográficoRESUMEN
Change history: In the HTML version of this Article, author 'Filipa Cox' had no affiliation in the author list, although she was correctly associated with affiliation 3 in the PDF. In addition, the blue circles for 'oak' were missing from Extended Data Fig. 1. These errors have been corrected online.
RESUMEN
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.
Asunto(s)
Ascomicetos , Compuestos de Calcio , Micorrizas , Óxidos , Pinus sylvestris , Pinus , PlantonesRESUMEN
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.
Asunto(s)
Fagus/microbiología , Micorrizas/fisiología , Hifa , Micorrizas/genética , Micorrizas/aislamiento & purificación , Fenotipo , Filogenia , SimbiosisRESUMEN
Restingomyces reticulatus gen. et sp. nov. is a recently discovered false truffle species from Atlantic "restinga" rainforest in northeastern Brazil. Molecular and morphological characters separate this new sequestrate species from other described taxa in the order Phallales (Phallomycetidae, Basidiomycota). In our phylogenetic analysis based on nuc 28S rDNA and atp6, R. reticulatus forms a sister clade to Trappea darkeri and Phallobata alba, with the three taxa forming the earliest diverging lineage within Phallales. Morphological and molecular data warrant the recognition of the new genus and species, described here, and we also amend the taxonomic description for the family Trappeaceae.
Asunto(s)
Basidiomycota/clasificación , Basidiomycota/aislamiento & purificación , Basidiomycota/citología , Basidiomycota/genética , Biometría , Brasil , Análisis por Conglomerados , ADN de Hongos/química , ADN de Hongos/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Microscopía , ATPasas de Translocación de Protón Mitocondriales/genética , Fotograbar , Filogenia , ARN Ribosómico 28S/genética , Bosque Lluvioso , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Basidiomycota/clasificación , Basidiomycota/genética , Micorrizas/clasificación , Micorrizas/genética , ADN de Hongos/genética , ADN Espaciador Ribosómico/genética , Europa (Continente) , Filogenia , Pinus/microbiología , América del SurRESUMEN
In total, 150 protein extracts from 94 different basidiomycete and ascomycete wild mushroom species were tested for antibacterial activity against the quarantine plant-pathogen bacterium Ralstonia solanacearum. In in vitro microtiter plate assays, 15 extracts with moderate to high antibacterial activities were identified: 11 completely inhibited bacterial growth and 4 showed partial inhibition. Of these 15 extracts, 5 were further tested and 3 extracts slowed disease progression and reduced disease severity in artificially inoculated tomato and potato plants. However, the in vitro activities of the extracts did not always correlate with their in vivo activities, which emphasizes the importance of performing early screening tests also in vivo. Testing of selected extracts against 12 R. solanacearum strains identified 6 with potential for broader applicability. Further analysis of extracts from Amanita phalloides and Clitocybe geotropa showed that the active substances are proteins with an approximate size of 180 kDa. To our knowledge, this is the first in vitro and in vivo study that demonstrates that mushroom protein extracts can be promising for treatment of bacterial wilt caused by R. solanacearum.
RESUMEN
· Much of the macroecological information about microorganisms is confounded by the lack of standardized methodology, paucity of metadata and sampling effect of a particular substrate or interacting host taxa. · This study aims to disentangle the relative effects of biological, geographical and edaphic variables on the distribution of Alnus-associated ectomycorrhizal (ECM) fungi at the global scale by using comparable sampling and analysis methods. · Ribosomal DNA sequence analysis revealed 146 taxa of ECM fungi from 22 Alnus species across 96 sites worldwide. Use of spatial and phylogenetic eigenvectors along with environmental variables in model selection indicated that phylogenetic relations among host plants and geographical links explained 43 and 10%, respectively,in ECM fungal community composition, whereas soil calcium concentration positively influenced taxonomic richness. · Intrageneric phylogenetic relations among host plants and regional processes largely account for the global biogeographic distribution of Alnus-associated ECM fungi. The biogeography of ECM fungi is consistent with ancient host migration patterns from Eurasia to North America and from southern Europe to northern Europe after the last glacial maximum, indicating codispersal of hosts and their mycobionts.
Asunto(s)
Alnus/microbiología , Geografía , Micorrizas/fisiología , Filogeografía , Biodiversidad , Calcio/metabolismo , Clima , Funciones de Verosimilitud , Filogenia , Lluvia , Suelo/químicaRESUMEN
The nuclear ribosomal internal transcribed spacer (ITS) region is the formal fungal barcode and in most cases the marker of choice for the exploration of fungal diversity in environmental samples. Two problems are particularly acute in the pursuit of satisfactory taxonomic assignment of newly generated ITS sequences: (i) the lack of an inclusive, reliable public reference data set and (ii) the lack of means to refer to fungal species, for which no Latin name is available in a standardized stable way. Here, we report on progress in these regards through further development of the UNITE database (http://unite.ut.ee) for molecular identification of fungi. All fungal species represented by at least two ITS sequences in the international nucleotide sequence databases are now given a unique, stable name of the accession number type (e.g. Hymenoscyphus pseudoalbidus|GU586904|SH133781.05FU), and their taxonomic and ecological annotations were corrected as far as possible through a distributed, third-party annotation effort. We introduce the term 'species hypothesis' (SH) for the taxa discovered in clustering on different similarity thresholds (97-99%). An automatically or manually designated sequence is chosen to represent each such SH. These reference sequences are released (http://unite.ut.ee/repository.php) for use by the scientific community in, for example, local sequence similarity searches and in the QIIME pipeline. The system and the data will be updated automatically as the number of public fungal ITS sequences grows. We invite everybody in the position to improve the annotation or metadata associated with their particular fungal lineages of expertise to do so through the new Web-based sequence management system in UNITE.
Asunto(s)
Bases de Datos de Ácidos Nucleicos , Hongos/clasificación , Filogenia , Código de Barras del ADN Taxonómico , ADN de Hongos/genética , ADN Espaciador Ribosómico/genética , Hongos/genética , InternetRESUMEN
Soil contamination is one of the main threats to ecosystem health and sustainability. Yet little is known about the extent to which soil contaminants differ between urban greenspaces and natural ecosystems. Here we show that urban greenspaces and adjacent natural areas (i.e., natural/semi-natural ecosystems) shared similar levels of multiple soil contaminants (metal(loid)s, pesticides, microplastics, and antibiotic resistance genes) across the globe. We reveal that human influence explained many forms of soil contamination worldwide. Socio-economic factors were integral to explaining the occurrence of soil contaminants worldwide. We further show that increased levels of multiple soil contaminants were linked with changes in microbial traits including genes associated with environmental stress resistance, nutrient cycling, and pathogenesis. Taken together, our work demonstrates that human-driven soil contamination in nearby natural areas mirrors that in urban greenspaces globally, and highlights that soil contaminants have the potential to cause dire consequences for ecosystem sustainability and human wellbeing.
Asunto(s)
Ciudades , Ecosistema , Internacionalidad , Parques Recreativos , Contaminantes del Suelo , Suelo , Microbiota , Factores Socioeconómicos , Suelo/química , Microbiología del Suelo , Contaminantes del Suelo/análisis , Contaminantes del Suelo/química , PlásticosRESUMEN
While the contribution of biodiversity to supporting multiple ecosystem functions is well established in natural ecosystems, the relationship of the above- and below-ground diversity with ecosystem multifunctionality remains virtually unknown in urban greenspaces. Here we conducted a standardized survey of urban greenspaces from 56 municipalities across six continents, aiming to investigate the relationships of plant and soil biodiversity (diversity of bacteria, fungi, protists and invertebrates, and metagenomics-based functional diversity) with 18 surrogates of ecosystem functions from nine ecosystem services. We found that soil biodiversity across biomes was significantly and positively correlated with multiple dimensions of ecosystem functions, and contributed to key ecosystem services such as microbially driven carbon pools, organic matter decomposition, plant productivity, nutrient cycling, water regulation, plant-soil mutualism, plant pathogen control and antibiotic resistance regulation. Plant diversity only indirectly influenced multifunctionality in urban greenspaces via changes in soil conditions that were associated with soil biodiversity. These findings were maintained after controlling for climate, spatial context, soil properties, vegetation and management practices. This study provides solid evidence that conserving soil biodiversity in urban greenspaces is key to supporting multiple dimensions of ecosystem functioning, which is critical for the sustainability of urban ecosystems and human wellbeing.
Asunto(s)
Ecosistema , Suelo , Humanos , Parques Recreativos , Biodiversidad , PlantasRESUMEN
Two new species of Hydnum, characterized by slender Hydnum rufescens-like basidiomes and ovoid to broadly ellipsoid basidiospores, are described from the Iberian Peninsula based on morphological and ITS molecular data. Hydnum ovoideisporum is distinguished by pilei with deep orange tones and strong preference for calcareous soil. It is widespread in the Iberian-Mediterranean area. Hydnum vesterholtii is characterized by its ocher to light ocher pileus, and nearly all the collections were made in the Pyrenees. Both ovoid-spored species are monophyletic well supported groups in the maximum parsimony and Bayesian ITS phylogenies, while the remainder of the samples assigned to H. rufescens s.l. and having globose basidiospores split into six well supported clades. The need to typify the name Hydnum rufescens is discussed, and a provisional key is given for the European taxa of Hydnum.
Asunto(s)
Basidiomycota/clasificación , Filogenia , Esporas Fúngicas/citología , Secuencia de Bases , Basidiomycota/citología , Basidiomycota/genética , Basidiomycota/aislamiento & purificación , ADN de Hongos/química , ADN de Hongos/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Cuerpos Fructíferos de los Hongos/clasificación , Cuerpos Fructíferos de los Hongos/citología , Cuerpos Fructíferos de los Hongos/aislamiento & purificación , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Esporas Fúngicas/clasificación , Esporas Fúngicas/aislamiento & purificaciónRESUMEN
Mattirolomyces is an edible commercial sequestrate genus that is globally distributed. From the five described taxa of this genus, Mattirolomyces terfezioides is the most common species in Asia. Our recent attempts to locate M. terfezioides outside its current distribution area in China documented its first records in areas of poplar trees with the lowest known temperature and precipitation averages ever recorded for this species. This peculiar ecology was not reflected on the species-morphological features nor on its phylogenetic position in the genus. The first attempt to apply the phylogenetic network approach to Mattirolomyces revealed its geographic origin in the Asian-Pacific areas prior to frequent long-distance migration events. Based on data from recent study areas, we found that the collections from Inner Mongolia and the Shanxi province were similar to European collections. Asian haplotypes were less distant from the outgroup comparing to collections from Europe, supporting the hypothesis that M. terfezioides was originated from this Chinese area and was subsequently transported to Europe. Exploring M. terfezioides ecology and its mycorrhiza potential to grow in association with poplars would be of great importance for planning cultivation projects of this valuable desert truffle species in Central and Eastern China, a currently underexploited economic sector that deserves further ecological and M. terfezioides mycorrhizal synthesis investigations.
Asunto(s)
Ascomicetos , Micorrizas , Filogeografía , Asia , China , Haplotipos/genética , FilogeniaRESUMEN
In the era of plastic pollution, plants have been discarded as a system that is not affected by micro and nanoplastics, but contrary to beliefs that plants cannot absorb plastic particles, recent research proved otherwise. The presented review gives insight into known aspects of plants' interplay with plastics and how plants' ability to absorb plastic particles can be utilized to remove plastics from water and soil systems. Microplastics usually cannot be absorbed by plant root systems due to their size, but some reports indicate they might enter plant tissues through stomata. On the other hand, nanoparticles can enter plant root systems, and reports of their transport via xylem to upper plant parts have been recorded. Bioaccumulation of nanoplastics in upper plant parts is still not confirmed. The prospects of using biosystems for the remediation of soils contaminated with plastics are still unknown. However, algae could be used to degrade plastic particles in water systems through enzyme facilitated degradation processes. Considering the amount of plastic pollution, especially in the oceans, further research is necessary on the utilization of algae in plastic degradation. Special attention should be given to the research concerning utilization of algae with restricted algal growth, ensuring that a different problem is not induced, "sea blooming", during the degradation of plastics.
Asunto(s)
Plásticos , Contaminantes Químicos del Agua , Contaminación Ambiental , Microplásticos/toxicidad , Suelo , Agua , Contaminantes Químicos del Agua/análisisRESUMEN
Increasingly, national space agencies are expanding their goals to include Mars exploration with sample return. To better protect Earth and its biosphere from potential extraterrestrial sources of contamination, as set forth in the Outer Space Treaty of 1967, international efforts to develop planetary protection measures strive to understand the danger of cross-contamination processes in Mars sample return missions. We aim to better understand the impact of the martian surface on microbial dormancy and survivability. Radiation resistance of microbes is a key parameter in considering survivability of microbes over geologic times on the frigid, arid surface of Mars that is bombarded by solar and galactic cosmic radiation. We tested the influence of desiccation and freezing on the ionizing radiation survival of six model microorganisms: vegetative cells of two bacteria (Deinococcus radiodurans, Escherichia coli) and a strain of budding yeast (Saccharomyces cerevisiae); and vegetative cells and endospores of three Bacillus bacteria (B. subtilis, B. megaterium, B. thuringiensis). Desiccation and freezing greatly increased radiation survival of vegetative polyploid microorganisms when applied separately, and when combined, desiccation and freezing increased radiation survival even more so. Thus, the radiation survival threshold of polyploid D. radiodurans cells can be extended from the already high value of 25 kGy in liquid culture to an astonishing 140 kGy when the cells are both desiccated and frozen. However, such synergistic radioprotective effects of desiccation and freezing were not observed in monogenomic or digenomic Bacillus cells and endospores, which are generally sterilized by 12 kGy. This difference is associated with a critical requirement for survivability under radiation, that is, repair of genome damage caused by radiation. Deinococcus radiodurans and S. cerevisiae accumulate similarly high levels of the Mn antioxidants that are required for extreme radiation resistance, as do endospores, though they greatly exceed spores in radioresistance because they contain multiple identical genome copies, which in D. radiodurans are joined by persistent Holliday junctions. We estimate ionizing radiation survival limits of polyploid DNA-based life-forms to be hundreds of millions of years of background radiation while buried in the martian subsurface. Our findings imply that forward contamination of Mars will essentially be permanent, and backward contamination is a possibility if life ever existed on Mars.
Asunto(s)
Medio Ambiente Extraterrestre , Marte , Humanos , Desecación , Congelación , Saccharomyces cerevisiae , Esporas Bacterianas/efectos de la radiación , Radiación Ionizante , PoliploidíaRESUMEN
BACKGROUND: Little is known about the global distribution and environmental drivers of key microbial functional traits such as antibiotic resistance genes (ARGs). Soils are one of Earth's largest reservoirs of ARGs, which are integral for soil microbial competition, and have potential implications for plant and human health. Yet, their diversity and global patterns remain poorly described. Here, we analyzed 285 ARGs in soils from 1012 sites across all continents and created the first global atlas with the distributions of topsoil ARGs. RESULTS: We show that ARGs peaked in high latitude cold and boreal forests. Climatic seasonality and mobile genetic elements, associated with the transmission of antibiotic resistance, were also key drivers of their global distribution. Dominant ARGs were mainly related to multidrug resistance genes and efflux pump machineries. We further pinpointed the global hotspots of the diversity and proportions of soil ARGs. CONCLUSIONS: Together, our work provides the foundation for a better understanding of the ecology and global distribution of the environmental soil antibiotic resistome. Video Abstract.
Asunto(s)
Antibacterianos , Suelo , Humanos , Antibacterianos/farmacología , Ecología , FenotipoRESUMEN
Species of the genus Tuber have gained a lot of attention in recent decades due to their aromatic hypogenous fruitbodies, which can bring high prices on the market. The tendency in truffle production is to infect oak, hazel, beech, etc. in greenhouse conditions. We aimed to show whether silver fir (Abies alba Mill.) can be an appropriate host partner for commercial mycorrhization with truffles, and how earthworms in the inoculation substrate would affect the mycorrhization dynamics. Silver fir seedlings inoculated with Tuber. aestivum were analyzed for root system parameters and mycorrhization, how earthworms affect the bare root system, and if mycorrhization parameters change when earthworms are added to the inoculation substrate. Seedlings were analyzed 6 and 12 months after spore inoculation. Mycorrhization with or without earthworms revealed contrasting effects on fine root biomass and morphology of silver fir seedlings. Only a few of the assessed fine root parameters showed statistically significant response, namely higher fine root biomass and fine root tip density in inoculated seedlings without earthworms 6 months after inoculation, lower fine root tip density when earthworms were added, the specific root tip density increased in inoculated seedlings without earthworms 12 months after inoculation, and general negative effect of earthworm on branching density. Silver fir was confirmed as a suitable host partner for commercial mycorrhization with truffles, with 6% and 35% mycorrhization 6 months after inoculation and between 36% and 55% mycorrhization 12 months after inoculation. The effect of earthworms on mycorrhization of silver fir with Tuber aestivum was positive only after 6 months of mycorrhization, while this effect disappeared and turned insignificantly negative after 12 months due to the secondary effect of grazing on ectomycorrhizal root tips.