Glyphosate treatments for weed control affect early stages of root colonization by Tuber melanosporum but not secondary colonization.

The cultivation of the ectomycorrhizal fungus Tuber melanosporum has considerably spread in recent years throughout the world. During the first years of truffle cultivation, weed control is a key practice to improve the establishment of host trees and the proliferation of the fungus in the soil. Glyphosate is nowadays the most commonly used herbicide in Spanish truffle orchards. We explored the effect of glyphosate on the proliferation of T. melanosporum mycorrhizae, on extraradical mycelium and on the inoculum potential of T. melanosporum spores in greenhouse experiments using Quercus ilex seedlings as host plants. No detrimental effect on the secondary infection of T. melanosporum was found after three sequential glyphosate applications in young seedlings during one vegetative period. Instead, a change in the distribution of fine roots and T. melanosporum mycorrhizae along soil depth was observed. On the other hand, results indicate that high application rates of glyphosate hinder the infectivity of T. melanosporum spore inoculum, without apparent impact on the host performance. Our results suggest that glyphosate has the potential to jeopardise the role of the soil spore bank as inoculum source for the colonisation of new roots, also raising the question of whether glyphosate could hinder the presumed role of spores in sexual mating.

Soil microclimate changes affect soil fungal communities in a Mediterranean pine forest.

Soil microclimate is a potentially important regulator of the composition of plant-associated fungal communities in climates with significant drought periods. Here, we investigated the spatio-temporal dynamics of soil fungal communities in a Mediterranean Pinus pinaster forest in relation to soil moisture and temperature. Fungal communities in 336 soil samples collected monthly over 1 year from 28 long-term experimental plots were assessed by PacBio sequencing of ITS2 amplicons. Total fungal biomass was estimated by analysing ergosterol. Community changes were analysed in the context of functional traits. Soil fungal biomass was lowest during summer and late winter and highest during autumn, concurrent with a greater relative abundance of mycorrhizal species. Intra-annual spatio-temporal changes in community composition correlated significantly with soil moisture and temperature. Mycorrhizal fungi were less affected by summer drought than free-living fungi. In particular, mycorrhizal species of the short-distance exploration type increased in relative abundance under dry conditions, whereas species of the long-distance exploration type were more abundant under wetter conditions. Our observations demonstrate a potential for compositional and functional shifts in fungal communities in response to changing climatic conditions. Free-living fungi and mycorrhizal species with extensive mycelia may be negatively affected by increasing drought periods in Mediterranean forest ecosystems.

Mushroom Emergence Detected by Combining Spore Trapping with Molecular Techniques.

Obtaining reliable and representative mushroom production data requires time-consuming sampling schemes. In this paper, we assessed a simple methodology to detect mushroom emergence by trapping the fungal spores of the fruiting body community in plots where mushroom production was determined weekly. We compared the performance of filter paper traps with that of funnel traps and combined these spore trapping methods with species-specific quantitative real-time PCR and Illumina MiSeq to determine the spore abundance. Significantly more MiSeq proportional reads were generated for both ectomycorrhizal and saprotrophic fungal species using filter traps than were obtained using funnel traps. The spores of 37 fungal species that produced fruiting bodies in the study plots were identified. Spore community composition changed considerably over time due to the emergence of ephemeral fruiting bodies and rapid spore deposition (lasting from 1 to 2 weeks), which occurred in the absence of rainfall events. For many species, the emergence of epigeous fruiting bodies was followed by a peak in the relative abundance of their airborne spores. There were significant positive relationships between fruiting body yields and spore abundance in time for five of seven fungal species. There was no relationship between fruiting body yields and their spore abundance at plot level, indicating that some of the spores captured in each plot were arriving from the surrounding areas. Differences in fungal detection capacity by spore trapping may indicate different dispersal ability between fungal species. Further research can help to identify the spore rain patterns for most common fungal species.IMPORTANCE Mushroom monitoring represents a serious challenge in economic and logistical terms because sampling approaches demand extensive field work at both the spatial and temporal scales. In addition, the identification of fungal taxa depends on the expertise of experienced fungal taxonomists. Similarly, the study of fungal dispersal has been constrained by technological limitations, especially because the morphological identification of spores is a challenging and time-consuming task. Here, we demonstrate that spores from ectomycorrhizal and saprotrophic fungal species can be identified using simple spore traps together with either MiSeq fungus-specific amplicon sequencing or species-specific quantitative real-time PCR. In addition, the proposed methodology can be used to characterize the airborne fungal community and to detect mushroom emergence in forest ecosystems.

Seasonal dynamics of extraradical mycelium and mycorrhizas in a black truffle (Tuber melanosporum) plantation.

Seasonal dynamics of black truffle (Tuber melanosporum) extraradical mycelium as well as the associated mycorrhizal community have been evaluated in a 16-year-old plantation with productive and non-productive trees. Mycelium biomass was seasonally quantified by real-time PCR over two consecutive years and the correlation with environmental variables explored. Extraradical mycelium biomass varied seasonally and between the two consecutive years, being correlated with the precipitation that occurred 1 month before sampling. In addition, productive trees had more mycelium in the brûlé area than non-productive trees did. The ectomycorrhizal community composition inside the burnt areas was seasonally evaluated during a year. Ten mycorrhizal morphotypes were detected; T. melanosporum was the most abundant in productive and non-productive trees. Black truffle mycorrhizas were more abundant (mycorrhizal tips per unit of soil volume) in productive trees, and no seasonal variation was observed. The occurrence of black truffle mycorrhizas was significantly and positively correlated with the biomass of extraradical mycelium. The mycorrhizal community within the brûlé areas was significantly different between productive and non-productive trees, and no variation was detected between seasons. The assessment of the fungal vegetative structures in a mature plantation is of paramount importance to develop trufficulture methods based on the knowledge of the biological cycle of the fungus and its relationships with the associated ectomycorrhizal communities.

Diversity of ectomycorrhizal Thelephoraceae in Tuber melanosporum-cultivated orchards of Northern Spain.

Truffles are edible hypogeous ascomycetes highly appreciated worldwide, especially the black truffle (Tuber melanosporum Vittad.). In recent decades, the cultivation of the black truffle has expanded across the Mediterranean climate regions in and outside its native range. Members of the Thelephoraceae (Thelephorales, Agaricomycetes, Basidiomycota) are commonly found in truffle plantations, but their co-occurrence with Tuber species and other members of the fungal community has been scarcely reported. Thelephoraceae is one of the most represented families of the ectomycorrhizal fungal community in boreal and Mediterranean forests. To reveal the diversity of these fungi in T. melanosporum-cultivated plantations, ten orchards located in the Navarra region (Northern Spain) were surveyed for 2 years. Morphological and molecular approaches were used to detect and identify the Thelephoraceae ectomycorrhizas present in those plantations. Ten different mycorrhizal types were detected and described. Four of them were morphologically identified as Tomentella galzinii, Quercirhiza cumulosa, Q. squamosa, and T39 Thelephoraceae type. Molecular analyses revealed 4-6 operational taxonomic units (OTUs), depending on the nucleotide database used, but similarities remained under 95 % and no clear species assignments could be done. The results confirm the diversity and abundance of this fungal family in the ectomycorrhizal community of black truffle plantations, generally established in Mediterranean areas. The occurrence and relative abundance of Thelephoraceae ectomycorrhizas is discussed in relation to their possible influence on truffle production.

Soil drying procedure affects the DNA quantification of Lactarius vinosus but does not change the fungal community composition.

Drying soil samples before DNA extraction is commonly used for specific fungal DNA quantification and metabarcoding studies, but the impact of different drying procedures on both the specific fungal DNA quantity and the fungal community composition has not been analyzed. We tested three different drying procedures (freeze-drying, oven-drying, and room temperature) on 12 different soil samples to determine (a) the soil mycelium biomass of the ectomycorrhizal species Lactarius vinosus using qPCR with a specifically designed TaqMan® probe and (b) the fungal community composition and diversity using the PacBio® RS II sequencing platform. Mycelium biomass of L. vinosus was significantly greater in the freeze-dried soil samples than in samples dried at oven and room temperature. However, drying procedures had no effect on fungal community composition or on fungal diversity. In addition, there were no significant differences in the proportions of fungi according to their functional roles (moulds vs. mycorrhizal species) in response to drying procedures. Only six out of 1139 operational taxonomic units (OTUs) had increased their relative proportions after soil drying at room temperature, with five of these OTUs classified as mould or yeast species. However, the magnitude of these changes was small, with an overall increase in relative abundance of these OTUs of approximately 2 %. These results suggest that DNA degradation may occur especially after drying soil samples at room temperature, but affecting equally nearly all fungi and therefore causing no significant differences in diversity and community composition. Despite the minimal effects caused by the drying procedures at the fungal community composition, freeze-drying resulted in higher concentrations of L. vinosus DNA and prevented potential colonization from opportunistic species.

Whose truffle is this? Distribution patterns of ectomycorrhizal fungal diversity in Tuber melanosporum brûlés developed in multi-host Mediterranean plant communities.

In the Mediterranean region, patches of vegetation recovering from disturbance and transiently dominated by shrubs produce one of the world's most prized fungi, the black truffle (Tuber melanosporum). In these successional plant communities, we have fragmentary knowledge of the distribution of T. melanosporum in space among ectomycorrhizal (ECM) host species and in time. Molecular identification of hosts (Restriction Fragment Length Polymorphism) and fungi (Internal Transcribed Spacer sequencing) and quantification of T. melanosporum mycelium (quantitative Polymerase Chain Reaction) were employed to evaluate the presence of T. melanosporum on four dominant ECM host species (Quercus ilex, Quercus  coccifera, Arbutus unedo, Cistus albidus) and the extent to which their respective ECM communities shared fungal diversity, over the course of development of truffle grounds, from recent unproductive brûlés to senescent ones where production has stopped. We found that truffle grounds host rich communities in which multi-host fungal species dominate in frequency. When considering both ECM tips and soil mycelia, we documented a dynamic and spatially heterogeneous pattern of T. melanosporum distribution in soils and a presence of ECM tips restricted to Q. ilex roots. This study advances our knowledge of the ecology of T. melanosporum, and provides insight into the extent of ECM fungal sharing among plant species that dominate Mediterranean landscapes.

Ectomycorrhizal fungus diversity and community structure with natural and cultivated truffle hosts: applying lessons learned to future truffle culture.

Since the first truffle plantations were established in France, Italy and other parts in the world, many studies have been carried out to improve their productivity and sustainability. Success of plantations is clearly related to the mycorrhizal status of the host trees over the years, from inoculated seedlings to truffle-producing trees. The experience gained in monitoring the ectomycorrhizal fungus status in cultivated truffle grounds has allowed us to develop an extensive catalogue of the ectomycorrhizal fungi present in truffle plantations. Herein, we summarize fungal community data from 85 references that represent different truffle studies in natural habitats and plantations. Approximately 25% of the ectomycorrhizae reported in the 85 references are common to most of the studies. In general, more fungal species are detected in productive plantations than in the non-productive ones. Truffle plantations display a diverse ectomycorrhizal fungal community, in which species of the genus Tuber are well represented. Tuber rufum and some members of Boletales are typically restricted to productive truffle plots. On the other hand, Hebeloma, Laccaria and Russula species are mostly associated with unproductive plots. Ectomycorrhizae belonging to Thelephoraceae are frequently found in mature truffle orchards but do not seem to affect sporocarp production. Several biotic and abiotic factors affect the ectomycorrhizal fungus communities associated with truffle orchards. Among them are plantation age, host species and its growth, the surrounding environment (particularly the presence of other ectomycorrhizal hosts), and plantation management. Understanding the ectomycorrhizal fungal communities inhabiting different plantations may give us clues about the dynamics of the targeted truffles and the possibility of identifying mycorrhizal fungal species that are good indicators of successful truffle plantations.

Quantification of extraradical mycelium of Tuber melanosporum in soils from truffle orchards in northern Spain.

Quantification of extraradical mycelium of black truffle (Tuber melanosporum) has been carried out in a natural truffle ground and in seven truffle orchards (around 20 years old) established in Tierra Estella and Valdorba sites, within the natural distribution area of the black truffles in Navarre (northern Spain). Specific primers and a Taqman® probe were designed to perform real-time PCR with DNA extracted from soil samples. Amplification of T. melanosporum DNA was obtained from 131 out of the 160 soil samples. The detection limit of the technique was 1.48 µg mycelium/g of soil. The extraradical mycelium biomass detected in the soil from the natural truffle ground was significantly greater (up to ten times higher) than the mycelium biomass detected in any of the orchards. Soil from productive, nonirrigated orchards in the Tierra Estella site contained significantly more extraradical mycelium than the rest of orchards irrigated, productive of T. brumale, or nonproductive. The comparison of soil mycelium biomass in nonirrigated evergreen oak orchards in both sites showed significantly more mycelium biomass in the Tierra Estella site. This study is the first attempt to quantify extraradical mycelium of T. melanosporum in the soil using Taqman® probes. The obtained quantitative results are of special interest to evaluate the fungal response to cultural treatments and to monitor the dynamics of the extraradical mycelium of T. melanosporum in the soil.

Seasonal dynamics of Boletus edulis and Lactarius deliciosus extraradical mycelium in pine forests of central Spain.

The annual belowground dynamics of extraradical soil mycelium and sporocarp production of two ectomycorrhizal fungi, Boletus edulis and Lactarius deliciosus, have been studied in two different pine forests (Pinar Grande and Pinares Llanos, respectively) in Soria (central Spain). Soil samples (five per plot) were taken monthly (from September 2009 to August 2010 in Pinar Grande and from September 2010 to September 2011 in Pinares Llanos) in eight permanent plots (four for each site). B. edulis and L. deliciosus extraradical soil mycelium was quantified by real-time polymerase chain reaction, with DNA extracted from soil samples, using specific primers and TaqMan® probes. The quantities of B. edulis soil mycelium did not differ significantly between plots, but there was a significant difference over time with a maximum in February (0.1576 mg mycelium/g soil) and a minimum in October (0.0170 mg mycelium/g soil). For L. deliciosus, significant differences were detected between plots and over time. The highest amount of mycelium was found in December (1.84 mg mycelium/g soil) and the minimum in February (0.0332 mg mycelium/g soil). B. edulis mycelium quantities were positively correlated with precipitation of the current month and negatively correlated with the mean temperature of the previous month. Mycelium biomass of L. deliciosus was positively correlated with relative humidity and negatively correlated with mean temperature and radiation. No significant correlation between productivity of the plots with the soil mycelium biomass was observed for any of the two species. No correlations were found between B. edulis sporocarp production and weather parameters. Sporocarp production of L. deliciosus was positively correlated with precipitation and relative humidity and negatively correlated with maximum and minimum temperatures. Both species have similar distribution over time, presenting an annual dynamics characterized by a seasonal variability, with a clear increase on the amounts of biomass during the coldest months of the year. Soil mycelial dynamics of both species are strongly dependent on the weather.

Aromatic Plants and Their Associated Arbuscular Mycorrhizal Fungi Outcompete Tuber melanosporum in Compatibility Assays with Truffle-Oaks.

The high value of black truffle recompenses the slow growth of the fungus when established in the field. Adding a secondary crop, such as medicinal and aromatic plants (MAPs), could further enhance the sustainability of truffle production agro-forest systems. The dual cultures of ectomycorrhizal truffle-oak seedlings and MAPs (lavender, thyme, and sage) previously inoculated and non-inoculated with native arbuscular mycorrhizal fungi (AMF), were established to evaluate plant-fungi relationships. After 12 months in a shadehouse, plants' growth, mycorrhizal colonization, and extraradical soil mycelium (both of Tuber melanosporum and AMF) were measured. Overall, truffle-oaks' growth was negatively affected by the presence of MAPs, especially when inoculated with AMF. In turn, the presence of truffle-oaks barely affected the co-cultured MAPs, and only lavenders showed a significant growth reduction. All AMF-inoculated MAPs showed higher shoot and root biomass than non-inoculated ones. Compared to truffle-oaks growing alone, the presence of co-cultured MAPs, especially when they were AMF-inoculated, significantly decreased both the ectomycorrhizas and soil mycelium of T. melanosporum. These results reveal the strong competition between AMF and T. melanosporum and warn about the need for the protection of intercropping plants and their associated symbiotic fungi to avoid reciprocal counterproductive effects in mixed truffle-oak-AMF-MAP plantations.

Interannual dynamics of Tuber melanosporum and fungal communities in productive black truffle orchards amended with truffle nests.

Truffle growers devote great efforts to improve black truffle productivity, developing agronomic practices such as 'truffle nests' (peat amendments that are supplemented with truffle spore inoculum). It has been hypothesized that improved fruiting associated with nests is linked to stimulation of truffle mycelia previously established in soil or to changes generated in soil fungal community. To assess this, we used real-time PCR to quantify black truffle extraradical mycelium during 2 years after nests installation. We also characterized the fungal community via high-throughput amplicon sequencing of the ITS region of rRNA genes. We found that neither the abundance of truffle mycelium in nests nor in the soil-nest interphase was higher than in the bulk soil, which indicates that nests do not improve mycelial growth. The fungal community in nests showed lower richness and Shannon index and was compositionally different from that of soil, which suggests that nests may act as an open niche for fungal colonization that facilitates truffle fruiting. The ectomycorrhizal fungal community showed lower richness in nests. However, no negative relationships between amount of truffle mycelium and reads of other ectomycorrhizal fungi were found, thus countering the hypothesis that ectomycorrhizal competition plays a role in the nest effect.

Quantification of extraradical soil mycelium and ectomycorrhizas of Boletus edulis in a Scots pine forest with variable sporocarp productivity.

The availability of most edible ectomycorrhizal mushrooms depends on their natural fructification. Sporocarp formation of these fungi is linked to habitat characteristics and climate conditions, but these data alone do not explain all the trends of fungal fruiting and dynamics. It could be hypothesized that the amount of soil mycelia could also be related to the production of carpophores. Soil samples (five cylinders of 250 cm(3) per plot) were taken monthly, from September to November, in five fenced permanent plots (5 × 5 m) in Pinar Grande (Soria, Spain), a Pinus sylvestris stand situated in the north of the Sistema Ibérico mountain range. Plots were chosen to establish a gradient of Boletus edulis productivity from 0 to 38.5 kg/ha year, according to the mean fresh weight of sporocarps collected during the last 10 years. B. edulis ectomycorrhizal root tips were identified in each soil sample according to its morphology and counted. DNA extractions were performed with the PowerSoil(TM) DNA Isolation Kit and quantification of extraradical soil mycelium by real-time polymerase chain reaction using specific primers and a TaqMan® probe. The concentration of soil mycelium of B. edulis (mg mycelium/g soil) did not differ significantly between plots (p = 0.1397), and sampling time (p = 0.7643) within the fructification period. The number of mycorrhizal short roots per soil volume showed significant differences between the plots (p = 0.0050) and the three sampling times (p < 0.0001). No significant correlation between the number of mycorrhizas and the productivity of the plot (kg of B. edulis/ha year) was detected (p = 0.615). A statistically significant positive correlation (p = 0.0481) was detected between the concentration of mycelia of B. edulis in the soil samples and the presence of short roots mycorrhizal with B. edulis in these samples. The productivity of the plots, in terms of sporocarps produced during the last 10 years, was not correlated either with the concentration of soil mycelium or with the presence or abundance of ectomycorrhizas.

Intraspecific variability of Lactarius deliciosus isolates: colonization ability and survival after cold storage.

Intraspecific variability in root colonization, extraradical growth pattern, and survival after cold storage of Lactarius deliciosus isolates was determined in pure culture conditions using Pinus pinaster as a host plant. The ectomycorrhizal ability of L. deliciosus at 30, 45, and 60 days from inoculation was highly variable among isolates and was negatively correlated to the age of the culture (time elapsed from isolation). The formation of rhizomorphs was related to colonization ability, but no relationship was found between colonization and formation of extraradical mycelium. The final colonization achieved at 60 days from inoculation was not related to the tree species under which the sporocarps were collected. However, isolates from sporocarps collected under P. pinaster colonized more rapidly the seedlings than those collected under other pine species. The climatic range of the sporocarps from which the isolates were obtained (maritime vs. continental) was not related to the formation of mycorrhizas at 60 days from inoculation. However, isolates from sporocarps collected from a maritime climate area colonized more rapidly the P. pinaster seedlings than those collected from a continental zone. Tolerance to cold water storage of L. deliciosus was also isolate dependent. Growth revival in agar was obtained from most of the isolates after 28 months of cold storage at 4°C, but only 10 out of 29 isolates showed unaffected growth. The ITS rDNA alignment of all the L. deliciosus isolates showed a low variability with identities over 99%. Most of the variation was detected in the ITS1 region and consisted in single nucleotide changes and/or punctual indel mutations. The number of base differences per sequence from averaging over all sequence pairs was 1.329, which is in the low range when compared with other ectomycorrhizal species. No ITS pattern due to geographical origin of the isolates could be discerned.

Sampling forest soils to describe fungal diversity and composition. Which is the optimal sampling size in mediterranean pure and mixed pine oak forests?

Soil sampling is a critical step affecting perceived fungal diversity, however sampling optimization for high-throughput-DNA sequencing studies have never been tested in Mediterranean forest ecosystems. We identified the minimum number of pooled samples needed to obtain a reliable description of fungal communities in terms of diversity and composition in three different Mediterranean forests (pine, oak, and mixed-pine-oak). Twenty soil samples were randomly selected in each of the three plots per type. Samples obtained in 100 m2 plots were pooled to obtain mixtures of 3, 6, 10, 15, 20 samples, and sequenced using Illumina MiSeq of fungal ITS2 amplicons. Pooling three soil samples in Pinus and Quercus stands provided consistent richness estimations, while at least six samples were needed in mixed-stands. ß-diversity decreased with increasing sample pools in monospecific-stands, while there was no effect of sample pool size on mixed-stands. Soil sample pooling had no effect over species composition. We estimate that three samples would be already optimal to describe fungal richness and composition in Mediterranean pure stands, while at least six samples would be needed in mixed stands.

Grifola frondosa (Maitake) Extract Reduces Fat Accumulation and Improves Health Span in C. elegans through the DAF-16/FOXO and SKN-1/NRF2 Signalling Pathways.

In recent years, food ingredients rich in bioactive compounds have emerged as candidates to prevent excess adiposity and other metabolic complications characteristic of obesity, such as low-grade inflammation and oxidative status. Among them, fungi have gained popularity for their high polysaccharide content and other bioactive components with beneficial activities. Here, we use the C. elegans model to investigate the potential activities of a Grifola frondosa extract (GE), together with the underlying mechanisms of action. Our study revealed that GE represents an important source of polysaccharides and phenolic compounds with in vitro antioxidant activity. Treatment with our GE extract, which was found to be nongenotoxic through a SOS/umu test, significantly reduced the fat content of C. elegans, decreased the production of intracellular ROS and aging-lipofuscin pigment, and increased the lifespan of nematodes. Gene expression and mutant analyses demonstrated that the in vivo anti-obesity and antioxidant activities of GE were mediated through the daf-2/daf-16 and skn-1/nrf-2 signalling pathways, respectively. Taken together, our results suggest that our GE extract could be considered a potential functional ingredient for the prevention of obesity-related disturbances.

Molecular identification of the edible ectomycorrhizal fungus Lactarius deliciosus in the symbiotic and extraradical mycelium stages.

Specific rDNA ITS amplifications, microsatellite-primed PCR and ITS-SSCP analysis were applied to identify and characterize pre-selected isolates of the edible ectomycorrhizal fungus Lactarius deliciosus in different stages of the life cycle. Sampling was performed from pure cultures, mycorrhizas and soil from experimental plots established with nursery-inoculated pine seedlings. A newly-designed reverse primer (LDITS2R) combined with the universal forward ITS1 allowed to perform specific amplifications of L. deliciosus from all the samples. Microsatellite-primed PCR using the (GTG)5 oligonucleotide as a primer showed clear polymorphisms among the different L. deliciosus isolates. The patterns of mycorrhiza samples showed additional bands corresponding to the plant DNA. Single strand conformation polymorphism (SSCP) analysis of the specific rDNA ITS fragment amplified from 18 L. deliciosus isolates showed nine clearly different patterns. Mycorrhiza and soil samples showed coincident patterns with their respective fungal isolates. Specific rDNA ITS amplifications had not been previously used for SSCP analysis of ectomycorrhizas and extraradical mycelium. This relatively simple and inexpensive technique allows tracking L. deliciosus isolates in different stages of the fungus development. Specific ITS-SSCP analysis is promising in studies of the persistence of inoculated L. deliciosus isolates and their competitiveness with native ectomycorrhizal fungi, especially at the extraradical mycelium stage.

Characterization and identification of field ectomycorrhizae of Boletus edulis and Cistus ladanifer.

Field ectomycorrhizae sampled under Boletus edulis and Cistus ladanifer have been characterized and described in detail based on standard morphological and anatomical characters. The described ectomycorrhiza has traits typical of Boletales: whitish with three differentiated plectenchymatous layers in the mantle in plan view forming ring-like structures and rhizomorphs with highly differentiated hyphae. The inflated, smooth cystidia-like clavate end cells on the surface of the rhizomorphs and their slightly twisted external hyphae are additional characterizing features. The Hartig net occupies 1 1/2 rows of cortical cells, partly reaching the endodermis. Not all hyphae have clamps. The identification of the fungal symbiont as B. edulis was confirmed by ITS rDNA sequence comparison between mycorrhizas and sporocarps. The singularity of this symbiotic association, as well as its ecological and practical implications, are discussed.

Field persistence of the edible ectomycorrhizal fungus Lactarius deliciosus: effects of inoculation strain, initial colonization level, and site characteristics.

Pinus pinea plants were inoculated with different strains of the edible ectomycorrhizal fungus Lactarius deliciosus. The inoculated plants were established in six experimental plantations in two sites located in the Mediterranean area to determine the effect of the initial colonization level and the inoculated strain on fungal persistence in the field. Ectomycorrhizal root colonization was determined at transplantation time and monitored at different times from uprooted plants. Extraradical soil mycelium biomass was determined from soil samples by TaqMan(R) real-time polymerase chain reaction (PCR). The results obtained indicate that the field site played a decisive role in the persistence of L. deliciosus after outplanting. The initial colonization level and the selection of the suitable strain were also significant factors but their effect on the persistence and spread of L. deliciosus was conditioned by the physical-chemical and biotic characteristics of the plantation soil and, possibly, by their influence in root growth. Molecular techniques based on real-time PCR allowed a precise quantification of extraradical mycelium of L. deliciosus in the field. The technique is promising for non-destructive assessment of fungal persistence since soil mycelium may be a good indicator of root colonization. However, the accuracy of the technique will ultimately depend on the development of appropriate soil sampling methods because of the high variability observed.

The influence of the ectomycorrhizal fungus Rhizopogon subareolatus on growth and nutrient element localisation in two varieties of Douglas fir (Pseudotsuga menziesii var. menziesii and var. glauca) in response to manganese stress.

Acidification of forest ecosystems leads to increased plant availability of the micronutrient manganese (Mn), which is toxic when taken up in excess. To investigate whether ectomycorrhizas protect against excessive Mn by improving plant growth and nutrition or by retention of excess Mn in the hyphal mantle, seedlings of two populations of Douglas fir (Pseudotsuga menziesii), two varieties, one being menziesii (DFM) and the other being glauca (DFG), were inoculated with the ectomycorrhizal fungus Rhizopogon subareolatus in sand cultures. Five months after inoculation, half of the inoculated and non-inoculated seedlings were exposed to excess Mn in the nutrient solution for further 5 months. At the end of this period, plant productivity, nutrient concentrations, Mn uptake and subcellular compartmentalisation were evaluated. Non-inoculated, non-stressed DFM plants produced about 2.5 times more biomass than similarly treated DFG. Excess Mn in the nutrient solution led to high accumulation of Mn in needles and roots but only to marginal loss in biomass. Colonisation with R. subareolatus slightly suppressed DFM growth but strongly reduced that of DFG (-50%) despite positive effects of mycorrhizas on plant phosphorus nutrition. Growth reductions of inoculated Douglas fir seedlings were unexpected since the degree of mycorrhization was not high, i.e. ca. 30% in DFM and 8% in DFG. Accumulation of high Mn was not prevented in inoculated seedlings. The hyphal mantle of mycorrhizal root tips accumulated divalent cations such as Ca, but not Mn, thus not providing a barrier against excessive Mn uptake into the plants associated with R. subareolatus.