Habitat is more important than climate for structuring soil fungal communities associated in truffle sites.

The ectomycorrhizal fungi Tuber melanosporum Vittad. and Tuber aestivum Vittad. produce highly valuable truffles, but little is known about the soil fungal communities associated with these truffle species in places where they co-occur. Here, we compared soil fungal communities present in wild and planted truffle sites, in which T. melanosporum and T. aestivum coexist, in Mediterranean and temperate regions over three sampling seasons spanning from 2018 to 2019. We showed that soil fungal community composition and ectomycorrhizal species composition are driven by habitat type rather than climate regions. Also, we observed the influence of soil pH, organic matter content and C:N ratio structuring total and ectomycorrhizal fungal assemblages. Soil fungal communities in wild sites revealed more compositional variability than those of plantations. Greater soil fungal diversity was found in temperate compared to Mediterranean sites when considering all fungal guilds. Ectomycorrhizal diversity was significantly higher in wild sites compared to plantations. Greater mould abundance at wild sites than those on plantation was observed while tree species and seasonal effects were not significant predictors in fungal community structure. Our results suggested a strong influence of both ecosystem age and management on the fungal taxa composition in truffle habitats.

The fall of the summer truffle: Recurring hot, dry summers result in declining fruitbody production of Tuber aestivum in Central Europe.

Global warming is pushing populations outside their range of physiological tolerance. According to the environmental envelope framework, the most vulnerable populations occur near the climatic edge of their species' distributions. In contrast, populations from the climatic center of the species range should be relatively buffered against climate warming. We tested this latter prediction using a combination of linear mixed effects and machine learning algorithms on an extensive, citizen-scientist generated dataset on the fruitbody productivity of the Burgundy (aka summer) truffle (Tuber aestivum Vittad.), a keystone, ectomycorrhizal tree-symbiont occurring on a wide range of temperate climates. T. aestivum's fruitbody productivity was monitored at 3-week resolution over up to 8 continuous years at 20 sites distributed in the climatic center of its European distribution in southwest Germany and Switzerland. We found that T. aestivum fruitbody production is more sensitive to summer drought than would be expected from the breadth of its species' climatic niche. The monitored populations occurring nearly 5°C colder than the edge of their species' climatic distribution. However, interannual fruitbody productivity (truffle mass year-1 ) fell by a median loss of 22% for every 1°C increase in summer temperature over a site's 30-year mean. Among the most productive monitored populations, the temperature sensitivity was even higher, with single summer temperature anomalies of 3°C sufficient to stop fruitbody production altogether. Interannual truffle productivity was also related to the phenology of host trees, with ~22 g less truffle mass for each 1-day reduction in the length of the tree growing season. Increasing summer drought extremes are therefore likely to reduce fruiting among summer truffle populations throughout Central Europe. Our results suggest that European T. aestivum may be a mosaic of vulnerable populations, sensitive to climate-driven declines at lower thresholds than implied by its species distribution model.

New Insights into the Complex Relationship between Weight and Maturity of Burgundy Truffles (Tuber aestivum).

Despite an increasing demand for Burgundy truffles (Tuber aestivum), gaps remain in our understanding of the fungus' overall lifecycle and ecology. Here, we compile evidence from three independent surveys in Hungary and Switzerland. First, we measured the weight and maturity of 2,656 T. aestivum fruit bodies from a three-day harvest in August 2014 in a highly productive orchard in Hungary. All specimens ranging between 2 and 755 g were almost evenly distributed through five maturation classes. Then, we measured the weight and maturity of another 4,795 T. aestivum fruit bodies harvested on four occasions between June and October 2015 in the same truffière. Again, different maturation stages occurred at varying fruit body size and during the entire fruiting season. Finally, the predominantly unrelated weight and maturity of 81 T. aestivum fruit bodies from four fruiting seasons between 2010 and 2013 in Switzerland confirmed the Hungarian results. The spatiotemporal coexistence of 7,532 small-ripe and large-unripe T. aestivum, which accumulate to ~182 kg, differs from species-specific associations between the size and ripeness that have been reported for other mushrooms. Although size-independent truffle maturation stages may possibly relate to the perpetual belowground environment, the role of mycelial connectivity, soil property, microclimatology, as well as other abiotic factors and a combination thereof, is still unclear. Despite its massive sample size and proof of concept, this study, together with existing literature, suggests consideration of a wider ecological and biogeographical range, as well as the complex symbiotic fungus-host interaction, to further illuminate the hidden development of belowground truffle fruit bodies.

Soil physical properties influence "black truffle" fructification in plantations.

Although the important effects of pH and carbonate content of soils on "black truffle" (Tuber melanosporum) production are well known, we poorly understand the influence of soil physical properties. This study focuses on physical soil characteristics that drive successful production of black truffles in plantations. Seventy-eight Quercus ilex ssp. ballota plantations older than 10 years were studied in the province of Teruel (eastern Spain). Soil samples were analyzed for various edaphic characteristics and to locate T. melanosporum ectomycorrhizae. The influence of cultivation practices, climatic features, and soil properties on sporocarp production was assessed using multivariate analyses. Low contents of fine earth and silt and high levels of bulk density, clay content, and water-holding capacity appear to promote fructification. Watering is also highly positive for truffle fructification. We develop and discuss a logistic model to predict the probability of truffle fructification in field sites under consideration for truffle plantation establishment. The balance between water availability and aeration plays a crucial role in achieving success in black truffle plantations.

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.

Edible sporocarp production by age class in a Scots pine stand in Northern Spain.

With the aim of increasing knowledge of community structure, dynamics and production of ectomycorrhizal fungi, edible sporocarp yields were monitored between 1995 and 2004 in a Pinus sylvestris stand in the northeast zone of the Iberian Peninsula. A random sampling design was performed by stand age class according to the forest management plan: 0-15, 16-30, 31-50, 51-70 and over 71-years-old. Eighteen 150 m plots were established and sampled weekly every year from September to December. One hundred and nineteen taxa belonging to 51 genera were collected, 40 of which were edible and represented 74% of the total biomass. Boletus edulis, Lactarius deliciosus, Cantharellus cibarius and Tricholoma portentosum sporocarps, which are considered to be of high commercial value, represented 34% of the total production. B. edulis and L. deliciosus were the most remarkable and abundant species, and both were collected in more than 60% of the samplings. B. edulis fructified every year of the experiment; its mean production was 40 kg/ha and year and its maximum productivity was more than 94 kg/ha in 1998. The age class with the largest production of this taxa was the fourth (51-70 years), with 70 kg/ha. L. deliciosus only failed to fructify one autumn (2000); its mean production was almost 10 kg/ha and its maximum productivity close to 30 kg/ha in 1997. The maximum productivity of this species was found in the second (16-30 years) and fifth (71-90 years) stand age classes, with 18 and 16 kg/ha, respectively. Advances in this field can certainly offer new insights into factors affecting sporocarp production.

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.

Tree age influences on the development of edible ectomycorrhizal fungi sporocarps in Pinus sylvestris stands.

The study of factors influencing the production and development of wild edible mushroom sporocarps is extremely important in the characterization of the fungi life cycle. The main objective of this work is to determine how tree age influences the speed of sporocarp growth of edible ectomycorrhizal fungi Boletus edulis and Lactarius deliciosus in a Pinus sylvestris stand. This study is based on information recorded on a weekly basis every autumn between 1995 and 2008 in a set of permanent plots in Spain. Sporocarps are collected weekly, and as a result, specimens may not have reached their maximum size. The study area is a monospecific P. sylvestris stand. Three age classes were considered: under 30 years, between 31 and 70 years, and over 70 years. Sporocarps of B. edulis and L. deliciosus grow faster in the first age class stands than in the other two, and in the second age class stands, sporocarps are more than 50% smaller. The average weight of the picked B. edulis sporocarps clearly varies in the three age classes considered, with its maximum in the first age class (127 g and 6.8 cm cap diameter), minimum in the second age class (68 g and 4.7 cm cap diameter), and showing a relative maximum in the third (79 and 4.3 cm cap diameter). L. deliciosus sporocarps are on average larger in the first age class (48 g and 7.4 cm cap diameter), decreasing in the second (20 g and 5.8 cm cap diameter) and also in the third (21 g and 5.3 cm cap diameter). The results show the influence of tree age in speed of sporocarp growth for the two ectomycorrhizal species.

Mycorrhizal synthesis between Boletus edulis species complex and rockroses (Cistus sp.).

Ectomycorrhizas of Boletus aereus, Boletus edulis, and Boletus reticulatus were synthesized with Cistus sp. under laboratory conditions using synthesis tubes filled with a mixture of sterilized peat-vermiculite and nutrient solution. The fungal strains isolated from sporocarps were identified by molecular techniques. The inoculated seedlings were grown for 4-5 months. The ectomycorrhizas formed were described based on standard morphological and anatomical characters. The three ectomycorrhizas described were very similar, with white monopodial-pinnate morphology, a three-layered plectenchymatous mantle on plan view and boletoid rhizomorphs.

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.