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.

Silvicultural management and altitude prevail on soil properties and fungal community in shaping understorey plant communities in a Mediterranean pine forest.

Understorey vegetation plays a key role in Mediterranean forest ecosystem functioning. However, we still lack a thorough understanding of the patterns and drivers of understorey composition and diversity. As a result, understoreys are often ignored during assessments of forest functioning under climate change. Here we studied the effect of silvicultural management, topography, soil fungal community composition and soil physical and chemical properties on understorey community composition and diversity. The plant cover and number of individuals of understorey perennial plants, shrubs and non-dominant trees was recorded on 24 plots (paired: control-thinned) in a Mediterranean pine-dominated mountainous area in Northeast Spain. The study area represented a broad thinning intensity gradient (from 0 to 70 % in removed stand basal area) along a 400-m altitudinal range (from 609 m to 1013 m). Our results showed that thinning intensity and topography explained the greatest proportion of the total variance in the understorey species composition, i.e., 18 % and 16 %, respectively. Interestingly, the effects of the silvicultural treatments were significant only when considering the altitudinal effect, so that, the main impacts of thinning on the understorey community composition occurred at low altitudes (between 609 m and 870 m). Moreover, we found a significant decrease in both richness and abundance of understorey species in both the control and thinned plots with increasing altitude, with thinned plots being significantly richer in species compared to the control plots. The difference in the understorey community sensitivity to forest thinning along the altitudinal gradient suggests changes in factors that limit plant growth. Low elevation plots were restrained by light availability while high altitudes plots limited by winter freezing temperature.

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.

Termite sensitivity to temperature affects global wood decay rates.

Deadwood is a large global carbon store with its store size partially determined by biotic decay. Microbial wood decay rates are known to respond to changing temperature and precipitation. Termites are also important decomposers in the tropics but are less well studied. An understanding of their climate sensitivities is needed to estimate climate change effects on wood carbon pools. Using data from 133 sites spanning six continents, we found that termite wood discovery and consumption were highly sensitive to temperature (with decay increasing >6.8 times per 10°C increase in temperature)-even more so than microbes. Termite decay effects were greatest in tropical seasonal forests, tropical savannas, and subtropical deserts. With tropicalization (i.e., warming shifts to tropical climates), termite wood decay will likely increase as termites access more of Earth's surface.

Lack of Phylogenetic Differences in Ectomycorrhizal Fungi among Distinct Mediterranean Pine Forest Habitats.

Understanding whether the occurrences of ectomycorrhizal species in a given tree host are phylogenetically determined can help in assessing different conservational needs for each fungal species. In this study, we characterized ectomycorrhizal phylogenetic composition and phylogenetic structure in 42 plots with five different Mediterranean pine forests: i.e., pure forests dominated by P. nigra, P. halepensis, and P. sylvestris, and mixed forests of P. nigra-P. halepensis and P. nigra-P. sylvestris, and tested whether the phylogenetic structure of ectomycorrhizal communities differs among these. We found that ectomycorrhizal communities were not different among pine tree hosts neither in phylogenetic composition nor in structure and phylogenetic diversity. Moreover, we detected a weak abiotic filtering effect (4%), with pH being the only significant variable influencing the phylogenetic ectomycorrhizal community, while the phylogenetic structure was slightly influenced by the shared effect of stand structure, soil, and geographic distance. However, the phylogenetic community similarity increased at lower pH values, supporting that fewer, closely related species were found at lower pH values. Also, no phylogenetic signal was detected among exploration types, although short and contact were the most abundant types in these forest ecosystems. Our results demonstrate that pH but not tree host, acts as a strong abiotic filter on ectomycorrhizal phylogenetic communities in Mediterranean pine forests at a local scale. Finally, our study shed light on dominant ectomycorrhizal foraging strategies in drought-prone ecosystems such as Mediterranean forests.

Climate change induced declines in fuel moisture may turn currently fire-free Pyrenean mountain forests into fire-prone ecosystems.

Fuel moisture limits the availability of fuel to wildfires in many forest areas worldwide, but the effects of climate change on moisture constraints remain largely unknown. Here we addressed how climate affects fuel moisture in pine stands from Catalonia, NE Spain, and the potential effects of increasing climate aridity on burned area in the Pyrenees, a mesic mountainous area where fire is currently rare. We first quantified variation in fuel moisture in six sites distributed across an altitudinal gradient where the long-term mean annual temperature and precipitation vary by 6-15 °C and 395-933 mm, respectively. We observed significant spatial variation in live (78-162%) and dead (10-15%) fuel moisture across sites. The pattern of variation was negatively linked (r = |0.6|-|0.9|) to increases in vapor pressure deficit (VPD) and in the Aridity Index. Using seasonal fire records over 2006-2020, we observed that summer burned area in the Mediterranean forests of Northeast Spain and Southern France was strongly dependent on VPD (r = 0.93), the major driver (and predictor) of dead fuel moisture content (DFMC) at our sites. Based on the difference between VPD thresholds associated with large wildfire seasons in the Mediterranean (3.6 kPa) and the maximum VPD observed in surrounding Pyrenean mountains (3.1 kPa), we quantified the "safety margin" for Pyrenean forests (difference between actual VPD and that associated with large wildfires) at 0.5 kPa. The effects of live fuel moisture content (LFMC) on burned area were not significant under current conditions, a situation that may change with projected increases in climate aridity. Overall, our results indicate that DFMC in currently fire-free areas in Europe, like the Pyrenees, with vast amounts of fuel in many forest stands, may reach critical dryness thresholds beyond the safety margin and experience large wildfires after only mild increases in VPD, although LFMC can modulate the response.

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.

White mulch and irrigation increase black truffle soil mycelium when competing with summer truffle in young truffle orchards.

The black truffle (Tuber melanosporum Vittad.) and the summer truffle (Tuber aestivum Vittad.) are two of the most appreciated edible fungi worldwide. The natural distributions of both species partially overlap. However, the interspecific interactions between these truffles and how irrigation and mulching techniques impact the dynamics between them are still unknown. Here, an experimental truffle plantation with Quercus ilex was established in Maials (Catalonia, Spain), combining three soil mulch treatments (white mulch, black mulch and bare soil as a control) and two irrigation regimes (irrigated and non-irrigated as a control) to investigate truffle mycelial dynamics in soil when both truffle species co-occur. The development of truffle mycelium in two different seasons (spring and autumn) in two consecutive years (2017 and 2018) was quantified using qPCR. Truffle mycelia of both species showed greatest development under white mulch. When mycelia of both truffle species co-occurred in soil, irrigation combined with white mulch resulted in greater quantities of T. melanosporum mycelial biomass, whereas the control irrigation treatment favoured the development of T. aestivum. Mulch treatments were also advantageous for seedling growth, which was expressed as root collar diameter and its increment during the study period. Significant relationships between root collar diameter and root growth and the amount of mycelial biomass in the soil were observed for both truffle species. Our results indicate the potential advantages of using white mulch to support irrigation in truffle plantations located in areas with dry Mediterranean climatic conditions to promote the development of Tuber mycelium.

Production and turnover of mycorrhizal soil mycelium relate to variation in drought conditions in Mediterranean Pinus pinaster, Pinus sylvestris and Quercus ilex forests.

In forests, ectomycorrhizal mycelium is pivotal for driving soil carbon and nutrient cycles, but how ectomycorrhizal mycelial dynamics vary in ecosystems with drought periods is unknown. We quantified the production and turnover of mycorrhizal mycelium in Mediterranean Pinus pinaster, Pinus sylvestris and Quercus ilex forests and related the estimates to standardised precipitation index (SPI), to study how mycelial dynamics relates to tree species and drought-moisture conditions. Production and turnover of mycelium was estimated between July and February, by quantifying the fungal biomass (ergosterol) in ingrowth mesh bags and using statistical modelling. SPI for time scales of 1-3 months was calculated from precipitation records and precipitation data over the study period. Forests dominated by Pinus trees displayed higher biomass but were seasonally more variable, as opposed to Q. ilex forests where the mycelial biomass remained lower and stable over the season. Production and turnover, respectively, varied between 1.4-5.9 kg ha-1  d-1 and 7.2-9.9 times yr-1 over the different forest types and were positively correlated with 2-month and 3-month SPI over the study period. Our results demonstrated that mycorrhizal mycelial biomass varied with season and tree species and we speculate that production and turnover are related to physiology and plant host performance during drought.

Insect defoliation is linked to a decrease in soil ectomycorrhizal biomass and shifts in needle endophytic communities.

Insect outbreaks of increasing frequency and severity in forests are predicted due to climate change. Insect herbivory is known to promote physiological changes in forest trees. However, little is known about whether these plant phenotypic adjustments have cascading effects on tree microbial symbionts such as fungi in roots and foliage. We studied the impact of defoliation by the pine processionary moth in two infested Pinus nigra forests through a multilevel sampling of defoliated and non-defoliated trees. We measured tree growth, nutritional status and carbon allocation to chemical defenses. Simultaneously, we analysed the putative impact of defoliation on the needle endophytes and on the soil fungal communities. Higher concentrations of chemical defenses were found in defoliated trees, likely as a response to defoliation; however, no differences in non-structural carbohydrate reserves were found. In parallel to the reductions in tree growth and changes in chemical defenses, we observed shifts in the composition of needle endophytic and soil fungal communities in defoliated trees. Defoliated trees consistently corresponded with a lower biomass of ectomycorrhizal fungi in both sites, and a higher alpha diversity and greater relative abundance of belowground saprotrophs and pathogens. However, ectomycorrhizal alpha diversity was similar between non-defoliated and defoliated trees. Specific needle endophytes in old needles were strongly associated with non-defoliated trees. The potential role of these endophytic fungi in pine resistance should be further investigated. Our study suggests that lower biomass of ectomycorrhizal fungi in defoliated trees might slow down tree recovery since fungal shifts might affect tree-mycorrhizal feedbacks and can potentially influence carbon and nitrogen cycling in forest soils.

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.

Mushroom biomass and diversity are driven by different spatio-temporal scales along Mediterranean elevation gradients.

Mushrooms are important non-wood-forest-products in many Mediterranean ecosystems, being highly vulnerable to climate change. However, the ecological scales of variation of mushroom productivity and diversity, and climate dependence has been usually overlooked due to a lack of available data. We determined the spatio-temporal variability of epigeous sporocarps and the climatic factors driving their fruiting to plan future sustainable management of wild mushrooms production. We collected fruiting bodies in Pinus sylvestris stands along an elevation gradient for 8 consecutive years. Overall, sporocarp biomass was mainly dependent on inter-annual variations, whereas richness was more spatial-scale dependent. Elevation was not significant, but there were clear elevational differences in biomass and richness patterns between ectomycorrhizal and saprotrophic guilds. The main driver of variation was late-summer-early-autumn precipitation. Thus, different scale processes (inter-annual vs. spatial-scale) drive sporocarp biomass and diversity patterns; temporal effects for biomass and ectomycorrhizal fungi vs. spatial scale for diversity and saprotrophic fungi. The significant role of precipitation across fungal guilds and spatio-temporal scales indicates that it is a limiting resource controlling sporocarp production and diversity in Mediterranean regions. The high spatial and temporal variability of mushrooms emphasize the need for long-term datasets of multiple spatial points to effectively characterize fungal fruiting patterns.

Structural social capital and local-level forest governance: Do they inter-relate? A mushroom permit case in Catalonia.

In diffuse forest uses, like non-timber forest products' harvesting, the behavioural alignment of pickers is crucial for avoiding a "tragedy of the commons". Moreover, the introduction of policy tools such as a harvest permit system may help in keeping the activity under control. Besides the official enforcement, pickers' engagement may also derive from the perceived legitimate decision of forest managers and the community pressure to behave according to the shared values. Framed within the social capital theory, this paper examines three types of relations of rural communities in a protected area in Catalonia (Spain) where a system of mushroom picking permits was recently introduced. Through social network analysis, we explore structural changes in relations within the policy network across the policy conception, design and implementation phases. We then test whether social links of the pickers' community relate to influential members of the policy network. Lastly, we assess whether pickers' bonding and bridging structures affect the rate of permit uptake. Our results show that the high degree of acceptance could be explained by an adequate consideration of pickers' preferences within the decision-making group: local pickers show proximity to members of the policy network with medium-high influence during the three policy phases. The policy network also evolves, with some members emerging as key actors during certain phases. Significant differences are found in pickers' relations among and across the involved municipalities following an urban-rural gradient. A preliminary relation is found between social structures and differential pickers' engagement. These results illustrate a case of positive social capital backing policy design and, probably, also implementation. This calls for a meticulous design of forest policy networks with respect to communities of affected forest users.

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.

Time and dose of irrigation impact Tuber melanosporum ectomycorrhiza proliferation and growth of Quercus ilex seedling hosts in young black truffle orchards.

In Mediterranean climate, young truffle-oak orchards are subjected to drought episodes that can compromise the development of Tuber melanosporum. We investigated the responses of T. melanosporum to water supply in three periods: May to July, August to October, and May to October. In each period, five water doses were established: 0, 25, 50, 75, and 100% of the reference evapotranspiration (ETo). Five orchards were planted with Quercus ilex inoculated with T. melanosporum, and in each orchard, we arranged a two-factorial design with irrigation period and irrigation dose as main factors to test their combined effects on the development of both T. melanosporum and Q. ilex after 3 years in the field. Irrigation period significantly interacted with irrigation doses for the absolute presence per seedling of T. melanosporum mycorrhizae. Irrigation in May-July increased significantly T. melanosporum colonization in seedlings irrigated with 50% ETo dose compared to the 0% ETo dose. A similar pattern with smaller differences in means was observed in August-October period, but the irrigation doses did not change T. melanosporum colonization when we watered from May to October. We found ectomycorrhizae different from T. melanosporum in 51% of the seedlings studied, but their presence was marginal. Our results suggest that a moderate irrigation dose promotes seedling growth and number of fine root tips per unit of fine root length, which may be potentially colonized by T. melanosporum.