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.

Pecan, Carya illinoinensis, is an economically important nut producing tree that can establish ectomycorrhizal symbiosis with a high diversity of fungi. In the southern USA, truffles (Tuber spp.) sometimes fruit prolifically in cultivated pecan orchards and regularly associate with pecan roots as ectomycorrhizae (ECMs). It has been demonstrated that some valuable European truffle species (e.g., Tuber borchii and Tuber aestivum) can form ECMs with pecan seedlings in nursery conditions. Thus, pecan may represent an attractive alternative host to forest trees for truffle growers given the potential for co-cropping truffles and pecans. To further explore the capacity of pecan to host truffle symbionts, pecan seedlings were inoculated with species of black truffles that are economically important in Europe, T. melanosporum and T. brumale. Ectomycorrhizae were characterized molecularly and their morphology was described in detail. Mycorrhization rates on pecan roots were assessed over a 2-year period. Tuber melanosporum and T. brumale produced well-formed ECMs with a level of root colonization in the first year of 37.3 and 34.5%, respectively. After 24 months, the level of mycorrhization increased for T. brumale (49.4%) and decreased for T. melanosporum (10.5%) inversely to that of non-target ECM greenhouse contaminants (e.g., Sphaerosporella brunnea, Trichophaea woolhopeia, Pulvinula constellatio). To assess whether mating types segregated in T. melanosporum as been reported for other host species, we amplified the mating-type locus from single T. melanosporum ECM belonging to different seedlings over a 2-year period. The two mating idiomorphs were nearly equally represented along the 2-year time span: MAT 1-1-1 decreased from 59.4% in the first year to 48.5% in the second year after inoculation. Data reported in this study add to knowledge on the mycorrhization of pecan trees with commercial truffles and has application to truffle and nut co-cropping systems.

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The black truffle (Tuber melanosporum Vittad.) is an important natural resource due to its relevance as a delicacy in gastronomy. Different aspects of this hypogeous fungus species have been studied, including population genetics of French and Italian distribution ranges. Although those studies include some Spanish populations, this is the first time that the genetic diversity and genetic structure of the wide geographical range of the natural Spanish populations have been analysed. To achieve this goal, 23 natural populations were sampled across the Spanish geographical distribution. ISSR technique demonstrated its reliability and capability to detect high levels of polymorphism in the species. Studied populations showed high levels of genetic diversity (h N  = 0.393, h S  = 0.678, Hs = 0.418), indicating a non threatened genetic conservation status. These high levels may be a consequence of the wide distribution range of the species, of its spore dispersion by animals, and by its evolutionary history. AMOVA analysis showed a high degree of genetic structure among populations (47.89%) and other partitions as geographical ranges. Bayesian genetic structure analyses differentiated two main Spanish groups separated by the Iberian Mountain System, and showed the genetic uniqueness of some populations. Our results suggest the survival of some of these populations during the last glaciation, the Spanish southern distribution range perhaps surviving as had occurred in France and Italy, but it is also likely that specific northern areas may have acted as a refugia for the later dispersion to other calcareous areas in the Iberian Peninsula and probably France.

The quality of seedlings colonized by Tuber melanosporum is one of the main factors that contributes to the success or failure of a truffle crop. Truffle cultivation has quickly grown in European countries and elsewhere, so a commonly shared seedling evaluation method is needed. Five evaluation methods are currently published in the literature: three are used in Spain and two in France and Italy. Although all estimate the percentage colonization by T. melanosporum mycorrhizae, they do it in different ways. Two methods also estimate total number of mycorrhizae per seedling. Most are destructive. In this work, ten batches of holm oak seedlings inoculated with T. melanosporum from two different nurseries were evaluated by means of the five methods noted above. Some similarity was detected between the percentages of T. melanosporum mycorrhizae estimated by each method but not in their ability to assess the suitability of each batch. We discuss the advantages and disadvantages for each method and suggest approaches to reach consensus within the truffle culture industry for certifying mycorrhizal colonization by T. melanosporum and seedling quality.

Tuber melanosporum cultivation, fruitbody traits are gaining relevance due to their increasing prominence on prices. We investigated the edaphic and temporal patterns of fruitbody traits and characterised the effect of truffle nests (localised peat-based amendment supplemented with T. melanosporum spores) on traits. We monitored fruitbody traits throughout two fruiting seasons in three blocks along a soil gradient. Each trait followed specific edaphic and temporal patterns. The number of fruitbodies per dig and spore maturity followed characteristic within-season trends, whereas fruitbody weight and infestation by truffle beetles were subject to complex interactions among edaphic and temporal variables, suggesting a relevant influence of annual environmental conditions. The application of truffle nests increased fruitbody depth, improved its shape and decreased infestation by truffle beetles. Nests increased the number of fruitbodies per dig, but only in two of the soils, suggesting a relevant role of the bulk soil/substrate interface in fruiting initiation. These results outline a complex scenario, with each trait being differently affected by environmental factors. In this scenario, nests proved to effectively modify several traits, although not always in the desired direction. The European black truffle (Tuber melanosporum) is an ectomycorrhizal fungus highly appreciated in "haute cuisine" due to its organoleptic properties. It grows wild in open oak forests of southern Europe, although nowadays most T. melanosporum production is harvested in orchards planted with seedlings previously inoculated in controlled conditions. Truffle cultivation has advanced greatly in recent decades and has expanded worldwide 1. This is leading to an increased and more stable supply of truffles to markets in recent years, thus encouraging price differentiation by quality 2. Commercial quality standards are defined by both pre-harvest fruitbody (FB) traits and post-harvest practices 3. The most important among the former are FB ripeness, gleba colour (associated to spore maturity), abiotic and biotic damages, fresh weight and shape 4. Despite recent advances, truffle cultivation is not completely domesticated yet, as the uncertainties around the mating process and the FB formation process remain 5,6. The environmental mechanisms triggering fruiting and influencing FB development and maturation are still poorly understood 6,7. Edaphic and climatic factors have a key role in ectomycorrhizal FB yields, phenology and morphology 8-10. This is particularly likely to apply to truffles, which grow below ground and require several months to develop and ripen 11,12. Information on the influence of environmental factors on FB traits could help to clarify the ecology of T. melanosporum and could be useful for improving productivity and sustainability in truffle cultivation. Unraveling the linkages between FB traits and edaphoclimatic conditions is difficult because: (1) the long period of formation of truffle FBs hinders the identification of ...

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The European black truffle is a mycorrhizal fungus native to Spanish Mediterranean forests. In most Spanish regions it was originally commercially harvested in the second half of the 20th century. Experts agree that wild truffle yields suffered a sharp decline during the 1970s and 1980s. However, official statistics for Spanish harvest are scarce and seemingly conflicting, and little attention has been paid to the regime for the exploitation of truffle-producing forests and its implications on the sustainability of this resource. Trends in harvest from 1969 to 2013 and current harvesting practices were analyzed as a case study, taking into account that Spain is a major truffle producer worldwide, but at the same time truffles have only recently been exploited. The available statistical sources, which include an increasing proportion of cultivated truffles since the mid-1990s, were explored, with estimates from Truffle Harvesters Federation showing higher consistency. Statistical sources were then compared with proxies for wild harvest (rents from truffle leases in public forests) to corroborate time trends in wild harvesting. Results suggest that black truffle production is recovering in recent years thanks to plantations, whereas wild harvest is still declining. The implications of Spanish legal and institutional framework on sustainability of wild truffle use are reviewed. In the current scenario, the decline of wild harvest is likely to continue and eventually make commercial harvesting economically unattractive, thus aggravating sustainability issues. Strengthening of property rights, rationalization of harvesting pressure, forest planning and involvement of public stakeholders are proposed as corrective measures.