First production of Italian white truffle (Tuber magnatum Pico) ascocarps in an orchard outside its natural range distribution in France.

Truffles are ectomycorrhizal species forming edible ascocarps. The Italian white truffle (Tuber magnatum Pico) is the most famous and expensive species harvested to date; it comes exclusively from natural habitats in European countries. The annual production of T. magnatum is generally insufficient to respond to the high demands making its cultivation a research hotspot. The first attempt to cultivate T. magnatum started in the 1970s without success; only recently have mycorrhized plants been successfully produced. The aims of this study were (1) to assess the persistence of T. magnatum in the soil of plantations realized with mycorrhized plants and (2) to characterize the first T. magnatum orchard that produced ascocarps outside the known natural geographic range of this species. In 2018, five orchards were sampled in France, and T. magnatum was investigated in the soil. We confirmed that T. magnatum survived in the soil 3 to 8 years after planting. The key finding of this study was the harvest of T. magnatum ascocarps in 2019 and 2020 from one orchard. The production of ascocarps started 4.5 years after planting, and the ascocarps were harvested under different trees and during two consecutive seasons. A detailed analysis of the productive orchards (i.e., soil features, soil water availability, cultivation techniques) is presented. These results demonstrate the feasibility of T. magnatum cultivation worldwide by planting mycorrhized plants. The cultivation of T. magnatum could therefore become a real opportunity for farmers and could respond to the high demand of this high-priced food.

Frequency of the two mating types in the soil under productive and non-productive trees in five French orchards of the Périgord black truffle (Tuber melanosporum Vittad.).

The Périgord black truffle (Tuber melanosporum Vittad.) is an ectomycorrhizal fungus forming edible fructifications. The production of T. melanosporum relies mainly on man-made plantations. T. melanosporum is a heterothallic species requiring the meeting of two partners of opposite mating types to fruit. It is common to have productive and non-productive trees in the same orchard. The aim of our study was to assess the distribution of T. melanosporum mating types in soil under productive and non-productive trees to test whether the presence or absence of one or two mating types could be an indicator of productivity. To achieve this aim, five orchards were selected in various French regions. Soils were harvested under productive and non-productive Quercus pubescens; soil characteristics and the distribution of the mating types in the soil were investigated. No significant differences between productive and non-productive soils according to soil parameters were detected. The total content of T. melanosporum DNA in the soil was significantly higher under productive trees compared with non-productive trees, and it was positively correlated only with soil available phosphorous. Under productive trees, it was more frequent to find both mating types than under non-productive trees. Soils with only one mating type were more frequent under non-productive trees than under productive ones. Moreover, no mating type was detected in the soil of 22% of the non-productive trees. These results suggest that the detection of T. melanosporum mating types in soil could be a tool to optimise the management of truffle orchards (e.g. by spore inoculation).

Two ectomycorrhizal truffles, Tuber melanosporum and T. aestivum, endophytically colonise roots of non-ectomycorrhizal plants in natural environments.

Serendipitous findings and studies on Tuber species suggest that some ectomycorrhizal fungi, beyond their complex interaction with ectomycorrhizal hosts, also colonise roots of nonectomycorrhizal plants in a loose way called endophytism. Here, we investigate endophytism of T. melanosporum and T. aestivum. We visualised endophytic T. melanosporum hyphae by fluorescent in situ hybridisation on nonectomycorrhizal plants. For the two Tuber species, microsatellite genotyping investigated the endophytic presence of the individuals whose mating produced nearby ascocarps. We quantified the expression of four T. aestivum genes in roots of endophyted, non-ectomycorrhizal plants. Tuber melanosporum hyphae colonised the apoplast of healthy roots, confirming endophytism. Endophytic Tuber melanosporum and T. aestivum contributed to nearby ascocarps, but only as maternal parents (forming the flesh). Paternal individuals (giving only genes found in meiotic spores of ascocarps) were not detected. Gene expression of T. aestivum in non-ectomycorrhizal plants confirmed a living status. Tuber species, and likely other ectomycorrhizal fungi found in nonectomycorrhizal plant roots in this study, can be root endophytes. This is relevant for the ecology (brûlé formation) and commercial production of truffles. Evolutionarily speaking, endophytism may be an ancestral trait in some ectomycorrhizal fungi that evolved from root endophytes.

Soil temperature and hydric potential influences the monthly variations of soil Tuber aestivum DNA in a highly productive orchard.

Tuber aestivum, also known as the summer or Burgundy truffle, is an ectomycorrhizal Ascomycete associated with numerous trees and shrubs. Its life cycle occurs in the soil, and thus soil parameters such as temperature and water availability could influence it. T. aestivum cultivation has started in several countries, but ecological and agronomic requirements for the establishment and management of orchards are largely unknown. The aims of this work were: 1) to design a specific qPCR protocol using genomic data to trace and quantify T. aestivum DNA in the soil; and 2) to assess the monthly soil DNA dynamic according to soil parameters (i.e. soil hydric potential and temperature) in this orchard. The study was conducted in a highly productive T. aestivum orchard (hazels, oaks, pines, lime and hornbeam). The production started five years after the plantation and then increased exponentially to reach a maximum of 320 kg/ha in 2017. The soil hydric potential and temperature partially explained the monthly T. aestivum soil DNA variability. The data presented here offer new insights into T. aestivum ecology and cultivation.

New insights into black truffle biology: discovery of the potential connecting structure between a Tuber aestivum ascocarp and its host root.

According to isotopic labeling experiments, most of the carbon used by truffle (Tuber sp.) fruiting bodies to develop underground is provided by host trees, suggesting that trees and truffles are physically connected. However, such physical link between trees and truffle fruiting bodies has never been observed. We discovered fruiting bodies of Tuber aestivum adhering to the walls of a belowground quarry and we took advantage of this unique situation to analyze the physical structure that supported these fruiting bodies in the open air. Observation of transversal sections of the attachment structure indicated that it was organized in ducts made of gleba-like tissue and connected to a network of hyphae traveling across soil particles. Only one mating type was detected by PCR in the gleba and in the attachment structure, suggesting that these two organs are from maternal origin, leaving open the question of the location of the opposite paternal mating type.

Five years investigation of female and male genotypes in périgord black truffle (Tuber melanosporum Vittad.) revealed contrasted reproduction strategies.

The Périgord black truffle (Tuber melanosporum Vittad.) is a heterothallic ascomycete that establishes ectomycorrhizal symbiosis with trees and shrubs. Small-scale genetic structures of female genotypes in truffle orchards are known, but it has not yet been studied in male genotypes. In this study, our aim was to characterize the small-scale genetic structure of both male and female genotypes over five years in an orchard to better understand the T. melanosporum sexual reproduction strategy, male genotype dynamics, and origins. Two-hundred forty-one ascocarps, 475 ectomycorrhizas, and 20 soil cores were harvested and genotyped using microsatellites and mating type genes. Isolation by distance analysis revealed pronounced small-scale genetic structures for both female and male genotypes. The genotypic diversity was higher for male than female genotypes with numerous small size genotypes suggesting an important turnover due to ascospore recruitment. Larger and perennial female and male genotypes were also detected. Only three genotypes (1.5%) were found as both female and male genotypes (hermaphrodites) while most were detected only as female or male genotype (dioecy). Our results suggest that germinating ascospores act as male genotypes, but we also proposed that soil mycelium could be a reservoir of male genotypes.