ORIGINAL ARTICLE Multi-cropping edible truffles and sweet chestnuts: production of high-quality Castanea sativa seedlings inoculated with Tuber aestivum, its ecotype T. uncinatum, T. brumale, and T. macrosporum Amaya Álvarez-Lafuente 1 & Luis F. Benito-Matías 2 & Juan L. Peñuelas-Rubira 1 & Laura M. Suz 3 Received: 29 June 2017 /Accepted: 23 October 2017 # Springer-Verlag GmbH Germany 2017 Abstract The plantation and management of sweet chestnut (Castanea sativa Mill.) orchards is a common and traditional land use system in many areas of Europe that offers the advan- tage of simultaneous production of nuts and timber. During the last decades, sweet chestnut has declined dramatically in many regions because of the profound social changes in rural areas coupled with pathogen attacks. Truffles, the hypogeous asco- carps of the ectomycorrhizal genus Tuber , are currently cultivat- ed using host trees inoculated with these fungi for improving production in truffle orchards. The production of good forestry quality chestnut seedlings inoculated with European truffles in nurseries is essential for multi-cropping plantation establish- ment, but so far, it has not been implemented in agroforestry practices. Moreover, it is necessary to assess the physiological condition of the seedlings due to the high calcium amendment needed for the growth of Tuber spp. mycelium that can become toxic for the host plants. In this study, seedlings of C. sativa were inoculated with Tuber aestivum and its ecotypes T. uncinatum, T. brumale, and T. macrosporum and were grown in a green- house using culture conditions favorable for the production of high-quality plants for forestry purposes. At the end of the assay, levels of root colonization and morphological and physiological parameters of the seedlings were measured. The colonization of C. sativa with T. aestivum, its ecotype T. uncinatum, and T. brumale was successful, and the seedlings showed normal growth. Inoculation protocols with T. macrosporum need to be improved. Tuber species formed well-developed ectomycorrhizae on C. sativa in nursery conditions. Keywords Castanea sativa . Tuber aestivum . Tuber uncinatum . Tuber brumale . Tuber macrosporum . Forestry quality seedlings . Multi-cropping Introduction The ectomycorrhizal (ECM) genus Tuber P. Micheli ex. F.H. Wigg. (Pezizales, Ascomycota) includes species that associate with ecologically important tree and shrub species in Mediterranean ecosystems (Benucci et al. 2012b). Truffles, the belowground fruiting bodies of Tuber spp., are edible and harvested for commercial purposes due to their exception- al organoleptic characteristics (Díaz et al. 2003; Pacioni et al. 2014). In Spain, mainly two Tuber species are collected in the wild (T. melanosporum Vittad. and T. aestivum Vittad.) due to the ease of their marketability; however, T. brumale Vittad., another edible species of economic interest, can also be found. Truffles grow in a wide range of habitats associated with a broad range of host species like oak, willow, poplar, hazel, and some shrubs like rockroses (Napoli et al. 2010). They all re- quire calcareous soils with pH 7–8 (Mello et al. 2006). The market price of truffles (€200–€3000 per kg) varies depending on the species and their geographic origin, and it is negatively correlated with the abundance of seasonal production. The Piedmont truffle (Tuber magnatum Pico) is the most prized, followed by T. melanosporum (Benucci et al. 2012c). * Amaya Álvarez-Lafuente dbvaal@gmail.com 1 TRAGSA CNRGF BEl Serranillo,^ Ministerio de Agricultura y Pesca, Alimentación y Medio Ambiente, 19080 Guadalajara, Spain 2 Departamento de Ingeniería y Gestión Forestal, ETSIMontes, 28040 Madrid, Spain 3 Comparative Plant and Fungal Biology, Royal Botanic Gardens, Kew, Richmond TW9 3DS, England Mycorrhiza https://doi.org/10.1007/s00572-017-0805-9