Snabl et al. Italian Journal of Mycology 48:16-25 (2019) DOI: https://doi.org/10.6092/issn.2531-7342/9374 Original paper Morels on the sand dunes of the Emilia-Romagna coast (Northwestern Adriatic Sea, Italy) Martin Snabl1, Urbano Guidori1, Carmelo Gianchino2, Mirco Iotti2, Alessandra Zambonelli3 1Italian Mycological Society (UMI), viale Fanin 44, 40127 Bologna, Italy 2Department of Health, Life and Environmental Sciences, University of L’Aquila, via Vetoio, Coppito 1, 67100 L’Aquila, Italy 3Department of Agricultural and Food Sciences, University of Bologna, via Fanin 44, 40127 Bologna, Italy Corresponding author e-mail: [email protected] Received 4/02/2019; accepted 18/04/2019. Abstract Morchella species are known as famous and prized edible fungi due to their culinary flavor and medicinal properties. The asomata are collected throughout the temperate regions of the northern hemisphere. Morchella spp. taxonomy has long been debated as a result of the high phenotypic plasticity charaterizing the genus. Most morels are considered saprobic but some species has been reported to interact with roots of many plant species forming different types of associations. In Emilia-Romagna (Italy), morels became a part of the culinary tradition, especially for the populations of the coastal areas. This work aimed to describe and identify the ascomata collected on the white dune habitat as well as to verify the extent of the interaction with plant species growing in the same area. All ascomata collected since 2001 shared a similar morphology with a range of variability mainly due to the harvesting period. Ascomata collected in 2017 were grouped within the Mes-17 clade, in the Esculenta group, based on their ITS rDNA sequences. Nine different plant species were found to be connected with morel ascomata through characteristic sand-mycelial structures never described before. The amount of the ascomata collected on the sand dunes demonstrates that the study area is particularly suitable for morel fruiting. Therefore, the protection of the dune system is imperative for preserving Morchella genetic diversity within the local population. Key words: Morchella vulgaris; Morchella esculenta; Morchella dunensis; white dunes; host plant Introduction True morels (Morchella, Ascomycetes) are among the most valuable edible mushrooms, which also have medicinal properties (Ajmal et al., 2015). Most of the morels in the market are harvested from the wild but recent attempts to cultivate these mushrooms were successful (Du et al., 2017). Before this success, other cultivation strategies were developed around the world but the low reproducibility and the high costs prevented large-scale cultivation (Peng et al., 2015). Species in the genus Morchella are collected throughout the temperate regions of the northern hemisphere where they produce ascomata during a few weeks’ time period in the springtime (Du et al., 2015). Taxonomy of morels has long been debated among mycologists due to the high phenotypic plasticity of their ascomata, which often do not reflect their phylogeny (Kellner et al., 2005). Moreover, these fungi exhibit a variety of trophic habits as well as a broad range of growth habitats. Most morels are considered saprobic because they can complete their life-cycle without host plants, but a number of species have been reported to interact with roots of many plant species forming different types of associations (Pilz et al., 2007). Morels can be found in many forest types, meadows, burned sites, coastal dunes and a broad range of disturbed soils and man-made environments (Pilz et al., 2007). In Italy, morels are locally appreciated but have not yet gained a significant market share among edible mushrooms. Most of the morels commercialized in Italy come from Hungary, Turkey, ________________________________________________________________________________________________________________________ ISSN 2531-7342 - Department of Agricultural and Food Sciences in partnership with the Italian Mycological Society (UMI) The journal is hosted and maintained by ABIS-AlmaDL © 2019 The Authors. This work is licensed under CC BY 4.0 Snabl et al. Italian Journal of Mycology 48 (2019) Romania and Bulgaria and their wholesale price is 20-28 € per kg whereas the retail price is 30-40 € per kg (Luigi Dattilo, Appennino Food Group SpA, personal communication). Unlike other high- priced edible mushrooms such as truffles (Tuber spp.) and porcini (Boletus edulis s.l.), the ecology and biology of morels in Italy have been poorly investigated. These mushrooms are particularly sought-after in the littoral area of the Emilia-Romagna region where their ascomata are collected either from the coast or from the inland pine and hardwood forests. In this work, we aimed i) to barcode the morels collected on the sand dunes of the Emilia-Romagna coast and ii) to investigate the type and extent of their interactions with the local plants. Materials and Methods The area under investigation consists of a series of sand dunes extended along the coastline from Punta Marina and Marina di Ravenna (Ravenna, northeastern Italy). The collection sites are approximately 100 m from the sea at an elevation of 2 to 5 m asl, within the EU habitat 2120 [Echinophoro spinosae-Ammophiletum australis (Br.-Bl. 1933) Géhu, Rivas-Martinez & R. Tx. 1972 (Géhu et al., 1984)]. In this area, the dune system is extremely fragmented and threatened by touristic facilities (Sytnik and Stecchi, 2015). The dune vegetation of the study area is dominated by Calamagrostis arenaria (L.) Roth [=Ammophila arenaria (L.) Link subsp. australis (Mabille) Laínz], Echinophora spinosa L., Eryngium maritimum L., Euphorbia paralias L., Medicago marina L., Cyperus capitatus Vand., Pancratium maritimum L., Stachys maritima Gouan and Spartina juncea auct., non (Michx.) Willd., often associated to allochthonous species such as Cenchrus incertus M.A.Curtis (=Cenchrus spinifex Cav.), Ambrosia psilostachya DC (=Ambrosia cronopifolia Torr. & A. Gray), Yucca gloriosa L. and Oenothera stucchii Soldano (Merloni et al., 2015). A coastal Pinus spp. forest extends in the inland area behind the dune system. Ascoma production was assessed every week during the fruiting season (from mid-March to mid- April) from 2001 to 2017. Voucher specimens were deposited in the private herbarium of the first to authors. The range of interactions between morels and host plant species was investigated by following the mycelial structures connecting the base of the stipe and the host roots. A trench was dug in proximity of each ascoma by using a trowel and the mycelial connecting structure was carefully unearthed with a paint brush. A total of 68 trenches were opened throughout the study period, most of them on the sand dunes but some also in the immediate surrounding anthropized areas (Table 1). Table 1 - List of the surveys in which sand-mycelial structures were unearthed. Host plant and number of ascomata collected in the same day of excavation are reported Survey date Locality Number of Host plant trenches 8 April 2001 Marina di Ravenna 3 Cyperus capitatus 1 Hypochaeris radicata 14 April 2001 Punta Marina 2 Yucca gloriosa 8 April 2004 Punta Marina 2 Reseda alba 7 April 2006 Punta Marina 6 Yucca gloriosa 8 Tamarix gallica 4 Elaeagnus angustifolia 30 March 2008 Punta Marina 1 Elaeagnus angustifolia 3 April 2008 Punta Marina 2 Pinus pinaster 5 April 2008 Punta Marina 5 Yucca gloriosa 11 April 2008 Punta Marina 4 Yucca gloriosa 3 Hypochaeris radicata 19 April 2010 Punta Marina 3 Yucca gloriosa 2 Sedum sp. 10 April 2015 Punta Marina 7 Hypochaeris radicata 2 Cyperus capitatus 1 April 2016 Punta Marina 4 Ambrosia cronopifolia 6 Hypochaeris radicata 24 March 2017 Marina di Ravenna 3 Cyperus capitatus 17 Snabl et al. Italian Journal of Mycology 48 (2019) Macro and micro-morphology of the specimens were assessed through the analysis of fresh ascomata. Micro-morphological analyses were carried out under a Laborlux K microscope (Leitz) on sections of the hymenia and the sterile ridges mounted either in water or Congo red. Measurements were made with an ocular micrometer at × 400 magnification for asci, paraphyses and elements of sterile ridges and × 1000 magnification for spores. One hundred measures were taken for each taxonomic character in two out of five ascomata collected in 2017. Five ascomata collected on March 2017 were barcoded by sequencing the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA). Few sterile hyphae were used directly as target material for DNA amplification through polymerase chain reaction (PCR), bypassing the need for DNA isolation (Iotti and Zambonelli, 2006). ITS region was amplified in a 50-μl volume reaction using the primer pair ITS1F-ITS4 (White et al., 1990; Gardes and Bruns, 1993) and a SimpliAmp thermal cycler (ThermoFisher). PCR reactions contained BioMix mastermix (Bioline), 400 nM for each primer and 30 μg of bovine serum albumin. Thermal cycler conditions were the following: 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with an initial denaturation at 94 °C for 8 min and a final extension at 72 °C for 10 min. Amplicons were purified with the NucleoSpin Extract II kit (Macherey-Nagel) and sequencing was performed with ITS5 and ITS4 as forward and reverse primers at GATC Biotech AG (Constance, Germany). ITS sequences were aligned and edited manually with BioEdit 7.0.5.3 (Hall, 1999) and consensus sequences were compared to those deposited in GenBank (www.ncbi.nlm.nih.gov) and Morchella MLST (http://www.cbs.knaw.nl/morchella/) databases. Genotyped specimens were deposited in Herbarium Mycologicum Aquilanum (AQUI9839 to AQUI9841) and their ITS sequences submitted to GenBank (accession numbers MK388505 to MK388509). The phylogenetic analysis of the genotyped ascomata was inferred by using the maximum likelihood (ML) estimation implemented in Raxml (version 8.2; Stamatakis, 2014) and MrBayes. Sequences generated in this study were analyzed together with other ITS sequences from GenBank. Alignment was performed by CLUSTAL W (Thompson et al., 1994) and gaps and ambiguous sites were excluded prior to phylogenetic analyses.