FISH technique to detect T. magnatum colonization in plant roots

Simone Graziosi ¹, Mélanie Januario ², Lara Deloche ², Mirco Iotti ³, Marc-André Selosse ², Claude Murat ⁴, Aurélie Deveau ⁴, Philippe Rech ², Alessandra Zambonelli ¹

Department of Agricultural and Food Sciences, University of Bologna, Viale G. Fanin 44, 40127, Bologna, Italy ¹, Institut de Systématique, Évolution, Biodiversité (ISYEB – UMR 7205 – CNRS, MNHN, SU, EPHE), Muséum national d’Histoire naturelle, 57 rue Cuvier, 75005 Paris, France ², Department of Life, Health and Environmental Science, University of L'Aquila, Via Vetoio, 67100, Coppito, L'Aquila, Italy ³, Université de Lorraine, INRAe, IAM, Nancy, France ⁴

Fluorescence in situ hybridization (FISH) is a powerful technique for visualizing target DNA sequences or messenger RNA (mRNA) transcripts in cultured cells, tissue sections, or whole-mount preparations. The high specificity of the probes used in FISH makes it an invaluable tool for examining the taxonomic distribution of organisms within microbiomes at small spatial scales. This method has also been employed to detect bacterial and fungal endophytism in plants. Recently, FISH was utilized to confirm the endophytic behavior of the ectomycorrhizal ascomycetes Tuber aestivum (Wulfen) Spreng. and Tuber melanosporum Vittad. (black truffle) in herbaceous plants (Schneider-Maunoury et al., 2020). Tuber magnatum Picco, one of the most prized truffles globally, forms ectomycorrhizal associations with specific broadleaf trees. However, its mycorrhizae are rarely observed in natural environments, even in productive sites, and are typically generated under controlled conditions (Hall et al., 2007). In this study, we investigated the suitability of FISH combined with Confocal Laser Scanning Microscopy (CLSM) to confirm Tuber magnatum colonization in typical ectomycorrhizal host plants and herbaceous plants. Specific 18S rRNA probes labeled with fluorescent chromophores were designed and tested alongside a generic fungal eukaryotic probe on mycelium and ectomycorrhizae of T. magnatum and closely related fungal species. The FISH-CLSM technique was then applied to herbaceous plants growing at T. magnatum fruiting sites, where the presence of T. magnatum in the soil was also confirmed using PCR with T. magnatum-specific primers. The roots of 100 host plants were collected and surface sterilized, followed by DNA amplification using T. magnatum-specific primers. Tuber magnatum was detected in 12 wild plant species and in 21 plant samples. FISH detected Tuber magnatum exclusively in the roots of Carex pendula Huds. Since the FISH technique highlights only viable cells, the discrepancy between the PCR and FISH results may stem from the short-term viability of the symbiotic hyphae, compared to mycelium in pure culture. This study highlights the potential of the FISH technique to enhance our understanding of plant-fungus interactions in natural environments. 1) I. Hall, B. Gordon, A. Zambonelli (2007) 1st ed., Timber Press: Portland, OR, USA, 2007, p. 304 2) L. Schneider-Maunoury, A. Deveau, M. Moreno, F. Todesco, S. Belmondo, C. Murat, P.E. Courty, M. Jakalski, M.A. Selosse (2020) New Phytol., 225, 2542–2556

Main author career stage: PhD student

Contribution type: Talk

First choice session: 5. Genetics, genomics, and bioinformatics

Second choice session: 6. Plants, Fungi and Society