Phytopathogenic fungi threaten the Quercus genus in a coastal forest: detection of fungal diversity by Nanopore Sequencing

Molecular characterization of potential fungal pathogens affecting Quercus spp. is the challenge to limit the expansion of the oak decline. The investigation was made on acorns and on Quercus seedlings grown in a forest nursery. The analysis aims to determine pathogens that can potentially infect seedlings and eventually cause plant death. Fungal detection by molecular tools was performed with two approaches: 1) by culture-dependent fungal profiling followed by Sanger sequencing and 2) by culture-independent fungal profiling using Oxford Nanopore Technology (ONT). In both cases, we sequenced the ITS region of ribosomal DNA. Both methods have proven effective, however, culture-independent methodology proved more advantageous; in fact, using culture independent approach, we were able to identify a larger variety of microorganism putatively involved in the oak decline syndrome. Using culture-dependent approach, several saprophytic fungal species were isolated in vitro and subsequently, characterized by ITS sequencing (Sanger). Nanopore ITS sequencing identified a broader variety of fungal species, with few associated to the oak decline such as Diplodia corticola and Discula quercina. Other fungal species identified like Epicoccum nigrum, Alternaria alternata, Alternaria sp., Lophiostoma corticola and Pleosporales sp. could play an important ecological role as they can change their lifestyles from endophytic to pathogenic, or saprobic, depending on the environmental conditions. Our research provides a phytosanitary protocol to rapidly diagnostic the presence of oak pathogens. Diagnosticated microbial genomes may be used to correlate the potential fungal infections to abiotic stresses.