Phenotypic characterization of cryptic Diplodia species by MALDI-TOF MS and the bias of mycelium age

Detection and identification of fungal cryptic species has been facilitated by DNA sequencing. However, the examination of some phenotypic traits is fundamental for the confirmation of genetic results. MALDI-TOF mass spectrometry has shown remarkable reliability in the recognition of species-specific phenotypic markers and has already been tested in several species of fungi. However, even though there is no direct evidence, the age of the mycelium appears to influence the composition of the metabolites detected. In this study, we demonstrate that MALDI-TOF is a reliable technique to identify suitable metabolites to distinguish three monophyletic species of Diplodia (D. pinea, D. seriata and D. scrobiculata), thus supporting recent DNA results. Nevertheless, different collections of samples over a period of 3 months after inoculation also revealed that the MALDI-TOF spectra are highly dependent on mycelial age. More importantly, the species-specific markers emerge only after 1 month of mycelial growth. The methodological and biological implications of these findings are discussed.