Extensive decline and mortality events of alder trees have recently been observed in several riparian ecosystems in Italy. Since there is little information about the aetiology of this disease and given the high ecological relevance of riparian ecosystems, an in-depth study was conducted in three sites spanning from the Mediterranean to Alpine regions. From spring 2019 to spring 2020, 261 samples of bleeding cankers, rhizosphere soil and leaves used as baits along waterways were collected and used for Phytophthora isolation. Based on morphology, colony appearance and DNA sequence data, 10 species belonging to 6 clades were identified. These included P. plurivora (84 isolates), P. pseudocryptogea (50), P. hydropathica (18), P. gonapodyides (14), P. bilorbang (13), P. pseudosyringae (12), P. lacustris (11), P. acerina (7), P. cactorum (1) and one isolate of the hybrid Phytophthora ×serendipita. In addition, two new Phytophthora species, one of which is described here as Phytophthora alpina sp. nov., were isolated. The pathogenicity of P. alpina and other species obtained from samples collected in the green alder stand was assessed on 3-year-old seedlings. All species proved to be pathogenic on green alder causing symptoms congruent with field observations. Results obtained have allowed us to expand knowledge about alder decline aetiology. The diversity of pathogenicity of Phytophthora species associated with symptomatic alder trees suggested that no single agent is responsible for the disease, but that it is the result of multiple infections of different Phytophthora species, variable in assemblages among sites.

A cork oak endophytic strain of Trichoderma citrinoviride, previously selected for its antagonistic potential against various fungal pathogens involved in oak decline, was screened for the production of bioactive secondary metabolites. From liquid culture a mixture of polypeptide antibiotics (peptaibols) belonging to the paracelsin family was isolated and characterized. This peptide mixture was purified by column chromatography and preparative TLC on silica gel, and separated by analytical HPLC. It was analysed by MALDI-TOF MS and nano-ESI-QTOF MS. Tandem mass experiments were performed to determine the amino acid sequences based on the fragmentation pattern of selected parent ions. The mixture comprised 20-residue peptides with C-terminal phenylalaninol and N-terminal acetylation. Twenty-eight amino acid sequences were identified, and amino acid exchanges were located in positions 6, 9, 12 and 17. Among them, seven sequences are new as compared to those reported in the database specifically for peptaibols and in the literature. In addition, we obtained experimental evidence suggesting the existence of non-covalent dimeric forms (homo- and hetero-) of the various peptaibol species. The peptide mixture showed strong antifungal activity toward seven important forest tree pathogens, and it was highly toxic in an Artemia salina (brine shrimp) bioassay. These results emphasize the cryptic role of endophytic fungi as a source of novel bioactive natural products and biocontrol agents.