Bioactive secondary metabolites produced by the emerging pathogen Diplodia olivarum

A new cleistanthane nor-diterpenoid, named olicleistanone (1), was isolated as a racemate from the culture filtrates of Diplodia olivarum an emerging pathogen involved in the aetiology of branch canker and dieback of in several plant species typical of the Mediterranean maquis in Sardinia, Italy. When the fungus was grown in vitro on Czapek medium olicleistanone was isolated together with some already known phytotoxic diterpenoids identified as sphaeropsidins A, C, and G and diplopimarane (2-5). Olicleistanone was characterized by spectroscopic methods (essentially 1D and 2D NMR and HR ESIMS) as 4-ethoxy-6a-methoxy-3,8,8-trimethyl-4,5,8,9,10,11-hexahydrodibenzo[de,g]chromen-7(6aH)-one. When the fungus was grown on mineral salt medium showed to produce (-)-mellein (6), sphaeropsidin A and a very low amount of sphaeropsidin G and diplopimarane. Olicleistanone (1) exhibited remarkable activity against Artemia salina L. (100% larval mortality) at 100 µg/mL. In addition, it did not exhibit phytotoxic, antifungal and antioomycetes activity. Among the metabolites isolated (1-6), the sphaeropsindin A (2) proved to be active in all bioassay performed exhibiting remarkable phytotoxicity on Phaseolus vulgaris L., Juglans regia L. and Quercus suber L. leaves at 1 mg/mL. Moreover, it completely inhibited the mycelial growth of Athelia rolfsii, Diplodia corticola, Phytophthora cambivora and P. lacustris at 200 µg/plug. It was also active in the Artemia salina assay. In this latter assay, diplopimarane (4) and sphaeropsidin G (4) were active (100% larval mortality). Diplopimarane also showed antifungal and antioomycetes activities. Athelia rolfsii was the most sensitive species to diplopimarane. Sphaeropsidin C (3) and (-)-mellein (6) were found to be inactive in all bioassays. Results obtained in this study have allowed us to expand the knowledge on the metabolic profile of Botryosphaeriaceae members and characterize the main secondary metabolites secreted in vitro by D. olivarum for the first time.