Integrazione di caratteri molecolari, micro-morfologici e chemiotassonomici per la caratterizzazione di Cladosporium tenuissimum, iperparassita di agenti di ruggine

The results of far-reaching investigations on strains of the rust hyperparasite Cladosporium tenuissimum are presented here. The micromorphology of three mycophylic Cladosporium species obtained from public culture collections was analyzed by means of SEM and compared with that of four C. tenuissimum strains isolated from aecidiospores of Cronartium flaccidum and Peridermium pini in different European areas. Such fungi were also compared for their ability to produce secondary metabolites in vitro. SEM observations revealed strong differences in shape and size among conidia of the investigated species: in particular, in C. herbarum and C. tenuissimum they were baculate, and in C. cladosporioides and C. sphaerospermum they presented a streaked-wrinkled ornamentation; moreover they were smaller and rather roundish in C. sphaerospermum, while in C. herbarum, C. cladosporioides and C.tenuissimum they were oblong. Unlike the other Cladosporium spp., C. tenuissimum strains produced in culture a typical yellow pigmentation, diffusible in the agar medium, constituted by a family of related meatbolites, the cladosporols A-E. Such compounds had antifungal activity as inhibitors of germination of Uromyces appendiculatus urediniospores, and reduced the radial growth of several other phytopathogens. The analysis of informative DNA regions has highlighted the occurrence of sequence variants suitable to distinguish C. tenuissimum from the congeneric species C. sphaerospermum, C. cladosporioides, C. oxysporum and C. herbarum. The hyperparasite displayed with these species a sequence homology of 94.6, 96.3, 94.4 and 95.2% respectively in a 540bp fragment from ITS1-5.8S-ITS2 region of rDNA. The internal 5.8 S gene turned out to have identical lenght (161bp) in all the species except C.herbarum (160bp). The only differences in the gene were a T-C transition at residue 13 in C. sphaerospermum, a G-T transversion at residue 21 in C. cladosporioides, another transversion at residue 81 in C.tenuissimum and a loss of a G residue at position 141 in C.herbarum. Microscopic observations already made at the interface between C.tenuissimum and Cronartium flaccidum or Uromyces appendiculatus confirmed an active hyperparasitism of rust spores by C. tenuissimum. These investigations need further in-depth studies taking into consideration the ecology of the hyperparasite and the favourable environmental conditions which seem to support this active intraction. The analysis of independent distinguishing features, like cultural, morphological and chemical characters, supplemented with biomolecular investigations, enabled us to verify the adequacy of taxonomic positioning of the hyperparasite strains in C. tenuissimum species and constitute a valuable approach to examine in detail the nature of the hyperparasite.