Plasmids as markers of intraspecific variability in Diaporthe helianthi

The stem canker caused by Diaporthe helianthi is one of the most serious diseases of sunflower in Europe, where the pathogen induces economical losses in several countries. The incidence levels of that disease are quite different in relation to the geographic origin of fungal populations: a severe epidemiology is reported in former Yugoslavia and in France, whereas in Italy it is recorded only sporadically although climatic conditions are favourable for infection. Therefore the genetic variability of D. helianthi isolates from different geographic origins (France, ex-Yugoslavia, Italy, Argentina and Romania) was investigated by several research groups, in order to disclose different genetic biotypes. The detection of plasmids in most isolates allowed the analysis of their distribution and intraspecific variability in the whole fungal collection. Their presence was limited to all French and Yugoslavian isolates and only to one Italian. They were identified as linear plasmids sized about 2.3 Kb. More detailed analysis was then performed on one plasmid (plasmid F) purified from a French isolate. Its intracellular location was proved to be mitochondrial. Plasmid F was then used as a probe in Southern hybridisation experiments on total DNAs derived from all D. helianthi isolates and it revealed a specificity towards all plasmids hosted by French and Yugoslavian isolates while no recognition was evident in Italian isolates, suggesting a molecular divergence of the Italian plasmid previously detected. An RFLP hybridisation analysis performed on genomic DNAs revealed a homogeneous restriction pattern in all French and Yugoslavian isolates, strongly supporting the hypothesis of a molecular relationship among plasmids present in those isolates. In conclusion plasmids harboured by French and Yugoslavian D. helianthi populations can be exploited as markers for isolates from those geographic regions where the disease is more dangerous. No information is yet available about the role of these plasmids upon pathogen virulence; anyway their presence should be investigated to choose isolates for selection of resistant sunflower varieties