Pepino mosaic virus and Tomato chlorosis virus causing mixed infection in protected tomato crops in Sicily

An unusual virus-like yellow leaf disorder associated to marbling on fruits, was observed during winter 2005 in some greenhouse tomato crops in Sicily (Southern Italy), in Ragusa province. Two leaves from 250 symptomatic tomato plants were serologically tested by DAS-ELISA technique using the sera to Tomato spotted wilt virus, Impatiens necrotic spot virus, Tobacco mosaic virus, Cucumber mosaic virus and Pepino mosaic virus (PepMV). Of the 250 samples tested, 215 were positive to PepMV presence. This virus was mechanically transmitted to Cucumis sativus “Cubit”, Datura metel, Nicotiana benthamiana and tomato “Rio Grande”. The experimental host range of PepMV-Ragusa differed from that of PepMV found in 2001 infecting tomato “Camone” in Sardinia. By applying RT-PCR technique to 25 PepMV-infected tomato plants, the expected 844 bp DNA fragment for PepMV and the expected 439 pb DNA fragment for Tomato chlororis virus (ToCV) were obtained from all the samples tested. These sequences were used to study phylogenetic relationships of the two viruses identified with isolates from other countries. To compare the PepMV-Ragusa isolate, 14 nucleotide sequences were retrieved from the GenBank entries: nucleotide sequences alignments of CP-PepMV-Ragusa showed homology of 99% with both isolates US2 and Spain-Murcia. To compare the ToCV-Ragusa isolate, 12 sequences were retrieved from GenBank entries: nucleotide sequences alignments of HSP70 of ToCV showed homology of 93% with isolate Florida and of 94% with isolate Cyprus. These results provide evidence that the unusual disorder found in protected tomato cultivated in Sicily can be associated to PeMV and ToMV, found for the first time in a mixed infection