First report of Maize rough dwarf virus (MRDV) on maize crops in Greece

In summer 2002, maize (Zea mays L.) crops grown in northern Greece (Macedonia), showed severe dwarfing, reduced corncob size and in some cases leaf reddening. These symptoms were different from those caused by Maize dwarf mosaic virus, which is endemic in maize crops in Macedonia. The dwarfing disease was only epidemic in 2002 and in some maize growing regions (Imathia and Serres), where crop losses were estimated to be over 70%. In contrast, in 2003 only a few cases were recorded. The symptoms were similar to those caused by two closely related members of the genus Fijivirus, Maize rough dwarf virus (MRDV) and Rice black-streaked dwarf virus (RBSDV) (Azuhata et al., 1993). The putative virus could not be transmitted mechanically (sap applied by rubbing the leaves) from maize-to-maize or to other indicator plants.

An RT-PCR was developed and optimised for the detection of both MRDV and RBSDV. Two primers ('MRDV-F1': 5'-AGCGGAGAACGTTtggatc-3' and 'MRDV-R2': 5'-ttaacaacagcagcttcacc-3') were designed from highly conserved regions within both viral genomes (segment 8 from MRDV and 9 from RBSDV). Total RNA was extracted, denatured at 95°C in the presence of 1μM primer 'MRDV-R2' and 10% DMSO, before being used as template for reverse transcription (RT). RT and subsequent PCR were performed according to standard protocols (Dovas et al., 2001) with an annealing temperature of 60°C used in PCR. RT-PCR using total RNA from 15 plants showing typical dwarfing symptoms gave the expected 568 bp product, which was subsequently cloned and sequenced. Sequence comparisons revealed 96% identity with genome segment 8 of an Italian isolate of MRDV (L76561) (Marzachi et al., 1996), whereas identity with genome segment 9 of two RBSDV isolates from China (AF459812, AY050486) was 85% (Bai et al., 2002). The presence of MRDV was further confirmed by ELISA using polyclonal antibodies (BioRad Phyto-Diagnostics, France). More than 50 samples collected from Imathia and Serres area, showing MRDV symptoms tested positive by ELISA. This is the first report of MRDV in Greece.