Identification of Maize dwarf mosaic virus in Maize in Poland

From 2005 to 2007 in Southern Wielkopolska, Lower Silesia, and Malopolska regions, maize (Zea mays) plants showing leaf mosaic and stunting symptoms were found. ELISA tests using commercial polyclonal antisera against Maize dwarf mosaic virus (MDMV) obtained from Bioreba (Basel, Switzerland) and Loewe (Munich, Germany) gave positive results in 71 samples. However, the ELISA response for symptomatic plants, in most cases, was low, with OD values ranging from 0.05 to 0.18. Therefore, only eight plants with relatively high virus concentration were chosen for further identification assays. Examination of leaf extracts with an electron microscope revealed the presence of potyvirus-like particles. Symptomatic leaves were positive for MDMV by using immunosorbent electron microscopy (ISEM) with antiserum raised against the Spanish isolate of MDMV (supplied as positive MDMV control from A. Achon, Centre Vdl-Irta, Lleida, Spain). A set of test plants, including sweet corn, dent corn, sorghum (Sorghum vulgare), and true millet (Panicum miliaceum), were mechanically inoculated with extracts from symptomatic plants in 0.05 M phosphate buffer plus 1% β-mercaptoethanol. Inoculated plants developed symptoms typical of MDMV in 2 to 5 weeks (1,2). For further investigations, three virus isolates were chosen. To confirm the identification of MDMV, reverse transcription (RT)-PCR was performed with total RNA isolated from infected plants with primers 3MDF (5' GAT GAG TTR AAY GTY TAT GCA CGA C 3'), a forward primer in the 3' region of NIb gene and either 1MDR (5' RTG CAT RAT TTG TCT GAA AGT TGG 3') or 3MDR (5' ACC AVA CCA TYA TWC CAC TC 3'), reverse primers in the 3' region of the coat protein gene (A. Zare, Shiraz University, personal communication). 3MDF corresponds to nucleotides 8306 to 8330, 3MDR is complementary to nucleotides 8791 to 8813, and 1MDR is complementary to nucleotides 8917 to 8939 of the MDMV genome (GenBank Accession No. AJ001691). The RT-PCR products obtained were analyzed by agarose gel electrophoresis. Amplicons of the expected sizes (635 and 560 bp) were obtained with RNA from symptomatic plants, but not from asymptomatic plants. The sequence of the 576-bp PCR product was deposited in GenBank (Accession No. EU240460). In alignments done with BlastN ( www.ncbi.nlm.nih.gov/blast ), the highest nucleotide sequence identities were 99% with Spanish MDMV isolates ("SP" AM110758, "SP" AJ416645, and "S1" AJ416635), 91% with the Hungarian isolate "Sc/H, sweet corn" AJ542536, 90% with "MDMV-A" U07216, and 87% with an Israeli MDMV (AF395135). On the basis of these findings, the virus isolated from diseased maize plants was identified as MDMV. The significance of MDMV detection is noteworthy because maize has become an important crop in Poland in recent years and acreage is increasing systematically. References: (1) M. A. Achon et al. Eur. J. Plant Pathol. 102:697, 1996. (2) A. J. Gibbs. Maize dwarf mosaic virus. Page 752 in: Viruses of Plants. Descriptions and Lists from the VIDE database. A. A. Brunt et al., eds. CAB International, Wallingford, UK, 1996.