First Report of Barley yellow dwarf virus-PAS in Wheat and Barley Grown in the Czech Republic.

Abstract

Barley yellow dwarf disease, an important, ubiquitous virus disease of cereal crops worldwide, is caused by a group of related single-stranded RNA viruses assigned to luteovirus (Barley yellow dwarf virus (BYDV) spp. PAV, PAS, MAV, and GAV) or polerovirus (Cereal yellow dwarf virus-RPV) genera or unassigned to a genera (BYDV-SGV, BYDV-RMV, and BYDV-GPV) in the family Luteoviridae (2). Incidence of BYDV in cereal crops (e.g., barley, wheat, and oats) was high and reached epidemic levels in recent years in many regions of the Czech Republic. Previously, only PAV isolates have been identified here on the basis of serological detection (4), although antibodies to differentiate between PAV, PAS, and MAV are not widely available. Field samples of cereal crops were routinely tested in 2006 and 2007 and BYDVs were detected by ELISA. One-step-reverse transcription (RT)-PCR (Qiagen, Hilden, Germany) was adapted for BYDV detection using primer pairs BYcpF (5'-CCACTAGAGAGGTGGTGAATG-3') and BYcpR (5'-CCGGTGTTGAGGAGTCTACC-3') designed from conserved sequences identified by aligning multiple BYDV sequences available in public databases. These primers amplify a 641-bp fragment spanning nucleotides 2839-3479 from PAV (GenBank Accession No. EF043235) or PAS (GenBank Accession No. NC_002160) that includes a region of the coat protein gene and the intergenic region. RT-PCR amplicons were generated from two field isolates, PS-RuJK (spring wheat isolate, cv. Granny, collected in July 2007 from experimental plots at the CRI in Prague) and JE-120JK (winter barley isolate, cv. Merlot, collected in January 2008 from a barley field in Rychnov), both of which induced severe BYD symptoms. Amplicons were sequenced in both directions in a CEQ2000XL sequencer (Beckman Coulter, Fullerton, CA). The partial coat protein gene sequence of 483 nt of PS-RuJK and JE-120JK was analyzed and compared with available sequences of 26 PAV, 17 PAS, and 13 MAV isolates by MEGA4 (3). PS-RuJK (GenBank Accession No. EU863652) nucleotide and amino acid sequence identities ranged from 96.3 to 99.2% and 93.7 to 98.7%, respectively, for available PAS isolates, and 89.9 to 90.5% and 85.5 to 86.9%, respectively, for available PAV isolates, and 78.3 to 79.5% and 70.0 to 72.5%, respectively, for available MAV isolates. Similarly, nucleotide and amino acid sequence identities JE-120JK (GenBank Accession No. EU863653) ranged from 95.2 to 98.6% and 90.6 to 96.9%, respectively, for PAS isolates, 88.8 to 90.1% and 83.1 to 84.4%, respectively, for PAV isolates, and 77.6 to 78.7% and 67.5 to 70.0%, respectively, for MAV isolates. Also, both of these isolates have the conserved amino acid motif "SIPGS" that is usually present in a variable region of the coat protein gene on the surface of virion (1) at position 52 to 56 of amino acid sequences of all published PAS-like isolates, including Vd29:AY167109, FH1:AJ223588, MA9516:AJ007926, FL2:AJ223586, ASL-1:AJ810418, and WS6603:DQ285680, contrary to "PVFRP" or "LISGP" motif in PAV or MAV, respectively. Therefore, the sequence data clearly confirm that these two isolates belong to the PAS species. To our knowledge, this is the first record of PAS detected in the Czech Republic.