Development of a one-step immunocapture real-time RT-PCR assay for the detection of barley stripe mosaic virus strains in barley seedlings.

A one-step immunocapture real-time RT-PCR (IC-real-time RT-PCR) was developed for efficient detection of barley stripe mosaic virus (BSMV) in barley seedlings. The novel detection system was designed using a primer set targeting the conserved region in the triple gene block 2 (TGB2) to expand its capacity to detect all BSMV strains. This assay was evaluated for its efficiency in detecting BSMV in barley seedlings. Using the immunocapture sample preparation, real-time RT-PCR was able to detect BSMV in samples, which were indicated as negative by ELISA. The sensitivity of detection in the real-time RT-PCR was as low as 50 fg/µl of total viral RNA under optimal reaction conditions. This level of sensitivity indicated that the one-step IC-real-time RT-PCR developed in the present study could be used for routine plant and seed health assays.

First Report of Sugarcane mosaic virus Infecting Maize in Poland.

Maize (Zea mays) has become an important crop in Poland with a constant increase in crop acreage since the 1990s. In 2007, maize plants with characteristic leaf mosaic were observed in two locations in the Wielkopolska Region near Poznan and Krotoszyn. Ninety-two samples from plants showing leaf mosaic, some leaf discoloration, stunting, or no symptoms were collected and tested for Maize dwarf mosaic virus (MDMV) and Sugarcane mosaic virus (SCMV) by double-antibody sandwich (DAS)-ELISA (Bioreba, Basel, Switzerland). SCMV was detected only in three samples with distinct leave mosaic symptoms. Electron microscopy of leaf extracts revealed numerous potyvirus-like particles. Immuno-specific electron microscopy (ISEM) with the SCMV antiserum gave positive results for all three samples. Each virus isolate was propagated by mechanical inoculation on five varieties of dent maize and three varieties of sweet maize, cockspur grass (Echinochloa crus-galli (L.) Beauv.), crab grass (Digitaria sanguinalis (L.) Scop.), and green bristle-grass (Setaria viridis (L.) P.B.). Leaf mosaic appeared 4 to 5 days postinoculation. ELISA detected all three isolates in the symptomless hosts of oat (Avena sativa L.), wheat (Triticum aestivum L.), and triticale (Triticale). The three isolates induced local leaf necrosis on sorghum (Sorghum vulgare L.), in which the virus occurred in low concentrations as determined by ELISA so infections of sorghum plants were confirmed by reverse transcription (RT)-PCR) with primers PS/PSC (1). Barley (Hordeum vulgare L.), true millet (Panicum miliaceum L.), and wind grass (Agrostis spica-venti (L.) P.B.) were not susceptible (2). Using the total RNA extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany) from leaves of inoculated maize plants, a one step RT-PCR (Qiagen) amplified a ~800-bp cDNA fragment of the coat protein gene with SCMV-specific primers PS/PSC (1). Six cDNA clones were sequenced for each isolate. Nucleotide sequences of the 823-bp cDNA clones of isolates SCMV-P1 and P2 (GenBank Accession Nos. EU761241 and EU761242, respectively) were 99% identical and each was 92% identical to the sequence of SCMV-P3 (FJ376609). The clones of SCMV-P1 and SCMV-P2 shared 99, 98, 90, and 87% nucleotide sequence identity with two German SCMV isolates (X981697 and X98168), a Spanish isolate (AM110759), the UT6 isolate from Thailand (AY630923), and the Nancheng isolate (EU346720) from China, respectively. The SCMV-P3 sequence was 98, 94, 92, 89, and 92% identical to the Mx isolate from Mexico (AY195610), a Bulgarian SCMV isolate (AJ006201), the German Seehausen (X98166) and Borsdorf (X98167) isolates, the SC-UD1 (DQ647661), the KL - Co86032 (DQ866744) isolates from Thailand and India, and the Chinese Nanchang (EU346720) and Pengze2 (EU346718) isolates, respectively. In 2008, SCMV was again detected by ELISA in mixed infections with MDMV in samples from the Wielkopolska Region, but only sporadically, and the virus is considered not to be important economically in maize production in Poland. References: (1) J. X. Jiang and X. P. Zhou. Arch. Virol. 147:2437, 2002. (2) D. M. Persley. Page: 1204 in: Viruses of Plants. Descriptions and Lists from the VIDE Database. CAB International, Wallingford, UK, 1996.

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% ß-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.