RESUMEN
Tropical grass and legume species used as pasture grasses for cattle feeding cover over 25% of the agricultural area in Brazil. In recent years, plants showing virus-like symptoms have been observed in the main pasture grass growing areas. Plants of Pennisetum purpureum line CNPGL 00211 showing typical virus mosaic symptoms on leaves and growth reduction were collected in Bahia State, Brazil. Flexuous elongated potyvirus-like particles were observed in the leaf-dip preparation of diseased plants by electron microscopy. In addition, the virus was mechanically transmitted using a standard procedure for potyviruses (4) and produced similar symptoms in inoculated P. purpureum plants. For further molecular identification, total RNA was extracted from frozen symptomatic leaves following the guanidine thiocyanate method (3). cDNA synthesis was performed using oligonucleotide, OligodT50M10 and PCR was carried out using Potyvirus degenerate primers PY11 (5'-GGNAAYAAYAGYGGNCARCC-3') (2) and M10 (5'-AAGCAGTGTTATCAACGCAGA-3'). The amplified fragments of the expected size (approximately 2 kb comprising part of the NIb protein gene, the entire coat protein [CP] gene, and the 3' nontranslated region) were separated using agarose gel electrophoresis, excised, and cloned into plasmid vector pGEMT-Easy (Promega) according to the manufacturer's instructions. Four selected clones were sequenced (Macrogen, South Korea). The sequenced 2.0-kb fragment (GenBank Accession No. KC333416) was compared with sequences available in GenBank and the highest nucleotide identity of 79% was observed with Johnsongrass mosaic virus (JGMV) isolated in Australia (4). According to the Potyvirus species demarcation convention based on CP identity (1), the virus isolate from P. purpureum belongs to the JGMV species. However, the amino acid sequence of the N-terminus of the CP of the Bahia isolate is distinct from JGMV sequences reported in GenBank. The phylogenetic analysis of the CP confirmed the difference since this Bahia isolate was located in a clearly distinct branch separate from all JGMV isolates. To our knowledge, this is the first report of a JGMV in Brazil infecting tropical grass in the main pasture areas. References: (1) M. J. Adams et al. Arch. Virol. 150: 459, 2005. (2) J. Chen et al. Arch. Virol. 146:757. 2001. (3) P. Chomczynski and N. Sacchi. Nature Protocols 1:581, 2006. (4) H. K. Laidlaw et al. Arch. Virol. 149:1633, 2004.
RESUMEN
Leaves of Cassia hoffmannseggii, a wild fabaceous species found in the Atlantic Forest, with a severe mosaic symptom were collected in Pernambuco State, Brazil. By transmission electron microscopy, two types of virus particles were found: the first was recognized as particles of a potyvirus, which was later identified as Cowpea aphid-borne mosaic virus; and the second was isometric and present in high concentration. The observation of vesicles at the periphery of chloroplasts suggested a tymovirus infection, which was confirmed by subsequent assays. A serological assay against several tymovirus antisera resulted in positive reaction of this tymo-like virus with an antiserum of Passion fruit yellow mosaic virus. By means of RT-PCR and using degenerated primers for the conserved region of RNA-dependent RNA polymerase (RdRp) gene of tymoviruses, a specific DNA fragment was amplified and sequenced. Based on this sequence, a specific forward primer was synthesized and successfully used to amplify the 3' terminal genome region, containing the partial RdRp gene and the complete coat protein (CP) sequences. The CP was 188 amino acids (aa) long, and the highest CP aa identity was observed with Kennedya yellow mosaic virus (61 %). Based on the current ICTV demarcation criterion, this isolate was considered as a distinct tymovirus and tentatively named as Cassia yellow mosaic-associated virus.