RESUMO
Macroptilium atropurpureum (siratro plants) is a perennial wild legume plant introduced to Taiwan as a forage crop in 1961 (3) and has become a naturalized weed found all over the island. In 2010, siratro plants with virus-like symptoms of mosaic and leaf deformation were observed on the campus of Da-Yeh University in central Taiwan. Flexuous virus-like particles about 750 × 12 nm were observed in the crude sap extracted from symptomatic leaves with a transmission electron microscope. Crude sap was mechanically inoculated to Chenopodium quinoa and local lesions can be observed on inoculated leaves 4 to 5 days after inoculation. Virus was purified from the leaves of inoculated C. quinoa with modified protocols of Gonsalves and Ishii (2). The virus coat protein (CP) consisted of a single major peptide with relative molecular weight of approximately 33 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Viral RNA extracted from the purified virus was used as a template and was primed with several primer sets corresponding to potyviruses and carlaviruses in reverse transcription-PCR to amplify possible corresponding cDNA fragments. After several attempts, a cDNA fragment of about 1,300 bp could be amplified with the degenerated primer set of BCMNV-F (5'CCDTGGACDGTWGGVATGAC3') and BCMNV-R (5'CACCAHACCATRAARCCATTCAT3'), which were designed on the basis of the conserved region of the nuclear inclusion b (NIb) and CP genes of some potyviruses including Bean common mosaic necrosis virus, Soybean mosaic virus, Blackeye cowpea mosaic virus, East Asian passiflora virus, and Passion fruit woodiness virus. BLAST analyses showed the amplicon was highly homologous to that of Passiflora virus Y (PaVY). Together with oligo dT, a specific primer (5'GATGACACTCAAATGGCTG3') corresponding to PaVY CP was used to amplify the cDNA fragment of the most 3' region of the viral RNA (about 800 bp). The assembled cDNA fragment of 1,958 bp (Accession No. AB679294) contains a partial NIb gene (877 nt), a complete CP gene (819 nt), and the 3' noncoding region (262 nt). The CP gene shared sequence identities of 89.4 to 98.9% and 92.7 to 98.9% in nucleotide and amino acid, respectively, to that of documented PaVY isolates. PaVY has also been found to be infecting Vigna trilobata, Rhynchosia minima, Clitoria ternatea, and Passiflora foetida in Australia (1). Here we present the first report to our knowledge of PaVY and its infection of siratro (M. atropurpureum) in Taiwan. Additional work is needed to investigate the spread of PaVY and its interaction with other legume plants in Taiwan. References: (1) B. A. Coutts et al. Arch. Virol. 156:1757, 2011. (2) D. Gonsalves and M. Ishii. Phytopathology 70:1028, 1980. (3) Y. Y. Lai et al. J. Taiwan Livestock Res. 42:19, 2009.
RESUMO
In order to establish a model system for introduction of foreign genes into papaya (Carica papaya L.) plants by Agrobacterium-mediated transformation, petioles from multishoots were used as explant source and bacterial neomycin phosphotransferase II (NPT II) gene and ß-glucuronidase (GUS) gene were used as a selection marker and a reporter, respectively. Cross sections of papaya petioles obtained from multishoots micropropagated in vitro were infected with A. tumefaciens LBA4404 containing NPTII and GUS genes and co-cultured for 2 d. The putative transformed calluses were identified by growth on the selective medium containing kanamycin and carbenicillin, and consequently regenerated to plants via somatic embryogenesis. Thirteen putative transgenic lines were obtained from a total of 415 petiole fragments treated. Strong GUS activity was detected in the selected putative transgenic calli or plants by fluorogenic assay. Western blot analysis using GUS antiserum confirmed that the GUS protein was expressed in putative transformed papaya cells and transgenic plants. The presence of the GUS gene in the papaya tissues was detected by PCR amplification coupled with Southern blot.
