First Report of Turnip mosaic virus on Watercress in Brazil.

Watercress (Nasturtium officinale L.), a member of the family Brassicaceae, is consumed mainly as salad. Medicinal properties have also been attributed to this species. In Brazil, watercress is grown mainly by very small farmers. The crop is primarily seed propagated and growers can harvest several times per year in an established planting. Very few diseases have been reported in this crop worldwide. In Brazil, watercress infection by Cauliflower mosaic virus (CaMV) (3), Cucumber mosaic virus (CMV) (1), and an unidentified potyvirus (2) were previously reported. In January 2009, 80% of watercress plants, cv. Gigante Redondo, exhibiting severe mosaic, leaf size reduction, and plant stunting were observed in a crop in Marechal Floriano Municipality, State of Espírito Santo, Brazil. Preliminary leaf dip analysis by transmission electron microscopy revealed the presence of potyvirus-like particles. Sap from five infected plants reacted in plate-trapped antigen (PTA)-ELISA with polyclonal antiserum against Turnip mosaic virus (TuMV), but not with antiserum against CMV. Both antisera were produced in the Plant Virology Laboratory, ESALQ/USP. Mechanically inoculated watercress plants developed similar systemic mosaic symptoms. The virus was also transmitted to Nicotiana benthamiana, which exhibited severe mosaic and stunting. The presence of TuMV on these inoculated plants was confirmed by PTA-ELISA and reverse transcription (RT)-PCR. Total RNA extracted from infected and healthy watercress and infected N. benthamiana was analyzed by RT-PCR using specific pairs of primers flanking the coat protein gene of TuMV. Degenerated anti-sense (5'-t/caacccctt/gaacgcca/cagt/ca-3') and sense (5'-gcaggtgaa/gacg/acttgat/ca/gc-3') primers were designed after analysis to an alignment of the nucleotide sequences for five isolates of TuMV available in the GenBank (Accession Nos. NC_002509, D10927, EU680574, AB362513, and D88614). One fragment of 838 bp was amplified from samples in the infected plants, but not in the healthy controls. Two amplicons were purified and directly sequenced in both directions. Comparisons of the 731-bp consensus nucleotide sequence (Accession No. HM008961) to several other isolates of TuMV revealed 94 to 95% identity in the coat protein region. To our knowledge, this is the first report of TuMV in watercress in Brazil. Management of the disease should include propagation by seeds instead of vegetative parts of the plants and rouging of diseased plants to prevent mechanical transmission during successive harvestings. References: (1) A. J. Boari et al. Fitopatol. Bras. 25:438, 2000. (2) A. J. Boari et al. Fitopatol. Bras. 27:S200, 2002. (3) M. L. R. Z. C. Lima et al. Fitopatol. Bras. 9:403, 1984.

First Report of Papaya ringspot virus-Type W and Zucchini yellow mosaic virus Infecting Trichosanthes cucumerina in Brazil.

Trichosanthes cucumerina L., known as snake gourd, is a cucurbitaceous plant that is probably native to and originally domesticated in India. It is cultivated in humid subtropical and tropical countries of Australia, Latin America, and Africa (2). Plants of this species exhibiting symptoms of mosaic and leaf malformation were found during November 2008 near an experimental field of the Departamento de Fitopatologia e Nematologia, Universidade de São Paulo, Piracicaba, State of São Paulo, Brazil. Electron microscopy examination of negatively stained extract of infected tissue showed the presence of filamentous potyvirus-like particles. Sap from these infected plants reacted in plate-trapped antigen (PTA)-ELISA with the antiserum against Papaya ringspot virus-type W (PRSV-W) or Zucchini yellow mosaic virus (ZYMV), but not with the antiserum against Cucumber mosaic virus (CMV) or Zucchini lethal chlorosis virus (ZLCV). PRSV-W and ZYMV were simultaneously transmitted by mechanical inoculation to four plants of Cucurbita pepo cv. Caserta and one plant of T. cucumerina, causing mosaic. In addition, PRSV-W and ZYMV isolates from our virus collection separately infected one plant of T. cucumerina after mechanical inoculation. Infections were confirmed by PTA-ELISA. Total RNA extracted from infected and healthy T. cucumerina was analyzed by reverse transcription (RT)-PCR using a primer pair specific to the coat protein (CP) gene of PRSV-W (4) or ZYMV (3). Fragments of 864 bp and 1,045 bp were amplified with each pair of primers, respectively. Nucleotide sequences directly obtained from purified PCR products were used for further identification of these potyviruses. The nucleotide and deduced amino acid sequences of part of the CP gene (792 nt) of PRSV-W (GenBank Accession No. GU586789) shared 99 and 98% identity, respectively, with that of the Brazilian isolate PRSV-W-C (GenBank Accession No. 4152). The nucleotide and deduced amino acid sequences of the entire CP gene (837 nt) of ZYMV (GenBank Accession No. 6790) shared 91 to 98% and 94 to 100% identity, respectively, with innumerous isolates of ZYMV deposited in the GenBank (e.g., Accession Nos. AB004640, D13914, AB004641, and AJ420019). Natural infection of T. cucumerina by PRSV-W was reported in Nepal (1). To our knowledge, this is the first report of T. cucumerina infected by PRSV-W and ZYMV in Brazil. References: (1) G. Dahal et al. Ann. Appl. Biol. 130:491, 1997. (2) R. W. Robinson and D. S. Decker-Walters. Cucurbits. CAB International, Wallingford, UK. 1997. (3) K. G. Thomson et al. J. Virol. Methods 55:83, 1995. (4) M. G. S. D. Vechia. Fitopatol. Bras. 28:678, 2003.

Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers.

Lettuce mottle virus (LeMoV) and dandelion yellow mosaic virus (DaYMV) infect lettuce in South America and Europe, respectively. LeMoV and DaYMV possess isometric particles, occur at low concentrations in plants and have narrow host ranges. Partial genome sequences of both viruses were obtained using purified viral preparations and universal primers for members of the family Sequiviridae. DaYMV and LeMoV sequences were analyzed and showed identity with other members of the family. Universal primers that detect both viruses and specific primers for LeMoV and DaYMV were designed and used in RT-PCR-based diagnostic assays. These results provide the first molecular data on the LeMoV and DaYMV genomes and suggest that LeMoV is a member of the genus Sequivirus, probably distinct from DaYMV.

First Report of a Lettuce-Infecting Sequivirus in Chile.

Sequiviruses are isometric aphidborne plant viruses. Dandelion yellow mosaic virus (DaYMV), genus Sequivirus, was isolated from dandelion and lettuce in Europe. Lettuce mottle virus (LeMoV), a putative sequivirus, is often found in mixed infections with Lettuce mosaic virus (LMV) in Brazil (3). DaYMV, LeMoV and LMV cause similar mosaics in field-grown lettuce. Differences in biology and sequence suggest that DaYMV and LeMoV are distinct species (2). Forty-two and 101 lettuce samples with mosaic symptoms collected from two locations near Santiago during a survey of lettuce viruses in Chile in 2002 and 2003, respectively, were analyzed for the presence of LeMoV using reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted (1) and used for RT-PCR with the specific LeMoV primers pairs Lmo3 (5' ACATGAGCACTAGTGAGG 3') and Lmo4 (5' AGATAGAGCCGTCT GGCG 3') (2). One of the 42 and three of the 101 samples produced the expected 300-bp fragment. Isometric particles of 30 nm diameter, typical of a sequivirus, were visualized by transmission electron microscopy. These samples were tested using RT-PCR for the presence of LMV and Cucumber mosaic virus (CMV), but no mixed infections were observed. One isolate, Ch36, was reamplified with the degenerate primer pairs DALE 1 (5' GARTTCAACATGCACGCCAG 3') and DALE 2 (5' TTTTTCTCCCCATYCGTCAT 3') which amplify part of the putative replicase gene (2) and produced a 563-bp fragment that was cloned on pGEM-T Easy (Promega, Madison, WI) and sequenced. The Ch36 product (EMBL Accession No. AM039965) showed 97% amino acid identity with LeMoV from Brazil, 79% with DaYMV, 72% with the sequivirus Parsnip yellow fleck virus, and 34% with the waikavirus Maize chlorotic dwarf virus. To our knowledge, this is the first report of a sequivirus in field lettuce in Chile, and although the virus was found at low incidence, this report extends the range of LeMoV to the western side of the Cordillera de Los Andes. The impact of LeMoV needs to be further analyzed in Chile, Brazil, and possibly other South American countries. References: (1) Y. D. Bertheau et al. DNA amplification by polymerase chain reaction (PCR) 1998. In: Methods for the Detection and Quantification of Erwinia carotovora subsp. atroseptica on potatoes. M. C. N. Perombelon and J. M. van der Wolff, eds. Scott. Crop Res. Inst. Occasional Publ., Dundee, 1998. (2) A. S. Jadão. Caracterização parcial e desenvolvimento de oligonucleotídeos específicos para detecção de sequivirus infectando alface. Ph.D. thesis. FCA-UNESP-Botucatu, Brazil, 2004. (3) O. Stangarlin et al. Plant Dis. 84:490, 2000.