First Report of Garlic virus X in Garlic Plants in Brazil.

Garlic is the fifth most economically important vegetable in Brazil and is frequently infected by a complex of different viruses that cause significant degeneration of the crop under field conditions. The species of the genus Allexivirus that infect garlic are: Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV-C), Garlic virus D (GarV-D), Garlic virus E (GarV-E), Garlic virus X (GarV-X), Garlic mite-borne filamentous viru s (GarMbFV), and Shallot virus X (ShVX). So far, only GarV-A, GarV-B, GarV-C, GarV-D, and GarMbFV have been reported in Brazil (3). During the 2010 through 2013 seasons, between April and October, 302 garlic plants with yellow mosaic strips and distorted leaves from the cultivars Caçador, Quitéria, Tropical Bergamota, and Tropical Shangai were collected in the states of Paraná, Minas Gerais, São Paulo, and Goiás and analyzed for the presence of allexiviruses. Total plant RNA was extracted with the Total RNA Purification kit (Norgen Biotek Corp., Canada) according to manufacturer's instructions. RT-PCR reactions were performed initially with the primer pair named Cpallexi-senso2 (5' CTACCACAAYGGNTCVTC 3') and Cpallexi-anti1 (5' CACNGCGTTRAAGAARTC 3') specifically designed to amplify a ~230-bp fragment from all currently known allexiviruses. Positive samples were then analyzed with specific primers for GarV-A, GarV-C, and GarV-D (2), GarMbFV (1) and GarV-B named CPBS2 (5' GCAGAATAARCCCCCYTC 3') and CPBA1 (5' RAAGGGTTTATTCTGTTG 3') obtained in this work. Among the plants analyzed, 50 were positive for the Cpallexi-senso2/Cpallexi-anti1 primers but negative for all the specific primers tested, indicating the presence of a different allexivirus. These samples were then analyzed by RT-PCR for the presence of GarV-X, GarV-E, and ShVX and an amplicon of ~550 bp was obtained only with primers CPXS2 (5' GCCTTCTGAAAATGACTTAG 3') and CPXA1 (5' CTAGGATTTGCTGTTGGG 3') designed in this work to amplify a fragment of the capsid protein gene for GarV-X. Since species demarcation in the genus Allexivirus is based on the coat protein (CP) gene (2), another set of primers, namely PIXS1 (GACGACGGYGCACTACTC) / PIXA1 (YGTGAATCGTGATGATCC) and PFXS2 (CRCTGAGACAATTYYGTGG) / PFXA2 (CAAAGCATCGGCCRTAGCG) derived from conserved regions of ORF4, ORF5 (CP), and ORF 6 sequences of allexiviruses available in the NCBI database, were used in RT-PCR to obtain the complete CP gene nucleotide sequence. A 1,071-nt sequence comprising 108 bp of ORF4 (partial), 732 bp of the CP, and 177 bp of ORF 6 was successfully amplified (GenBank Accession No. KF530328). The complete CP gene showed 98% nucleotide sequence identity with GarV-X from Australia (JQ807994.1). In summary, GarV-X was detected in the 50 samples collected from Minas Gerais, São Paulo, and Paraná, indicating widespread distribution in Brazil. To our knowledge, this is the first report of GarV-X in garlic in Brazil. References: (1) M. S. Fayad-Andre et al. Trop. Plant Pathol. 36:341, 2011. (2) P. A. Melo Filho et al. Pesq. Agropec. Bras. 39:735, 2004. (3) R. J. Nascimento et al. Summa Phytopathol. 34:267, 2008.

First Report of Shallot latent virus in Garlic in Brazil.

Garlic (Allium sativum L.) can be infected by several viruses of the genera Allexivirus, Carlavirus, and Potyvirus (3). Garlic common latent virus (GarCLV) and Shallot latent virus (SLV) are the most important Carlavirus species infecting garlic, but only GarCLV has been described on garlic in Brazil. Seven hundred thirty-one samples of garlic showing mosaic symptoms and chlorotic streaking were collected from the states of São Paulo (São Manuel), Minas Gerais (Santa Juliana and São Gotardo), Goiás (Campo Alegre and Ipameri), and Paraná (Guarapuava and Piraquara) from April 2008 to July 2009 and analyzed by double-antibody sandwich (DAS)-ELISA for the presence of GarCLV and SLV using specific antiserum for SLV and GarCLV according to the manufacturer's protocol (Agdia Inc., Elkhart, IN). Cultivars sampled were Caçador, Chonan, Ito, Jonas, Quitéria, and Tropical. Fifty-five samples (7.5% of 731) tested positive for GarCLV, and none of the samples tested positive for SLV. Total RNA was extracted (2) from 15 samples that represented different states of production and used with primers SLV 7044 (5'-CTTTTGGTTCACTTTAGG-3') and SLV 8004 (5'-GCACGCAATAGTCTACGG-3'), designed in this study, to detect SLV in a one-step reverse transcription (RT)-PCR assay. Only 3 of the 15 samples, two from São Paulo and one from Paraná State, produced a 960-bp fragment covering the putative coat protein gene (ORF 5) (1) of SLV. The amplicons of the three isolates were sequenced. A nucleotide sequence identity of 91 to 92% was detected in comparison with two strains of SLV (GenBank Accession Nos. AB004567 and DQ520093), indicating the presence of two isolates of SLV in São Manuel (São Paulo State) and one in Piraquara, Paraná State (submitted to GenBank as Accession Nos. GU120176, HQ128602, and GU120175, respectively). To confirm identity of the virus, another pair of primers was constructed and tested (SLV 6737: 5'-YCCSGCCARGAAYTTCCC-3', and SLV 7060: 5'-TTAGAGCGCTGTWAACC-3'), from which a 340-bp fragment covering a portion of TGB2 (ORF 3) and TGB3 (ORF 4) (1) was amplified using the two isolates from São Paulo (GenBank Accession Nos. HQ123181 and HQ123182, respectively). The amplicon sequences shared 87% identity with that of an SLV isolate (Accession No. AJ292226), which confirmed the presence of SLV. The low titer of SLV in garlic might account for the false negative results by DAS-ELISA. The source of cultivated garlic bulbs in these regions of Brazil is unknown. Garlic cloves have been cultivated in São Manuel for approximately 15 years, indicating that SLV may have been present in Brazil for many years. To our knowledge, this is the first report of SLV in Brazil. References: (1) M. J. Adams et al. Virus Taxonomy: 8th Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, 2005. (2) Y. D. Bertheau et al. Methods for the Detection and Quantification of Erwinia carotovora subsp. atroseptica on Potatoes. M. C. N. Perombelon and J. M. van der Wolff, eds. Scottish Crop Research Institute, Dundee, U.K., 1998. (3) T. V. M. Fajardo et al. Fitopatol. Bras. 26:619, 2001.

First Report of Tomato chlorotic spot virus Infecting Spilanthes oleracea in Brazil.

Spilanthes oleracea L., popularly known as toothache plant, belongs to the family Asteraceae and is a South American native plant. Fresh leaves can be eaten for their medicinal properties or used by the cosmetics industry for their spilol contents. Plants showing leaf deformation that were collected in a field in São Paulo State, Brazil in March 2005 were suspected to be infected by a virus. Electron microscopy of leaf dip preparations of symptomatic plants revealed pleiomorphic particles typical of tospoviruses. Extracts from these plants prepared with 0.01 M sodium phosphate buffer, pH 7.0, containing 1% sodium sulfite were mechanically inoculated to indicator plants. Chenopodium amaranticolor and Gomphrena globosa were symptomless. Necrotic local lesions were observed on C. quinoa. Necrotic local lesions followed by a systemic necrosis that caused the death of the plants were observed on Datura stramonium, Nicotiana glutinosa, and N. tabacum 'TNN' and 'Turkish'. Concentric rings followed by systemic necrosis and plant death were induced on N. rustica, N. tabacum 'Havana 425', N. clevelandii, Physalis floridana, Capsicum annum 'Magda', and Solanum lycopersicum 'Santa Clara'. Total RNA was extracted (1) from infected S. oleracea and N. rustica plants for reverse transcription-PCR amplification with tospovirus specific primers BR60 (5' CCCGGATCCTGCAGAGCAATTGTGTCA 3') and BR65 (5' ATCAAGCCTTCTGAAAGTCAT 3') (2), which amplified an approximate 440-bp fragment covering part of the nucleocapsid protein gene. This fragment was sequenced (EMBL Accession No. AM887766) and showed 99% nt sequence identity with Tomato chlorotic spot virus (TCSV) (GenBank Accession No. AF521102), a tospovirus species (3). To our knowledge, this is the first report of a tospovirus infecting S. oleracea in Brazil and indicates that this plant might constitute a reservoir of TCSV or other tospoviruses that could also infect tomato and pepper plants. 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 Wolf, eds. Scott. Crop Res. Inst. Occas. Publ. Dundee, Scotland, 1998. (2) M. Eiras et al. Fitopatol. Bras. 26:170, 2001. (3) F. Lovato et al. Virus Genes 29:321, 2004.

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.

A naturally occurring recombinant isolate of Lettuce mosaic virus.

LMV-Common and LMV-Most are two seed-borne types of Lettuce mosaic virus (LMV), genus Potyvirus. LMV-Most, but not LMV-Common, overcomes the resistance afforded to lettuce by two recessive genes, mo11 and mo12. An RT-PCR-based assay thought to be specific for LMV-Most also amplified LMV-Tn2, previously typified as LMV-Common. The sequence of selected regions along the genome indicated that LMV-Tn2 is a natural recombinant between LMV-Most and LMV-Common isolates, with a putative recombination site located within the P3 coding region. This is the first evidence of a naturally occurring LMV recombinant isolate.

Occurrence of a New Pathotype of Lettuce mosaic virus on Lettuce in Brazil.

Since 1970 lettuce mosaic has not been an important disease of lettuce in Brazil, due to the growing of cultivars that contain gene g, derived from cv. Gallega de Invierno, for tolerance. Recently, however, a widespread, serious outbreak of lettuce mosaic occurred in the State of Sao Paulo. Both lettuce cultivars that lack and those that contain gene g have been affected, suggesting the emergence of a new pathotype of Lettuce mosaic virus (LMV). Commercial lettuce fields were visited, and 20 samples were collected for virus identification by bioassay on differential hosts, serological tests, and electron microscopy. Of these, 12 were infected only by LMV, and 8 contained mixed infections of LMV and a possible new sequivirus, Lettuce mottle virus. Seedlings of susceptible cv. White Boston were inoculated with LMV from tolerant cultivars, and plants were allowed to flower. Seeds were collected and sown, and seedlings with mosaic symptoms were recovered. Twenty monolesional isolates, obtained by passing the virus three times through Chenopodium amaranticolor, were inoculated individually on lettuce differential cvs. Salinas and White Boston (susceptible) and cvs. Salinas 88, Vanguard 75, Ithaca, Malika, and Gallega de Invierno (tolerant) to previously described pathotypes of LMV (1). Considering the susceptibility of all test differentials, we concluded that a LMV pathotype IV exists that has overcome tolerance conferred by genes m and g and is responsible for the new outbreak of LMV in Brazil. This is the first report of LMV-IV in Latin America. Reference: (1) D. A. C. Pink et al. Plant Pathol. 41:5, 1992.

Occurrence, Distribution, and Relative Incidence of Five Viruses Infecting Cucurbits in the State of São Paulo, Brazil.

Cucurbits species grown in 38 of 40 agricultural regions in the state of São Paulo, Brazil, were surveyed for the relative incidence of Cucumber mosaic virus (CMV), Papaya ringspot virus-type W (PRSV-W), Watermelon mosaic virus-2 (WMV-2), Zucchini lethal chlorosis virus(ZLCV), and Zucchini yellow mosaic virus (ZYMV) during May 1997 and June 1999. Samples from 621 plants, representing eight cultivated species, six wild species, and one commercial hybrid (Cucurbita moschata × C. maxima), were analyzed by plate trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA). PRSV-W and ZYMV were the most frequently found viruses, accounting for 49.1 and 24.8%, respectively, of 605 samples tested. ZLCV, CMV, and WMV-2 were detected in 7.8, 6.0, and 4.5% of 612, 497, and 423 samples tested, respectively. Double infection was found in 97 samples, and triple infection was found in 10 samples. Quadruple infection was detected in one C. pepo sample. Plants that were symptomatic but negative by PTA-ELISA might be due to abiotic agents, infection by virus for which antiserum was not available, such as Squash mosaic virus, or infection with an as yet uncharacterized virus.

Cayaponia tibiricae: New Host of Zucchini Yellow Mosaic Virus in Brazil.

Cayaponia tibiricae Cogn. (CT) is a wild Cucurbitaceae species found in secondary forests in the State of São Paulo, Brazil. The species has indefinite growth and bears oblong dark green fruits, 15 to 20 mm long (1,2). CT plants showing yellow mosaic symptoms were found in Atibaia County. Extracts from symptomatic plants were rub inoculated to zucchini squash (Cucurbita pepo L.) and Chenopodium amaranticolor Coste & Reyn. Zucchini squash plants developed severe yellow mosaic with intense leaf malformation, while C. amaranticolor showed necrotic local lesions. Extracts from naturally infected CT, zucchini squash, and C. amaranticolor were tested by plate trapped antigen-enzyme-linked immunosorbent assay (PTA-ELISA) with antisera against papaya ringspot virus type W (PRSV-W), zucchini yellow mosaic virus (ZYMV), zucchini lethal chlorosis virus (ZLCV), watermelon mosaic virus 2 (WMV-2), and cucumber mosaic virus (CMV). All samples were positive in PTA-ELISA only with ZYMV antiserum. Also, in Western blot (immunoblot) assay, ZYMV antiserum labeled a protein of approximately 36 kDa. Electron microscopic examination of ultrathin sections from infected CT tissue revealed the presence of pinwheel inclusions typical of potyvirus (type 1) infection in the cytoplasm of the cell. CT seedlings were susceptible to mechanical inoculation with the ZYMV isolated from this species. This is the first report of CT as a natural host for ZYMV in Brazil. References: (1) A. Cogniaux. Flora Brasiliensis 6:1, 1878. (2) M. Pio Corrêa. 1926. Diccionário das plantas úteis do Brasil e das exóticas cultivadas. Vol 1. Ministério da Agricultura, Rio de Janeiro, Brazil.