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Hippeastrum Mosaic Virus Diagnosed in Hippeastrum and Eucharis in Brazil

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Hippeastrum Mosaic Virus Diagnosed in Hippeastrum and Eucharis in Brazil 010a_JPP761RP(Alexandre)_643 15-11-2011 18:02 Pagina 643 Journal of Plant Pathology (2011), 93 (3), 643-649 Edizioni ETS Pisa, 2011 643 HIPPEASTRUM MOSAIC VIRUS DIAGNOSED IN HIPPEASTRUM AND EUCHARIS IN BRAZIL M.A.V. Alexandre1, L.M.L. Duarte1, E.B. Rivas1, A. Cilli2, R. Harakava1, S.R. Galleti1 and E.W. Kitajima3 1 LFF/Centro de Pesquisa e Desenvolvimento de Sanidade Vegetal (CPDSV), Instituto Biologico, Avenida Conselheiro Rodrigues Alves, 1252, CEP 04014-002 São Paulo, SP, Brazil 2 Instituto Adolfo Lutz, Avenida Dr. Arnaldo, 355,CEP 01246-902 São Paulo, SP, Brazil 3 Escola Superior de Agricultura Luiz de Queiróz, USP, CP 09, 13418-900 Piracicaba, SP, Brazil SUMMARY INTRODUCTION A potyvirus associated with foliar mosaic symptoms Amaryllidaceae Jaume St.-Hil., former Liliaceae, cf. displayed by Hippeastrum (Hip-Br) and two Eucharis Amaryllidaceae subfamily Amaryllidoideae (APG, 2009) samples (Euch-B and Euch-C) from three different comprises several genera including Amaryllis, Eucharis cities of Brazil was detected and identified by transmis- and Hippeastrum. These plants have been economically sion tests, electron microscopy, reverse-transcription, exploited as ornamentals and are therefore included in RT-PCR and sequencing. Transmission electron mi- various breeding programs (Bruyn et al., 1992). Amaryl- croscopy revealed the presence of elongated, flexuous lis (Cape belladonna, Belladonna lily or “true” amaryl- viral particles in the extracts of symptomatic leaves, lis) also known as “naked lily” because of the lovely while in thin sections of symptomatic leaves, cytoplas- trumpet-shaped flower, is indigenous to Southern mic aggregates of type II lamellar inclusions (Edward- Africa and is a monotypic genus. Hippeastrum son’s classification) were observed. The experimental (Leopoldii hybrid or Dutch amaryllis) is a genus of host range was restricted. RT-PCR using universal po- about 90 species and 600 hybrids and cultivars, origi- tyvirus primers amplified DNA products of 1,499 bp nating from a much wider geographical area extending corresponding to the partial nuclear inclusion body from Argentina to Mexico and into the Caribbean. Cur- (NIb) gene, full-length coat protein (CP) gene and rently it is grown extensively in tropical countries (Graf, 3’end untranslated region (UTR). Pairwise analyses 1992). The first hybrids made in the Netherlands were from CP and NIb nucleotide sequences of Hip-Br, those between H. reginae and H. vittatum both native to Euch-B and Euch-C viruses and other potyviruses re- Brazil (Tombolato et al., 2004). Eucharis (Amazon lily) vealed high identities with previously reported Alaska is found in the tropical rain forest and is adapted to low (GQ857550) and Rilona (EF203685) Hippeastrum mo- light conditions. The bulbous plants with broad, basal saic virus (HiMV) isolates, Amazon lily mosaic virus leaves narrowed into petioles, are distributed from (ALiMV, AY590143), and Amaryllis potyvirus (AmaPV, Guatemala to Bolivia, mainly in the western Amazon AY566239). The phylogeny calculated by a maximum basin and adjacent lower slopes of the eastern Andes likelihood method showed that Hip-Br, Euch-B and (Meerow, 1989; Graf, 1992). Eucharis amazonica Linden Euch-C formed a monophyletic group supported by ex Planchon is erroneously know as E. x grandiflora 100% bootstrap with HiMV. This is the first report of Planchon et Linden (Meerow and Dehgan, 1984) by HiMV in Hippeastrum sp. and Eucharis sp. from Brazil. horticulturists, although they are two taxonomically dis- Our data support the taxonomic position of HiMV and tinct species (Meerow, 1989), both of which occur and other potyviruses isolated from amaryllis and Amazon are widely cultivated in Brazil. lily in a separated cluster of Amazon lily mosaic virus Viruses do not usually kill ornamental plants, but (AB158523). they often reduce their value. Several have been report- ed to cause foliar symptoms in Hippeastrum/Amaryllis Key words: potyvirus, Amaryllis, Amazon lily, orna- such as irregular light and dark green mosaic pattern as- mentals, phylogeny, cytopathology. sociated with Hippeastrum mosaic virus (HiMV) (Duarte et al., 2009; Raj et al., 2009) and yellow concen- tric ring and line patterns associated with Cucumber mo- saic virus (CMV) and Tomato spotted wilt virus (TSWV) (Derks, 1995). In Brazil, Groundnut ringspot virus (GRSV) was found associated with yellow spot and rings (Duarte et al., 2001). Some viruses such as Nerine Corresponding author: M.A.V. Alexandre latent virus (NeLV) induce symptomless infections Fax: +55.11.50871793 E-mail: [email protected] (Derks, 1995). In Eucharis, a mosaic symptom is fre- 010a_JPP761RP(Alexandre)_643 15-11-2011 18:02 Pagina 644 644 Hippeastrum mosaic virus in Brazil Journal of Plant Pathology (2011), 93 (3), 643-649 quently attributed to Amazon lily mosaic virus (AL- Transmission tests. Extracts of symptomatic Hippeas- iMV), HiMV, NeLV, CMV (Jayasinghe and Dijkstra, trum and Eucharis were obtained by grinding infected 1979; Terami et al., 1995, 2004; Cilli et al., 2002; Fuji et leaves in a mortar with 0.05 M phosphate-buffered al., 2004) and to other uncharacterized potyviruses saline (PBS) pH 7.4 + 0.5% Na2SO3 or 1% K2HPO4, (Brandes, 1964; Wu and Chang, 2003). Other symptoms and inoculated mechanically, using a carborundum abra- observed include: mottle caused by Tobacco ringspot sive, on different herbaceous plants: Amaranthaceae virus (TRSV) (Kahn and Scott, 1962) and stunting, leaf (Gomphrena globosa), Chenopodiaceae (Chenopodium mottling, streaks and malformations induced by an un- amaranticolor, C. quinoa, C. murale), Gentianaceae (Eu- named carlavirus (Kaminska and Szyndel, 1995). A stoma sp.), Solanaceae (Datura stramonium, Nicotiana virus identical or related to NeLV was also reported in benthamiana, N. nigrum, N. rustica, N. tabacum ‘Samsun’ Eucharis, in single or mixed infection with HiMV and ‘TNN’). Inoculated plants were maintained in an in- (Jayasinghe and Dijkstra, 1979). The present paper de- sect-proof greenhouse for up to 30 days to allow symp- scribes the biological properties, molecular analyses and tom development. The sanitary status of Eucharis and phylogenetic relationships of Brazilian isolates of HiMV Hippeastrum plants was verified by electron microscope found in Hippeastrum and Eucharis. observations. Asymptomatic Hippeastrum plants were not included in the assay because virus-like particles were observed (data not shown). MATERIAL AND METHODS RNA extraction, RT-PCR and sequencing. Total Virus source. The virus sources used in this study RNA was extracted from infected leaf tissues by a phe- were two potted Eucharis sp. displaying mosaic symp- nol method (Chomczynski and Sacchi, 1987) and pre- toms on leaves and tepals as well as leaf deformation, cipitated with ethanol. A fragment corresponding to the and one potted Hippeastrum x hybridum (Hip-Br) with 3’-terminal region of nuclear inclusion b (NIb) gene, the foliar mosaic (Fig. 1B) purchased in São Paulo/SP entire coat protein (CP) gene and 3’-untranslated region (Brazil). One of the two Eucharis plants was from a was amplified by RT-PCR, employing oligo dT and an florist shop in Campinas/SP (Euch-C) and the other universal primer for potyvirids (Gibbs and Mackenzie, (Euch-B) was from a nursery in NOVACAP Brasília/DF 1997) under the conditions described by the authors. (Fig. 1A). The amplified PCR products were subject to elec- trophoresis in a 1.5% agarose gels and Tris-acetate Electron microscopy. Leaf extracts from sympto- EDTA (TAE) buffer, purified, concentrated (CONCERT matic Eucharis and Hippeastrum were negatively stained Rapid Gel Extraction System, GIBCO-BRL, UK) and with 1% uranyl acetate. For ultrastructural observa- cloned into the pGEM-T Easy vector (Promega, USA). tions, small fragments of symptomatic leaves were fixed Three independent clones were sequenced in both for- and processed for transmission electron microscopy as ward and reverse directions, using an automated DNA described elsewhere (Kitajima and Nome, 1999). Exam- sequencer (ABI PRISM 377 Sequencer, Applied Biosys- inations were made with a Zeiss EM 900 transmission tems, USA). electron microscope. Fig. 1. A. Severe mosaic symptoms accompanied with light-green to striped symptoms on leaves and tepals (insert) of naturally in- fected Eucharis. B. Mosaic symptoms on naturally infected Hippeastrum leaves. 010a_JPP761RP(Alexandre)_643 15-11-2011 18:02 Pagina 645 Journal of Plant Pathology (2011), 93 (3), 643-649 Alexandre et al. 645 Sequence and phylogenetic analyses. Obtained se- Table 1. Nucleotide sequence identity (%) among Hippeas- quences were compared with those of other potyviruses trum mosaic virus, isolated from Hippeastrum (Hip-Br) and retrieved from GenBank using the Blast algorithm Eucharis (Euch-B) and (Euch-C), and 34 potyvirus species. (Altschul et al., 1997). Sequences of each region (NIb Potyvirus/Acession no. Hip-Br Euch-B Euch-C and CP) were aligned independently by eye using the CP Core NIb CP Core NIb CP* Core NIb program Se-Al, version 1.0 alpha 1 (Rambaut, 1996). PVY0 / U09509 58 58 47 57 58 47 58 49 The program PAUP*, version 4.0b10 for Macintosh SuCMoV /AF255677 58 56 56 58 58 55 57 55 (Swofford, 2002) was used to determine sequence iden- PepMoV/ NC001517 59 58 52 56 57 50 59 49 tities and to infer the phylogenetic relationship between TEV/ NC001555 61 58 58 58 57 57 59 56 BYMV/ AY192568 59 57 58 56 57 55 57 55 the viruses from Eucharis and Hippeastrum. Rooted ClYVV/ NC003536 58 55 55 56 54 57 56 54 trees were constructed by using maximum parsimony LMV/ NC003605 57 55 58 53 54 57 54 57 (MP) and maximum likelihood (ML) criteria, after esti- ZYMV/ AY279000 58 56 52 57 56 51 57 52 mating the nucleotide substitution, the proportion of in- BCMV/ NC003397 60 59 48 57 57 49 57 48 variables sites (I) and gamma distribution (G) with the TuMV/ AB093602 57 53 53 57 54 54 53 54 OrMV/ AB079650 58 56 51 55 56 48 57 48 software modeltest v.3.06 (Posada and Crandall, 1998).
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