1 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE CIÊNCIAS AGRONÔMICAS CAMPUS DE BOTUCATU CARACTERIZAÇÃO E IDENTIFICAÇÃO DE VÍRUS EM Allium spp. DAIANA BAMPI Tese apresentada à Faculdade de Ciências Agronômicas da UNESP - Campus de Botucatu, para obtenção do título de Doutor em Agronomia (Proteção de Plantas). BOTUCATU – SP Agosto – 2015 2 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE CIÊNCIAS AGRONÔMICAS CAMPUS DE BOTUCATU CARACTERIZAÇÃO E IDENTIFICAÇÃO DE VÍRUS EM Allium spp. DAIANA BAMPI Orientadora: Prof. Dra. Renate Krause Sakate Co-orientador: Prof. Dr. Marcelo Agenor Pavan Tese apresentada à Faculdade de Ciências Agronômicas da UNESP - Campus de Botucatu, para obtenção do título de Doutor em Agronomia (Proteção de Plantas). BOTUCATU – SP Agosto – 2015 3 4 5 Aos meus pais Arlindo e Clecy que apesar da distâcia, sempre estiveram ao meu lado apoiando e incentivando a realização deste trabalho! Dedico! III 6 A sabedoria de um homem não está em não errar, chorar, se angustiar e se fragilizar, mas em usar seu sofrimento como alicerce de sua maturidade. Augusto Cury IV 7 AGRADECIMENTOS À CAPES, pela concessão da bolsa de estudos ao longo do meu doutorado no Brasil. À CAPES, pela concessão da bolsa de estudos PDSE que possibilitou executar parte do meu doutorado no United States Department of Agriculture (USDA). À Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) pelo financiamento do projeto. À Universidade Estadual Paulista “Júlio de Mesquita Filho”, assim como ao Departamento de Defesa Fitossanitária pela oportunidade da realização do curso de doutorado Ao United States Department of Agriculture pela concessão do uso das suas dependências para efetuar os experimentos do projeto. À minha orientadora, Prof. Dra. Renate Krause Sakate, pela sabedoria, paciência e pelos ensinamentos que contribuíram para o meu crescimento profissional. Ao Dr. John Hammond pelo aceite do estágio em seu laboratório e por todo o carinho, paciência, confiaça e pelos ensinamentos durante o período em que estive sob sua orientação em Beltsville. Ao Prof. Dr. Marcelo Agenor Pavan pela ajuda e contribuições neste trabalho. Ao Dr. Valdir Yuki (IAC-Campinas) pela disponibilidade e contribuições com o trabalho. Aos técnicos de laboratório do USDA, Michael and Mary Ann pelo auxílio e ensinamentos. Aos professores Regiane Bueno, Antonio Carlos Maringoni, Marcelo Agenor Pavan, Renate Krause-Sakate e Carlos Gilberto Raetano pela formação. Aos colegas do laboratório Leonardo, Kelly, Mônika, Leyzimar, Milena, Bruno, Tatiana, Gerson, Letícia , Vinícius, Guilherme, Luiz, David e Júlio pela ajuda e amizade. V 8 Às amigas do laboratório de Virologia, Evelynne, Késsia e Cristiane, aos amigos da Pós- Graduação Ana Karolyna, Gilmar e Evandro e aos amigos de Beltsville Giseli, Thays, Rheam, Rodrigo, Shaguer, Willian, Judith, Sheron e Eleanor pelo companheirismo e pelos momentos de discontrações. À minha família, em especial meus pais pela educação, por todo incentivo e carinho. A Deus por me acompanhar todos os momentos desta caminhada. Por fim, agradeço sem exceção a todos que de certa forma contribuíram direta ou indiretamente com o desenvolvimento deste trabalho. Muito Obrigada! VI 9 SUMÁRIO Página LISTA DE TABELAS................................................................................................. IX LISTA DE FIGURAS................................................................................................. X 1 RESUMO.................................................................................................................. 1 2 SUMMARY............................................................................................................... 3 3 INTRODUÇÃO........................................................................................................ 5 4 REVISÃO BIBLIOGRÁFICA................................................................................ 7 4.1 O gênero Allium.................................................................................................... 7 4.2 A cultura do alho e o cenário produtivo. ............................................................. 7 4.3. Vírus infectando o gênero Allium......................................................................... 9 4.3.1. Gênero Allexivirus....................................................................................... 9 4.3.2. Gênero Carlavirus........................................................................................ 10 4.3.3. Gênero Potyvirus.......................................................................................... 11 4.4. Métodos para identificação e detecção de vírus..................................................... 4.5. Sintomatologia e controle de viroses em Allium spp............................................. 4.6. Danos provocados por viroses na cultura do alho.................................................. 13 14 15 5 REFERÊNCIAS ...................................................................................................... 17 CAPÍTULO I Leek yellow stripe virus isolates from Brazil form a distinct clade based on the P1 gene………………………………...………………………………………………… 24 ABSTRACT................................................................................................................. 24 1 INTRODUCTION.................................................................................................... 25 2 MATERIAL AND METHODS............................................................................... 28 3 RESULTS AND DISCUSSION............................................................................... 30 4 REFERENCES......................................................................................................... 34 CAPÍTULO II Narcissus yellow stripe virus and different species of the genera Allexivirus, Carlavirus and Potyvirus are present in ornamental Allium species available in the United States.................................................................................................................. 44 VII 10 ABSTRACT................................................................................................................. 1 INTRODUCTION………………………………………………………………… 2 MATERIAL AND METHODS…..………………………………………………. 3 RESULTS……………………………………………………………….…..……. 4 DISCUSSION……………………………………………………………………… 44 45 46 49 51 5 REFERENCES......................................................................................................... 55 CONCLUSÕES GERAIS........................................................................................... 62 APÊNDICE.................................................................................................................. 63 VIII 1 LISTA DE TABELAS Página Tabela 1. Oligonucleotídeos utilizados para identificação de vírus em Allium spp................................................................................................................................. 14 CAPÍTULO I Table 1. Comparison of the full genomic nucleotide (nt) and amino acid (aa) sequences between Brazilian Leek yellow stripe virus isolate MG (LYSV-MG) and representative LYSV isolates from China (NC_004011) Australia (JX429967.1) Australia (JX429965) and Japan (AB194623)…………………................................. 39 CAPÍTULO II Table 1. Allexivirus, Carlavirus and Potyvirus detected by RT-PCR and ELISA in ornamental Allium........................................................................................................ 59 Table 2. Percentage of each virus detected in ornamental Allium by RT-PCR.......... 60 IX 2 LISTA DE FIGURAS Página Figura 1. Organização genômica do vírus Tobbaco etch virus (TEV), gênero Potyvirus. A linha escura representa o RNA sentido positivo e os retângulos coloridos representam as ORF com suas respectivas massas moleculares (kDa). Os símbolos acima das ORF representam os sítios de clivagem das proteases (BERGER et al., 2005)………………......................................................................... 11 CAPÍTULO I Figure 1. Schematic representation of the genome organization of Leek yellow stripe virus (LYSV-MG). The terminal untranslated regions (UTR) are depicted by lines, and the open reading frame is shown as an open box with the different polyprotein domains (proteins). The small box above P3 represents the ORF PIPO. The numbers above the genome indicate the start of each region. The predicted proteinase cleavage sites in the polyprotein are indicated below the genome. The primers used to amplify the genome are indicated below the arrows with the estimated size of amplicon indicated above lines (not to scale)…………………………. 40 Figure 2. Phylogenetic analysis using the complete nucleotide sequences of Leek yellow stripe virus MG-isolate (LYSV-MG) and LYSV isolates from China, Australia and Japan .The tree was constructed the Neighbor-joining method implemented in the MEGA 6.0 software. Bootstrap values of 1000 replicates are shown. OYDV (AJ510223) was used as an outgroup………………………………. 41 Figure 3. Phylogenetic relationships of LYSV isolates based on the partial P1 gene. The tree was constructed the Neighbor-joining method implemented in the MEGA 6.0 software. Bootstrap values of 1000 replicates are shown. OYDV (AB219834) was used as an outgroup. Brazilian isolates used: BAN from Bandeirantes city, IPM from Ipomeri, SGO from São gotardo, SJU from Santa Juliana, SP from São Paulo, GUA from Guarapuava and LYSV-MG isolate from Santa Juliana selected for complete genome sequence………………………………………………………….. 42 X 3 CAPÍTULO II Figure 1. Particles of Allexivirus in Allium caeruleum (A) and Allium sphaerocephalon (B). Particles stained with uranyl acetate. Bar represents 500 nm................................................................................................................................. 61 Figure 2. Particles of Narcissus yellow stripe virus (NYSV) in Allium carinatum (A) and of Shallot latent virus (SLV) in Allium moly (B). Particles stained with uranyl acetate. Bar represents 500 nm.......................................................................... 61 XI 1 1. RESUMO O gênero Allium inclui espécies de importância para o consumo humano como o alho, cebola e espécies ornamentais. Algumas destas espécies por serem propagadas vegetativamente, podem ser infectadas por vírus pertencentes aos gêneros Allexivirus, Carlavirus e Potyvirus, frequentemente em infecções mistas. Leek yellow stripe virus (LYSV) pertence ao gênero Potyvirus e é considerada a espécie de maior importância na cultura do alho, de modo que foi sequenciado o genoma completo de um isolado de LYSV brasileiro (LYSV-MG) e avaliada sua diversidade genética. Seu genoma possui 10.341 nucleotídeos e codifica uma poliproteína de 3221 aminoácidos. Baseado na análise da região codificadora para a proteína P1, o isolado LYSV-MG, bem como demais coletados em regiões produtoras de alho, não foram classificados como pertencentes aos grupos S e N. Os isolados brasileiros apesar de não apresentarem a deleção na região da P1 (típica do grupo S), formaram um grupo monofilético muito próximo deste grupo e de um isolado de Okinawa, Japão. Em relação ao gene P1, os isolados brasileiros compartilharam 97-99% de identidade de nucleotídeos entre si e 51-64% com isolados de LYSV de outros países. Os dados sugerem que os isolados brasileiros de LYSV, o isolado de Okinawa e os isolados pertencentes ao grupo S podem ser provenientes de um ancestral comum antes da ocorrência da deleção na P1 e divergência dos isolados no grupo S. Como parte do doutoramento sanduíche, realizado na United States Department of Agriculture, sob orientação do Dr. John Hammond, foram estudados os vírus presentes em quatorze diferentes espécies de Allium ornamental nos Estados Unidos. As plantas foram adquiridas em floriculturas e plantadas a campo no outono de 2013 na região de Beltsville, MD. Folhas sintomáticas foram coletadas após a floração durante a primavera de 2014 e 2 avaliadas por PCR usando primers universais para os gêneros Allexivirus, Carlavirus e Potyvirus, e específicos para as espécies em cada gênero. Após a floração os bulbos foram colhidos e armazenados a 4º C durante 60 dias. Os bulbos foram plantados em vasos e mantidos sob temperatura controlada de 4º C até a emergência das folhas e posteriormente transferidos para casa-de-vegetação. As plantas foram testadas por ELISA utilizando antissoro específico para os vírus: Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV-C), Shallot virus X (ShV-X), Garlic common latent virus (GarCLV), Shallot latent virus (SLV), Leek yellow stripe virus (LYSV), Onion yellow dwarf virus (OYDV) e Shallot yellow stripe virus (SYSV). Amostras positivas para presença de vírus foram visualizadas ao microscopio eletrônico de transmissão. Nas plantas de Allium ornamental avaliadas por PCR foi detectada a presença de GarVB, GarVC, Garlic virus D (GarVD), Garlic virus E (GarVE) em Allium sphaerocephalon, GarVB, GarVC e ShVX em Allium caeruleum, LYSV em Allium bulgaricum, Allium flavum e Allium atropurpureum, SLV em Allium moly e Narcissus yellow stripe virus (NYSV) em Allium carinatum. Os testes de ELISA confirmaram a presença de GarVB, GarVC, ShVX, SLV e LYSV. Testes de microscopia eletrônica revelaram a presença de partículas flexuosas de 650-800 nm, típicas de allexivirus, carlavirus e potyvirus. Este foi o primeiro relato de NYSV em espécies de Allium spp. Além disto, A. sphaerocephalon e A. caeruleum não haviam sido previamente descritas como hospedeiras de allexiviruses nos Estados Unidos. Palavras-chave: Leek yellow stripe virus, alho, potyvirus, allexivirus, Allium ornamental, Narcissus yellow stripe virus. 3 CHARACTERIZATION AND IDENTIFICATION OF VIRUS IN Allium spp. Botucatu, 2015, 73p., Thesis (Doutorado em Agronomia/Proteção de Plantas) – Faculdade de Ciências Agronômicas, Universidade Estadual Paulista. Author: DAIANA BAMPI Adviser: RENATE KRAUSE SAKATE Co-adviser: MARCELO AGENOR PAVAN 2. SUMMARY The genus Allium includes important species used for human consumption, as garlic,onion and many other species grown as ornamentals. Some of these species are propagated vegetatively and can be infected by viruses belonging to the genera Allexivirus, Carlavirus and Potyvirus, often in mixed infections. Leek yellow stripe virus (LYSV) belongs to the genus Potyvirus and is considered the most important virus in Allium species. The complete genomic sequence of Leek yellow stripe virus garlic isolate from Brazil (LYSV- MG) has been determined. The LYSV-MG genome consists of 10,341 nucleotides and encodes a deduced polyprotein of 3,221 amino acids. Based on the analysis of the coding region for P1 protein, isolate LYSV-MG and others collected in garlic producing regions, could not be classified as belonging to the groups S and N. Brazilian isolates do not have the deletion present in the P1 from the S-type group but are more closely related to S-type than to N-type isolates. The Brazilian isolates formed a monophyletic group closer to S- type and one isolate from Okinawa, Japan. Brazilian isolates share 97-99% of P1 region nucleotide identity with each other, and 51-64% with different isolates from around the 4 world. The data suggest that Brazilian LYSV isolates are derived from an ancestral source of the Okinawa and S-type isolates, prior to the P1 deletion and divergence in the S-type isolates. As part of the doctorate sandwich in United States Department of Agriculture, under the supervision of Dr. John Hammond, viruses present in ornamental Allium from Beltsville, MD were detected. Bulbs of fourteen different species of Allium were purchased from retail nurseries and planted in the field during the fall of 2013. Leaf tissue from the flowering symptomatic plants were collected during spring 2014, and tested by PCR using generic primers for the genus Allexivirus, Carlavirus and Potyvirus. PCR-positive samples were re-tested using specific primers for specific species described in each genus. After flowering and foliage dieback, the bulbs were harvested and stored at 4º C for 60 days, before being planted in pots and maintained in a cooler until leaves emerged, and then transferred to a greenhouse. The sprouted plants were tested by ELISA using specific antibodie for the Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV- C), Shallot virus X (ShV-X), Garlic common latent virus (GarCLV), Shallot latent virus (SLV), Leek yellow stripe virus (LYSV), Onion yellow dwarf virus (OYDV) and Shallot yellow stripe virus (SYSV). Electron microscopy was performed for positive samples. The presence of GarV-B, GarV-C, Garlic virus D (GarV-D), Garlic virus E (GarV-E) in Allium sphaerocephalon, GarV-B, GarV-C and ShV-X in Allium caeruleum, LYSV in Allium bulgaricum, Allium flavum and Allium atropurpureum, SLV in Allium moly and Narcissus yellow stripe virus (NYSV) in Allium carinatum was confirmed by PCR. ELISA test was positive for GarV-B, GarV-C, ShV-X, SLV and LYSV. Electron microscopy revealed the presence of flexuous particles of 650-800 nm, typical of Allexivirus, Carlavirus and Potyvirus.This was the first report of NYSV in Allium spp. Moreover, A. sphaerocephalon and A. caeruleum had not previously been described as allexiviruses host in the United States. Keywords: Leek yellow stripe virus, garlic, potyvirus, allexivirus, Ornamental Allium, Narcissus yellow stripe virus. 5 3. INTRODUÇÃO O gênero Allium é o mais numeroso da família Alliaceae e inclui cerca de 750.000 espécies (BLOCK, 2010). Parte destas espécies são de importância na alimentação humana (alho, celoba, cebolinha e alho poró), outras possuem potencial medicinal e ainda há aquelas utilizadas como plantas ornamentais (KATIS et al., 2012). O alho é considerado a segunda principal espécie do gênero Allium mais amplamente consumida no mundo (FAO, 2015). O alho é propagado de forma vegetativa, propiciando o acúmulo de vírus pertencentes aos gêneros Potyvirus, Carlavirus e Allexivirus (FAJARDO et al., 2001), que podem ocorrer de forma isolada ou em infecções mistas. As espécies descritas em Allium spp. são: Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Turnip mosaic virus (TuMV) e Shallot yellow stripe virus (SYSV) pertencentes ao gênero Potyvirus; Garlic common latent virus (GarCLV) e Shallot latent virus (SLV) pertencentes ao gênero Carlavirus e 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 virus (GarMbFV) e Shallot virus X (ShVX) pertencentes ao gênero Allexivirus (ADAMS et al., 2011a; KATIS et al., 2012). Esses vírus causam sintomas de mosaico e estrias brancas ou amarelas. Os maiores danos na produção de alho têm sido atribuídos às infecções causadas por OYDV e LYSV. LYSV é considerado um dos vírus mais importantes na cultura devido sua ampla distribuição e pela redução do rendimento e qualidade dos bulbos (LOT et al., 1998). No Brasil LYSV é considerado um dos vírus predominantes em campos comerciais de produção de alho (FAYAD-ANDRE et al., 2011). 6 Os isolados de LYSV, segundo classificação de Yoshida et al., 2012, podem ser classificados em dois grandes grupos, S e N, analisando-se a região codificadora para a proteína P1. O grupo S possui uma deleção na P1 em torno de 201 nucleotídeos em relação ao grupo N (TAKAKI et al., 2005 e YOSHIDA et al., 2012) e está presente na China, Sul do Japão, Coréia e Austrália, enquanto que os isolados do grupo N foram relatados no Norte do Japão, Europa, Uruguai e Austrália (YOSHIDA et al., 2012). No Brasil, apesar da importância das infecções com LYSV, não há dados moleculares sobre os isolados de LYSV brasileiros. Portanto, o genoma completo de um isolado de LYSV proveniente de alho do Brasil foi sequenciado e a região para a proteína P1 avaliada para este isolado bem como demais coletados em diversas regiões produtoras de alho. Durante o doutorado sanduíche no United States Department of Agriculture, sob coordenação do Dr. John Hammond, também foram avaliadas as espécies de vírus presentes em plantas de Allium ornamental nos Estados Unidos. Foi identificada a presença de diversos vírus relatados em espécies de alho e cebola e a presença de um potyvirus até então não relatado em Allium spp, o Narcissus yellow stripe virus. O presente trabalho foi dividido em dois capítulos intitulados: 1) “Leek yellow stripe virus isolates from Brazil form a distinct clade based on the P1 gene.” redigido em inglês conforme as normas da revista Journal of Plant Pathology e 2) “Narcissus yellow stripe virus and different species of the genera Allexivirus, Carlavirus and Potyvirus are present in ornamental Allium species available in the United States” redigido em Inglês conforme as normas da revista Plant Disease. 7 4. REVISÃO BIBLIOGRÁFICA 4.1 O Gênero Allium O gênero Allium inclui cerca de 750.000 espécies (BLOCK, 2010), caracterizadas por apresentarem bulbos envoltos por túnicas membranosas (algumas vezes fibrosas), tépalas livres ou semi-livres (FRITSCH et al., 2006). Algumas dessas espécies podem ser usadas na alimentação, outras possuem potencial medicinal e um maior grupo de espécies são usadas como plantas ornamentais (KATIS et al., 2012). As utilizadas na alimentação humana são: cebola (Allium cepa), cebolinha (Allium schoenoprasum), alho (Allium sativum) e alho poró (Allium porrum) (BREWSTER, 1994). As espécies ornamentais distinguem-se das demais pela diversidade de flores e tamanhos (MIGLINO et al., 2011). Evidências sugerem que o alho e cebola foram domesticadas na Ásia central e então propagadas para o Oriente Médio e Mediterraneo, que é considerado o centro secundário destas espécies (BLOCK, 2010). Porém o centro de origem de todas as espécies de Allium spp não foi completamente elucidada. 4.2 A cultura do alho e o cenário produtivo O alho, é considerado a segunda principal espécie do gênero Allium mais amplamente consumida no mundo (FAO, 2015). Sua propagação se dá através do plantio dos bulbilhos (FILGUEIRA, 2007). É uma hortaliça que possui alto valor nutricional, composta por 8 vitaminas A, B2, B6, C, aminoácidos, adenosina, sais minerais (ferro, silício, iodo), enzimas, como a aliniase (YOSHIDA et al.,1987; RESS et al., 1993; BIANCHI et al., 1997). O alho é uma cultura de clima temperado, portanto, as temperaturas baixas são ideais para que a bulbificação ocorra (SOUZA e MACEDO, 2009). As temperaturas médias mensais exigidas para formação dos bulbos são de aproximadamente 13 °C e para um bom desenvolvimento das plantas é de 24 °C, sendo que a falta destas temperaturas pode afetar a formação dos bulbos (MASCARENHAS, 1978). No Brasil pesquisadores adaptaram a técnica de vernalização pré-plantio, que consiste em armazenar o alho- semente em câmara com temperatura de 3 a 5 °C, por um período de 40 a 60 dias possibilitando o plantio de cultivares nobres de alho originárias da Argentina e do Sul do Brasil, em regiões onde as condições termo-fotoperiódicas não satisfazem as exigências da planta (SOUZA e MACEDO, 2009). Atualmente existem no mercado brasileiro basicamente três grupos de cultivares que foram desenvolvidas para atender a demanda do consumidor. Essas cultivares são diferenciadas pela duração do ciclo, exigências de fotoperíodo e de temperatura (FILGUEIRA, 2000). As cultivares precoces ou comuns possuem um ciclo mais curto, em torno de quatro meses, são cultivares menos exigentes em fotoperíodo e temperatura, porém apresentam bulbilhos pequenos e de menor valor comercial (Ex: Branco mineiro e Cateto roxo). As cultivares de ciclo mediano ou semi-nobre, possuem um ciclo de aproximadamente cinco meses, sendo essas cultivares mais exigentes em fotoperíodo e temperatura, porém produzem bulbilhos mais graúdos e alcançam melhor cotação comercial (Ex: Amarante). Para as cultivares nobres ou tardias: o ciclo é mais longo, em torno de seis meses, e são cultivares muito exigentes em fotoperíodo (mínimo de 13 horas) e exigem temperaturas baixas, produzindo bulbilhos graúdos de alta qualidade comercial, portanto obtém alta cotação comercial, o que as torna competitivas ao alho importado (Ex: Chonan, Roxo-Pérola-de-Caçador e Quitéria) (FILGUEIRA, 2007; FAYAD-ANDRÉ, 2011). O cultivo do alho nobre roxo no Brasil se iniciou no ano de 1977 no estado de Santa Catarina. Sem conhecimento tecnológico, as primeiras produtividades de alho nobre roxo no estado foram em torno de 3.000Kg/ha. (LUCINI, 2008). Hoje com o avanço da tecnologia muitas lavouras conseguem produtividades em torno de 13.000 Kg/ha, mesmo assim, a produção brasileira não é suficiente para suprir o consumo anual. A 9 produção brasileira representa 33% do abastecimento interno, o restante é importado da China (45%) e Argentina (25%) (CARVALHO, 2013). Nos anos de 1990, a produção de alho nacional atingiu quase que a totalidade da demanda consumida no Brasil. Porém, com o aumento do consumo, a importação cresceu em números exorbitantes, devastando a produção interna, devido a desvalorização do alho nacional (CARVALHO et al., 2013). De acordo com dados obtidos pelo Levantamento Sistemático da Produção Agrícola (2015), em 2014 o Brasil produziu 93.859 toneladas, com rendimento médio de 9.629 Kg/ha, em uma área de 9.748 hectares. Na safra 2014 o Estado de Santa Catarina foi o que mais produziu no País, em uma área de 2.230 hectares, produziu 21.449 toneladas. Em seguida veio o Estado de Minas Gerais, com produção de 21.173 toneladas, seguido de Goias, com 21.040 toneladas e Rio Grande do Sul com 16.614 toneladas. O estado de Minas Gerais apresentou um rendimento médio de 13.538 Kg/ha, seguido de Santa Catarina com rendimento de 9.618 Kg/ha, Goiás 9.277 Kg/ha e Rio Grande do Sul 7,6Kg/ha (Levantamento Sistemático de Produção Agricola, 2015). A China é o país com maior produção, sendo que produz em média 76% da produção mundial (FAO 2015). 4.3 Vírus infectando o gênero Allium Grande parte das espécies do gênero Allium são propagadas de forma vegetativa, propiciando o acúmulo de vírus pertencente aos gêneros Allexivirus Carlavirus e Potyvirus (KATIS et al., 2012). 4.3.1 Gênero Allexivirus O gênero Allexivirus pertence à família Alphaflexiviridae. As espécies possuem partículas filamentosas flexuosas com 800 nm de comprimento e 12 nm de diâmetro, uma molécula de RNA linear de fita simples senso positiva com 9.0 kb de tamanho, incluindo a cauda poly (A) na extremidade 3‟ e a CAP no terminal 5‟. A organização genômica baseia-se em seis ORFs (“open reading frame” ou fase aberta de leitura). A ORF1 codifica a polimerase viral; a ORF2 e 3 codificam a TGB (Triple gene block) que está envolvida no movimento; a ORF4 codifica uma proteína que não apresenta homologia com nenhuma proteína conhecida; ORF5 correspondente à proteína capsidial e a ORF6 codifica uma proteína com função ainda desconhecida, e possui uma estrutura denominada “dedo de zinco” que tem a capacidade de se ligar a ácidos nucléicos (ADAMS 10 et al., 2011a; KING et al., 2012). A transmissão dos vírus pertencentes a esse gênero ocorre através de ácaros da espécie Aceria tulipae (KANG et al., 2007) e além disto podem ser transmitidos por extrato vegetal para espécies de Allium e para Chenopodium quinoa, C. amaranticolor e C. murale que mostram sintomas de lesões locais (KATIS et al., 2012). Oito são as espécies pertencentes ao gênero que podem infectar Allium spp.: GarV-A, GarV-B, GarV-C, GarV-D, GarV-E, GarV-X, GarMbFV e ShVX, porém prevalecem na cultura do alho (KATIS et al., 2012). Os allexivirus estão amplamente distribuídos em todo mundo e podem causar sintomas mais severos quando associados com um potyvirus (TAKAICHI et al., 2001). Entretanto, há falta de informação dos efeitos causados pela infecção, devido à dificuldade de se isolar cada espécie do complexo viral. Segundo Cafrune et al. (2006), GarVA seria a espécie que causa maiores danos à cultura do alho e afeta a sua produção. 4.3.2 Gênero Carlavirus O gênero Carlavirus pertence a família Betaflexiviridae. São vírus constituídos por uma molécula de RNA fita simples, senso positiva, com partículas virais flexuosas e alongadas com 610 a 700 nm de comprimento e 12 a 13 nm de diâmetro. cotendo seis ORFs: a ORF1 codifica a replicase viral; a ORF2, 3 e 4 formam o Triple Gene Block (TGB), conjunto de proteínas envolvidas no movimento célula a célula (CHEN et al., 2004). A ORF5 codifica a proteína capsidial e a ORF6 codifica uma proteína com função ainda desconhecida, talvez responsável pela transmissão por afídeos, envolvimento na replicação do RNA, e/ou silenciamento gênico (KING et al. 2012). As espécies de carlavirus mais comuns em Allium spp. são GarCLV e SLV (KATIS et al., 2012). Estes virus são transmitidos por afídeos de forma não persistente, sendo as principais espécies transmissoras do SLV: Aulacorthum solani, Myzus ascalonicus, M. persicae, Neutoxoptera formosana e Aphis gossypii (BOS et al., 1978). Para GarCLV não há relatos de espécies vetoras (KATIS et al., 2012). Celosia argentea, C. amaranticolor, C. quinoa e Nicotiana occidentalis foram relatadas como hospedeiras do SLV e GarCLV (VAN DIJK, 1993a). Infecções com GarCLV e SLV são geralmente latentes, porém os sintomas são mais severos quando ocorrem em infecções mistas com 11 potyvirus ou allexivirus (TAKAICHI et al., 2001). 4.3.3 Gênero Potyvirus O gênero Potyvirus pertence à família Potyviridae, que inclui seis gêneros (Potyvirus, Ipomovirus, Maclunavirus, Rymovirus, Tritimovirus, Brambyvirus e Bymovirus) (KING et al., 2012). A família Potyviridae constitui o maior e mais importante grupo de vírus de plantas do ponto de vista econômico, sendo que aproximadamente 30% dos vírus de plantas já caracterizados pertencem a essa família (KING et al., 2012). O genoma é composto por uma molécula de RNA fita simples, senso positiva e possuem partículas filamentosas flexuosas com 680-900 nm de comprimento e 11-13 nm de diâmetro. A organização genômica baseia-se em uma única ORF responsável por codificar uma poliproteína de aproximadamente 345 kDa. Apresentam a proteína VPg ligada covalentemente ao terminal 5‟, o genoma possui cerca de 9,7 Kb de extensão e apresentam uma cauda poli (A) no terminal 3‟ (ADAMS et. al., 2011b). Possuem uma pequena ORF incorporada na região codificadora para a proteína P3 denominada de Pretty Interesting Potyviridae ORF - PIPO (CHUNG et al., 2008). O RNA genômico é envolto por um capsídeo formado por aproximadamente 2.200 cópias. A poliproteína codificada sofre autoproteólise dando origem a cada uma das proteínas virais: P1, HC-Pro, P3, 6KI, CI, 6K2, VPg, NIa, NIb e CP (Fig. 1) (ADAMS et al., 2011b). Figura 1. Organização genômica do vírus Tobbaco etch virus (TEV), gênero Potyvirus. A linha escura representa o RNA sentido positivo e os retângulos coloridos representam as ORFs com suas respectivas massas moleculares (kDa). Os símbolos acima das ORF representam os sítios de clivagem das proteases (Adaptado de BERGER et al., 2005). Uma importante característica dos potyvirus é que a maioria das proteínas codificadas são multifuncionais (URCUQUI-INCHIMA et al., 2001). A proteína 12 P1 é a mais variável tanto em tamanho quanto em sequência de nucleotídeos e aminoácidos do genoma dos potyvirus e considerada a proteína mais apropriada para estudos de relacionamento filogenético e estudos de história evolutiva dos potyvirus. (VALLI et al., 2007). O papel da proteína P1 no processo infeccioso não é totalmente conhecido. Estudos relataram seu envolvimento no aumento da patogenicidade (PRUSS et al., 1997), amplificação do genoma (VERCHOT e CARRINGTON, 1995) , acumulação de proteínas (JOHANSEN e CARRINGTON, 2001), autoproteólise (VERCHOT et al., 1991), auxilia a HC-Pro na supressão do silenciamento gênico (RAJAMAKI et al., 2005; VALLI et al., 2007) e interfere na sintomatologia (RYAN & FLINT, 1997). A proteína HC-Pro está envolvida na transmissão por afídeos, proteinase e autoclivagem do carboxi-terminal, movimento sistêmico, supressão do silenciamento gênico, sinergismo e desenvolvimento de sintomas (MAIA et al., 1996). A proteína P3 apresenta função relacionada à patogenicidade e a proteína 6K1 apresenta função desconhecida (RODRIGUEZ-CEREZO et al., 1993; LANGENBERG e ZHANG, 1997). A proteína CI tem função de helicase e movimento célula-a-célula (LAIN et al., 1990). A proteína 6K2 tem função de ancorar o complexo replicativo às membranas da célula (RESTREPO-HARTWIG e CARRINGTON, 1992). A proteína NIa (VPg-Pro) tem função de localização celular, proteinase e interações proteína-proteína (HONG et al., 1995). A proteína NIb possui função de RNA polimerase dependente de RNA (RdRp) (HONG e HUNT, 1996) e a proteína CP (proteína capsidial) está envolvida na transmissão por afídeos, movimento célula-a-célula e sistêmico e na montagem da partícula viral (SHUKLA e WARD, 1988). O ICTV (Comite internacional de taxonomia de vírus) utiliza como critérios para demarcação de novas espécies no gênero Potyvirus a identidade de aminoácidos inferior a 80% e sequência de nucleotídeos menor que 76% para a proteína capsidial ou o genoma completo (KING et al., 2012). Além disto, diferentes sítios de clivagem da poliproteína, gama de hospedeiros, ausência de proteção cruzada, transmissão ou não por sementes, reações de hospedeiros, diferenças sorológicas e vetores primários diferentes (BERGER et al., 2005). As espécies frequentemente relatadas em Allium spp são OYDV e LYSV, sendo estas consideradas economicamente importantes pela sua ampla distribuição geográfica e pelos danos na produção (CONCI et al., 1992; VAN DIJK, 1993b; CHEN et al., 2001). Os potyvirus são transmitidos por afídeos em uma relação de transmissão do 13 tipo não persistente (COSTA, 1998). As principais espécies de afídeos que transmitem LYSV são M. persicae, A. fabae, A. gossypii, A. nerii, Hyperomyzus carduellinus, Rhopalosiphum maidis, R. padi, Schizaphis graminum, e Uroleucon sonchi ( LUNELLO et al., 2002). Para OYDV existem mais de 50 espécies descritas como vetoras, porém as mais importantes são M. ascalonicus, M. persicae, R. maidis, e Acyrthosiphon pisum (VAN DIJK, 1993a). Alguns isolados de LYSV causam lesões locais necróticas em C. album, C. murale, C. amaranticolor e C. quinoa (KATIS et al., 2012). 4.4 Métodos para identificação e detecção de vírus Os vírus que infectam o gênero Allium ocorrem muitas vezes em infecções mistas dificultando a determinação dos sintomas relacionados a cada vírus (WARD et al., 2009). Várias técnicas podem ser utilizadas na detecção de vírus, incluindo microscopia eletrônica, hibridização de ácidos nucléicos, ELISA (Enzyme Linked Immuno Sorbent Assay), RT-PCR (Transcriptase Reverse - Polymerase Chain Reaction) e métodos biológicos, sendo as técnicas moleculares as mais utilizadas e precisas (MARTIM et al., 2000). As técnicas moleculares, como de RT-PCR são altamente específicas e sensíveis, permitindo a detecção de vírus a partir de uma pequena quantidade do RNA alvo (LUNELLO et al., 2004). Para os principais vírus de Allium spp. já estão disponível na literatura oligonucleotídeos para identificação dos gêneros e específicos para as espécies (Tabela 1). Além da técnica de PCR tradicional, o PCR quantitativo já tem demonstrado alta eficiência para algumas espécies (LUNELLO et al., 2004; MITUTI, 2013). Também foram relatadas algumas plantas indicadoras para vírus que infectam alho como C. quinoa, C. murale, C. amaranticolor e N. occidentalis (VERBEEK et al., 1995). O teste ELISA é o principal método para o diagnóstico utilizado em larga escala na detecção de vírus em espécies de Allium. Antissoros específicos comerciais são disponíveis para as duas espécies de carlavirus (GarCLV e SLV), além de ser empregado também o antissoro universal para potyvirus comercializado pela empresa Agdia (MITUTI, 2013). 14 Tabela 1: Oligonucleotídeos utilizados para identificação de vírus em Allium spp. Primer Sequência (5’- 3’) Gênero ou espécie Referência SLV/GCLV 7303 SLV/GCLV 7665 GGNTKKGAAWCTGGGAGDCC CATKTMATTCCAAACAACNGGYGC Carlavirus Mituti et al., 2015 PV1 WCIEN GAT TTA GGT GAC ACT ATA GT16- ATG GTT TGG TGY ATY GAR AAT Potyvirus Gibbs and Mackenzie, 1997 Mota et al., 2004 Cpallexi-senso2 Cpallexi-anti 1 CTACCACAATGGTTCCTC GATTTCTTTAACGCAGTG Allexivirus Oliveira, 2013 OYDV-F OYDV-R CRCCARTTCTGGATAAYGC CTCCGTGTCCTCATCCG OYDV Mituti et al., 2015 P1-1170 LY5P CTTCMTCRCASTCATGKTCC AATCTCAACACAACTTATRC LYSV Mituti et al., 2015 Yoshida et al., 2012 SLV 7044 SLV 8004 CTTTTGGTTCACTTTAGG GCACGCAATAGTCTACGG SLV Mituti et al., 2011 GCLV 7303 SLV/GCLV 7665 GGSTTTGARACTGGGAGGCC CATKTMATTCCAAACAACNGGYGC GarCLV Mituti et al., 2015 GarV-A1 GarV-A2 CCCAAGCTTACTGGAAGGGTGAATTAGAT CCCAAGCTTAGGATATTAAAGTCTTGAGG GarV-A Melo Filho et al., 2004 CPBS2 CPBA1 GCAGAATAARCCCCCYTC RAAGGGTTTATTCTGTTG GarV-B Oliveira, 2013 GarV-C1 GarV-C2 CCCAAGCTTCATCTACAACAACAAAGGCG CCCAAGCTTATAAGGGTGCATGATTGTGG GarV-C Melo Filho et al., 2004 GarV-D1 GarV-D2 CCAAGCTTAAGCAAGTGAAGAGTGTAAG CCAAGCTTTTTGGAAGAGGAGGTTGAGA GarV-D Melo Filho et al., 2004 CPShS1 CPShA1 GAATGCATCAGGRGAYCTC GCRGGRGGTTTCTTCTG ShV-X Oliveira, 2013 CPES1 CPEA1 CPXS2 CPXA1 GGRTCGTCACGATTYGTTAC YTTGAACCTCATACCYCC GCCTTCTGAAAATGACTTAG CTAGGATTTGCTGTTGGG GarV-E GarV-X Oliveira, 2013 Oliveira et al., 2013 4.5 Sintomatologia e controle de viroses em Allium spp. Os vírus interferem diretamente nos fenômenos vitais e nos processos bioquímicos e fisiológicos da planta, na síntese de proteínas, na fotossíntese, no transporte de água e de elementos minerais através do sistema vascular ocasionando reduções significativas na concentração de clorofila, vigor vegetativo e prejudicando a formação dos bulbos, devido a redução na produção de carboidratos (ZAMBOLIM et al., 2007). 15 Alguns vírus formam inclusões citoplasmáticas afetando diretamente os componentes celulares, como nucléolo, núcleo, citoplasma e suas organelas (EL-ELA et al., 2006), como os potyvirus que formam inclusões citoplasmáticas do tipo cata-vento no citoplasma das plantas infectadas (HULL, 2002). Os vírus em alho causam sintomas de mosaico, amarelecimento, estrias e clorose levando a grandes perdas econômicas e danos na produção, principalmente quando há infecção por OYDV e LYSV (CONCI et al., 1992; VAN DIJK, 1993a; CHEN et al., 2001; LUNELLO et al., 2007). Sintomas típicos de infecção viral por allexivirus em alho incluem o subdesenvolvimento da planta e folhas retorcidas devido o ataque de ácaros (KANG et al. 2007). Os carlavirus apresentam-se aparentemente assintomáticos (PALUDAN, 1980). Os allexivirus e carlavirus quando em infecção mista com potyvirus induzem sintomas mais severos e maiores perdas de produção (CONCI et al., 2003; LOT et al., 1998; CAFRUNE et al., 2006). Os vírus em alho devem ser controlados preventivamente, principalmente pela utilização de bulbos livres de vírus. A associação da termoterapia e a cultura de tecido propicia a obtenção de bulbos livres de vírus e tem sido empregada com sucesso (PAVAN, 1998). Evitar cultivos sucessivos e próximos à lavouras mais velhas, realizar o plantio em áreas com altitudes elevadas, controlar plantas hospedeiras de vírus e dos afídeos, eliminar restos culturais também são medidas preventivas indicadas para o controle dos vírus em alho (DUSI, 1995). O controle de afídeos através da aplicação de inseticidas, visando redução de incidência de potyvirus e carlavirus não é uma prática recomendada. Os afídeos transmitem os vírus de maneira não persistente, desta forma o vírus é transmitido em poucos segundos após a aquisição e muitas vezes a aplicação do inseticida acelera o metabolismo dos pulgões, aumentando a transmissão logo após a aplicação (ZAMBOLIM et al., 2007). Para o gênero Allexivirus, o ácaro vetor pode ser controlado através do tratamento por imersão dos bulbos ou bulbilhos em acaricidas antes do plantio (MENEZES SOBRINHO, 1997), bem como durante seu armazenamento (OLIVEIRA et al., 2014). 4.6 Danos provocados por viroses na cultura do alho As viroses são consideradas um dos principais problemas na cultura do alho devido sua propagação vegetativa (VAN DIJK 1993a; CHEN et al., 2001; FAJARDO et al., 2001). Os maiores danos na produção em alho têm sido atribuídos às infecções causadas por OYDV e LYSV, porém estes se agravam na presença de infecções 16 mistas entre os diferentes vírus do complexo viral que infectam o alho (FAYAD-ANDRÉ, 2011). De acordo com Mituti et al. (2015), 81 % das plantas de alho nobre coletadas em campos comerciais nas safras de 2007 a 2011 nas principais regiões produtoras do Brasil encontraram-se infectadas por potyvirus. Destas, 41 % estavam infectadas somente com potyvirus, 16% apresentaram infecção mista com LYSV e OYDV, 25 % apresentaram infecção mista de potyvirus e allexivirus, 6 % estavam infectadas com potyvirus e carlavirus e 9% apresentaram infecção mista com os tres gêneros, LYSV e OYDV foram encontrados com a mesma frequência. A ocorrência de infecções mistas torna difícil determinar as perdas e danos atribuidos a cada vírus (WARD et al., 2009). No entanto, alguns estudos mostram que infecções com LYSV reduziram o peso de bulbos em até 28 %. Enquanto que com o OYDV a redução foi de 48 a 65%. Já em infecções mistas com LYSV e OYDV houve uma redução no peso de bulbos de 56% a 84% (LOT et al., 1998). Os Allexivirus são o segundo gênero com maior prevalência em campos comerciais no Brasil, tendo sido detectados em 49 % das amostras avaliadas de 2007 a 2011 com predominância das espécies GarVD e GarVA (MITUTI et al., 2015). 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Variability in the P1 gene helps to refine phylogenetic relationships among Leek yellow stripe virus isolates from garlic. Archives of Virology, v. 157, p. 147– 153, 2012. ZAMBOLIN, L. et al. Manejo Integrado de Doenças e Pragas Hortaliças. Editora UFV: Viçosa - MG, 2007, p. 627. 23 CAPÍTULO I Leek yellow stripe virus ISOLATES FROM BRAZIL FORM A DISTINCT CLADE BASED ON THE P1 GENE1 1 Aceito para publicação na revista Journal of Plant Phatology 24 Leek yellow stripe virus ISOLATES FROM BRAZIL FORM A DISTINCT CLADE BASED ON THE P1 GENE. Daiana Bampi1,3, Tatiana Mituti2, Marcelo Agenor Pavan1, John Hammond3, Renate Krause-Sakate1 1 Faculdade de Ciências Agronômicas, FCA-UNESP, Departamento Proteção Vegetal, Rua José Barbosa de Barros, 1780, CEP: 18610-307, Botucatu, SP, Brazil; 2Escola Superior de Agricultura Luiz de Queiroz – Universidade de São Paulo, ESALQ/USP, Departamento de Fitopatologia e Nematologia, Av. Pádua Dias, 11, CEP 13418-900, Piracicaba – SP, Brazil; 3USDA-ARS, USNA, Floral and Nursery Plants Research Unit, 10300 Baltimore Avenue, B-010A, Beltsville, Maryland, United States, 20705. Daiana Bampi Fax number: +55 (14) 3880-7132 Email: daiana@fca.unesp.br Abstract The complete genome sequence of a garlic isolate of Leek yellow stripe virus (LYSV) from Brazil (LYSV-MG) was determined and its phylogenetic relationships with other LYSV isolates from other parts of the world were inferred. The LYSV-MG genome consists of 10,341 nucleotides and encodes a polyprotein of 3,221 amino acids. Brazilian LYSV isolates are more closely related to S-type than to N-type isolates but do not have a deletion in the P1 gene. Partial nucleotide sequence analysis of the P1 gene revealed 97-99% identity among Brazilian isolates, and 51-64% identity between Brazilian isolates and isolates from other countries for which sequence information is available in Genbank. The Brazilian isolates formed a monophyletic group closest to the S-type isolates and one isolate from Okinawa (O-type). These data suggest that Brazilian LYSV isolates are derived from an ancestral source of the Okinawa and S-type isolates, prior to the occurrence of the P1 deletion and divergence of the S-type isolates. Keywords: Allium sativum, Potyvirus, variability. mailto:daiana@fca.unesp.br 25 Introduction Garlic (Allium sativum L.) can be infected by multiple viruses, mainly of the genera Allexivirus, Carlavirus and Potyvirus (Conci et al., 2003; Melo-Filho et al., 2006; Lunello et al., 2007; Katis et al., 2012). Leek yellow stripe virus (LYSV) belongs to the genus Potyvirus in the family Potyviridae, with flexuous and filamentous particles containing a single-stranded, positive sense RNA genome. It was first reported in 1987 in garlic by Walkey et al. (1987). LYSV is considered the most important virus in garlic due to the wide distribution and severe symptoms (Van Dijk, 1993, 1994). Infected garlic plants exhibit mosaic and yellow stripe symptoms on leaves and may produce smaller, malformed bulbs resulting in severe losses of yield and quality that vary depending on the cultivar (Lot et al., 1998; Takaki et al., 2005). LYSV is transmitted by aphids and may easily re-infect virus-free garlic plants in the fields (Yoshida et al., 2012). Mituti et al. (2015) found that 81 % of field-grown noble garlic in Brazil were infected with potyviruses of which 41% were infected only by potyviruses (Onion yellow dwarf virus (OYDV) either LYSV), 16% were mixed infected with OYDV and LYSV, 25% were infected with potyviruses and allexiviruses, 6% were infected with potyviruses plus carlaviruses, and 9% were infected with potyviruses, allexiviruses, and carlaviruses. Previous studies showed that LYSV was the most prevalent virus from garlic in Brazil (Fayad-Andre et al., 2011). The occurrence of multiple virus infections in the same plant makes it difficult to determine and predict yield loss attributed to each virus (Ward et al., 2009). Single infections of LYSV showed a maximum reduction in bulb weight of 17- 59%, depending on the cultivar; single infections of OYDV reduced bulb weight by up to 48-65% and mixed infections with OYDV and LYDV had a synergistic effect with bulb weight reduced by as much as 56- 84% in different cultivars (Lot et al., 1998). 26 The P1 coding region has been demonstrated to be the most variable of the potyvirus genes. The roles of P1 in potyvirus infection are still emerging, although there are indications that P1 may enhance the ability of HC-Pro to suppress RNA silencing (Rajamäki et al., 2005; Valli et al., 2006), and to increase viral pathogenicity (Pruss et al., 1997). One of the first functions identified was self-cleavage at the P1/HC-Pro junction (Verchot et al., 1991); further work identified stimulation of genome amplification (Verchot and Carrington, 1995), and increasing mRNA and protein accumulation (Johansen and Carrington, 2001). RNA binding activity has been reported for the P1 protein of Turnip mosaic virus (Soumounou and Laliberte, 1994) and Potato virus A (Merits et al., 1998), while RNA binding has been attributed to involvement of P1 in viral movement (Arbatova et al., 1998). More recent work has shown that two single amino acid mutations in the P1 of Plum pox virus (PPV) were associated with milder symptoms, and that one mutation (W29R) reduced both virus accumulation and symptom severity in Prunus persica, while the other (V139E) caused symptom attenuation in herbaceous hosts (Maliogka et al., 2012); this, combined with the studies on P1 diversity of Valli et al. (2007), indicates that P1 variability is associated with host adaptation, and that particular viral variants are adapted to specific hosts or even specific host genotypes. The N-terminal region of P1 shows a high variability in both length and amino acid sequence, and host-dependent regulation of P1 self-cleavage may function to modulate the efficiency of establishment of infection, and to minimize induction of host defense responses; Pasin et al. (2014) showed that an infectious clone of PPV from which P1 was deleted was still able to replicate and spread systemically in Arabidopsis, and that this was unrelated to any direct effects on RNA silencing suppression. However, P1 was shown to be indirectly involved in RNA silencing suppression, because when P1 was not able to self-cleave, HC-Pro function was limited and infectivity adversely affected in wild-type 27 Arabidopsis, but not in an Arabidopsis mutant defective in RNA silencing; these authors also demonstrated that the N-terminal 164 residues of PPV P1 are not required for P1 self- processing, but act as a negative regulator of self-cleavage in an in vitro assay (Pasin et al., 2014). Deletion of this regulatory region led to enhanced early amplification of the PPV genome, but reduced overall viral accumulation; it was therefore proposed that P1 acts to fine-tune potyviral regulation by interacting with specific host-plant effectors and activating the P1 protease activity to release HC-Pro with its RNA silencing suppression activity. As HC-Pro may itself induce further host defenses, the combined effects of P1 negative regulation and HC-Pro silencing suppression activity may maintain higher long- term replication in hosts to which the virus is adapted (Pasin et al., 2014). The P1 protein of Tobacco etch virus (TEV) has both a nucleolar localization signal and a nuclear export signal, and to traffic in and out of the nucleus and nucleolus during infection (Martinez and Daròs, 2014). When an infectious clone of TEV was separately labeled with different fluorescent proteins fused to P1 and NIb, the labeled P1 was only detected close to the infection front, whereas NIb was detected in cells from early to late stages if infection, suggesting that P1 is rapidly degraded. P1 was also shown to bind specifically to 60S ribosomal subunits during infection, and it was suggested that P1 stimulates protein translation, whereas HC-Pro inhibits translation, potentially suppressing cap-dependent translation of host mRNAs in favor of viral synthesis (Martinez and Daròs, 2014). P1 may thus play multiple roles in adaptation of potyviruses to specific hosts, and by virtue of this role in host adaptation, it is therefore an appropriate sequence to utilize for phylogenetic comparisons to understand the evolutionary history of a potyvirus species (Valli et al., 2007). In this work, we determined the complete nucleotide sequence of a representative LYSV isolate collected from garlic crops in Brazil and compared it with the complete 28 sequences of LYSV isolates from China, Japan and Australia. We have further analyzed the nucleotide sequence of the partial P1 gene of various LYSV isolates from garlic collected in several locations in Brazil, and compared these with other isolates for which sequence information is available in Genbank. Our results indicate that LYSV isolates from Brazil form a monophyletic clade distinct from isolates from other countries. Material and Methods LYSV-infected garlic plants from various regions of Brazil were initially identified by PCR using the WCIEN/PV1 generic potyvirus primers (Mota et al., 2004; Gibbs and Mackenzie, 2007) to amplify part of the coat protein (CP) gene, followed by sequencing. Identification of LYSV was confirmed by PCR with the LY5P/LY2M specific primers to amplify part of the 5ʹ untranslated region and the N-terminal domain of the P1 gene as described by Yoshida et al. (2012). Plants were also tested by PCR for viruses of the genera Allexivirus and Carlavirus, and for the presence of Onion yellow dwarf virus (OYDV) using the primers described by Oliviera et al. (2013), Mituti et al. (2011), and Mituti et al. (2015), respectively. LYSV isolate MG (LYSV-MG) was identified from a singly-infected garlic plant from Santa Juliana, Minas Gerais State, and was selected for full genome sequencing. The variability analysis was carried out with isolates from garlic plants collected in the most important production areas of Brazil, located in the South, Southeast and West Central regions. Total RNA was extracted from infected leaves using the Total RNA Purification Kit (Norgen Biotek). The cDNA was prepared using the PV1 primer (Gibbs and Mackenzie, 1997) with AMV reverse transcriptase (Promega), following the manufacturer‟s instructions. The PCR reactions were performed using either degenerate or specific upstream and downstream primers, with a total of 5 μL of 10x High 29 Fidelity PCR Buffer, 2 mM of MgSO4, 0.4 μM of each primer, 0.2 mM of dNTPs (2.5 mM each), 1 μL of Platinum Taq High Fidelity, 3 μL of cDNA and water per complete 50 μL reaction. Partial CI and HC-Pro genes were amplified using the universal primers described for the family Potyviridae (Ha et al., 2008). Partial CP and nuclear inclusion b (NIb) genes were amplified using universal primer WCIEN paired with PV1 (Mota et al., 2004; Gibbs and Mackenzie, 2007) and with NIB2F/NIB3R primers described by Zheng et al. (2010) respectively. The partial P1 gene was amplified using LY5P/LY2M specific primers for LYSV (Yoshida et al., 2012). The gaps between these regions were covered using specific primers designed from the sequences previously generated (Fig. 1). The amplified fragments were cloned using the p-GEM T Easy Vector system (Promega), and the cloned fragments were sequenced in both directions by Macrogen Inc. (Republic of Korea). To obtain a consensus sequence three clones of each fragment were sequenced. The final LYSV-MG genome sequence and the partial P1 gene sequences of various Brazilian isolates were deposited in the DDBJ/ EMBL/ GenBank database under the accession numbers KP258216 (full genome of LYSV-MG), KP236094, KP236095, KP236096, KP236097, KP236098, KP236099, KP236100, KP236101, KP236102 and KP236103. Comparisons were made between the LYSV-MG sequence and the available complete sequence of other garlic isolates as follows: Chinese (NC_004011); (DD462959), Australian (JX429967); (JX429965) and Japanese (AB194623); (AB194621) and (AB194622) isolates. Variability between isolates was evaluated by phylogenetic analysis of the P1 amino acid sequence encoded by the 846 nucleotides corresponding to part of the P1 gene of LYSV-MG, and the corresponding regions of other isolates. OYDV (AB219834) was used as the outgroup sequence for the phylogenetic analysis. The sequences were aligned using ClustalW 30 (Thompson et al., 1994) with default parameters. Neighbor-joining phylogenetic trees were prepared using MEGA 6.0 (Tamura et al., 2013) with 1,000 bootstrap replicates. Results and Discussion LYSV-MG was selected for cloning and sequencing of the full genome because the source plant was determined by PCR to be infected by LYSV, but free of any detectable allexivirus, carlavirus, or OYDV infections. The complete genome of LYSV-MG consisted of 10,341 nucleotides (nt), excluding the 3‟ terminal poly (A) tail (Fig. 1). The overall ssRNA nt composition of LYSV-MG was adenine (A) 32.2%, cytosine (C) 19.4%, guanine (G) 22.8%, and uracil (U) 25.5%. The AUG codon located at nt position 83-85 is likely to be the translation initiation codon, since it was in a context (AACAUGGCT) that is similar to the context of the initiation codon of other isolates of LYSV. The 5ʹ non-coding region (NCR) was 82 nt in length, and AU-rich. The 3‟-NCR was 592 nt in length and also AU- rich. The stop codon (UGA) was at position 9,746–9,748. The putative translation product contained 3,221 amino acids (aa). According to the conserved cleavage sites predicted by Adams et al. (2005a), a total of ten proteins are expected: P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa, NIb and CP (Fig. 1). The predicted cleavage sites of LYSV-MG were consistent with the known sites of other potyviruses (Adams et al., 2005a). Several previously identified, highly conserved amino acid sequence motifs described in potyviruses (Shukla et al., 1994) were identified in LYSV-MG, including PTK, IGN and CSC motifs of HC- Pro, which are known to be required for aphid transmission, RNA amplification and systemic movement, respectively. The GDD motif in the NIb region associated with viral RNA replication (Urcuqui-Inchima et al., 2001; Ha et al., 2008) and a DAG motif in the CP region associated with aphid transmission (Lopez-Moya et al., 1999) were also 31 identified. The PIPO (pretty interesting potyviridae ORF) with the G1-2A6-7 motif (Chung et al., 2008) was found at nucleotides 3230 to 3470 and contains 80aa. Phylogenetic analysis based on the full genome sequence of garlic isolates of LYSV showed that LYSV-MG falls into a clade with one Australian (JX429965) and two Chinese isolates (including NC_004011), whereas three other Australian isolates (including JX429967) and three Japanese isolates (including AB194623) form a separate clade, with both clades supported by bootstrap values of 100% (Fig. 2). The P1 region of the two Chinese isolates and one Australian isolate (JX429965) contains a deletion of 201- nucleotides (67 aa) with respect to the isolates in the other clade (including JX429967 and AB194623). LYSV-MG does not have this deletion, but shows the highest genomic nt and aa identities with the isolates which do have the deletion. We therefore compared the complete genomic nt and aa sequences, and those of the individual genome segments of LYSV-MG and Chinese (NC_004011), Australian (JX429965 and JX429967) and Japanese (AB194623) isolates from the two clades. LYSV- MG shares highest identity of both the complete genomic nt and polyprotein aa sequences with the Chinese isolate (NC-004011) and one Australian isolate (JX429965) having the deletion in P1 (Table 1). Both the whole genome identities (76-77% nt; 82-84% aa) and CP identities (79-83% nt; 83-85% aa) of all isolates were above the species discrimination values for the genus Potyvirus (Adams et al., 2005b). Comparison of individual LYSV-MG cistrons with those of other isolates revealed that the regions encoding CP and CI were the most highly conserved, whereas the P1 region was the most variable (Table 1). However, despite the absence of a deletion in the LYSV-MG P1, the P1 region was most closely related to those of NC_004011 and JX429965, both of which have a 201 nt/67 aa deletion (Table 1). In addition, while the cleavage sites between all proteins (Fig. 1) were identical between LYSV-MG and these two most closely-related isolates, there were differences at 32 the sites 6K1/CI, CI/6K2, 6K2/VPg and NIb/CP when compared to the less-related Australian (JX429967) and Japanese (AB194623) isolates (data not shown). Because differences were found among the P1 coding regions of LYSV-MG and isolates from Japan, China and Australia, we decided to analyze the partial P1 coding region from a wider range of garlic isolates collected from different locations in Brazil and compare their nucleotide sequence with that of other garlic isolates that is available in Genbank. We analyzed ten isolates of LYSV from Brazil, together with 25 LYSV partial P1 sequences obtained from GenBank. The phylogenetic analysis showed that Brazilian isolates form a clade distinct from the other isolates of other countries, but are phylogenetically closest to the S-type isolates and an isolate from Okinawa (AB636327) (Fig. 3). Brazilian isolates share 97-99% nt identity and 51-64% with isolates from other geographic origin. The position of LYSV-MG within the clade of Brazilian isolates demonstrates that this fully-sequenced isolate is representative of the garlic LYSV population in Brazil. In agreement with the previous reports by Chen et al. (2001), Takaki et al. (2005), and Yoshida et al. (2012), our phylogenetic analysis revealed that LYSV partial P1 nt sequences from garlic formed two main groups, the N-type (including isolates from Northern Japan, and some from Uruguay and Australia) and the S-type (Southern Japan, China, Korea and other Australian isolates). The Sp-type clade (Spanish isolates) and monotypic U-type clade (Uruguay) of Yoshida et al. (2012) were on the same major branch as the N-type isolates (Fig. 3). As the Sp-type and U-type isolates group closely with the N-type isolates, and also lack the deletion present in the S-type isolates, we considered this major branch as essentially „N-type‟. The S-type group has large P1 sequence deletions relative to the N-type group (around 201 nt; Yoshida et al., 2012). None of the isolates 33 from Brazil (B-type) have this deletion, but are phylogenetically closer to S-type isolates than N-type isolates. There is evidence that garlic originated in Central Asia, then spread in two directions, to China and India and to Europe and Russia (Yoshida et al., 2012). It is likely that an ancestral LYSV population was similarly divided resulting in the emergence of the N-type group and the S-type group during the evolution of LYSV, with two separate introductions into both Japan and Australia. It is possible that the S-type group derived from the N-type group after extensive deletion because insertions are much rarer than deletions (Takaki et al., 2005). It is likely that the distinct variability of those LYSV isolates can be explained by both their host‟s geographical origin and unique adaptation to a particular garlic cultivar, resulting in formation of the various clades observed (Takaki et al., 2005; Yoshida et al., 2012). The P1 protein has been demonstrated to be the most variable protein among those encoded by the Potyvirus genome (Shukla et al., 1994; Valli et al., 2007). As P1 diversity may be associated with adaptation to particular hosts or even specific host genotypes, the P1 region is therefore an appropriate sequence for use in studies of phylogenetic relationships to understand the evolutionary history of LYSV isolates in garlic (Takaki et al., 2005; Yoshida et al., 2012). We believe that the Brazilian isolates have a common origin due to the high identity (97-99%) observed among them. The data suggest a possible common origin for Brazilian and O-type Okinawa (AB636327) LYSV isolates, suggesting that Brazilian LYSV isolates are derived from an ancestral source of the Okinawa and S- type isolates, prior to the occurrence of the P1 deletion and divergence in the S-type isolates. A second possibility is that isolates from Brazil, Australia and Okinawa were introduced via China prior to the divergence of the S-type. 34 Acknowledgments This work was supported by grants received from FAPESP (Sao Paulo State Foundation for Research Support), protocol number 2010/16148-9. And the first author was supported by a fellowship from CAPES PDSE 99999.011577/2013-04 (Coordination for the Improvement of Higher Level Personnel). References Adams M.J., Antoniw J.F., Beaudoin F., 2005a. Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. 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Plant Pathology 59: 211-220. 39 Table 1 Comparison of the full genomic nucleotide (nt) and amino acid (aa) sequences between Brazilian Leek yellow stripe virus isolate MG (LYSV-MG) and representative LYSV isolates from China (NC_004011), Australia (JX429965), Australia (JX429967), and Japan (AB194623). Isolated compared 5’UTR Whole genome P1 HC- Pro P3 CI NIa- VPg NIa- Pro NIb CP 3‟UTR Range of nt and aa identity (%) Chinaa nt 68 77 73 76 77 81 81 79 81 83 89 China aa 84 63 81 78 94 93 93 93 85 Australiab nt 40 77 70 76 78 82 81 79 81 81 90 Australia aa 84 63 82 80 95 93 92 92 84 Australiac nt 50 76 65 78 75 80 76 78 79 80 83 Australia aa 82 55 87 71 93 84 90 90 83 Japand nt 53 76 64 77 75 81 76 78 79 79 83 Japan aa 82 55 87 71 92 84 91 91 83 a NC_004011, isolate Yugang GYH; b JX429965, isolate SG2; c JX429967, isolate AG1; d AB194623, isolate 1A31 40 HPFor HPRev 700bp CIFor CIRev 700bp Wcien PV1 800bp P1HCFor P1HCRev HCCIFor1 HCCIRev1 NIBCPFor1 NIBCPRev1 500bp900bp NIB2F NIB3RLY5P LY2M 1600bp 2000bp 500bp 3500bp CINIBFor2 CINIBRev2 CINIBFor2 5.964Rev 1600bp 500bp 5.812For 6.285Rev Fig. 1 Schematic representation of the genome organization of Leek yellow stripe virus (LYSV-MG). The terminal untranslated regions (UTR) are depicted by lines, and the open reading frame is shown as an open box with the different polyprotein domains (proteins). The small box above P3 represents the ORF PIPO. The numbers above the genome indicate the start of each region. The predicted proteinase cleavage sites in the polyprotein are indicated below the genome. The primers used to amplify the genome are indicated below the arrows with the estimated size of amplicon indicated above lines (not to scale). 41 Japan (AB194623.1) Japan (AB194621) Japan (AB194622) Australia (JX429967.1) Australia (KF597283) Australia (KF597285) LYSV-MG (KP258216) Australia (JX429965) China (NC_004011) China (DD462959) OYDV (AB219834) 100 100 100 100 99 100 100 52 0.1 Fig. 2 Phylogenetic analysis using the complete nucleotide sequences of Leek yellow stripe virus MG-isolate (LYSV-MG) and garlic LYSV isolates from China, Australia and Japan.The tree was constructed the Neighbor-joining method implemented in the MEGA 6.0 software. Bootstrap values of 1000 replicates are shown. OYDV (AJ510223) was used as an outgroup. 42 AB194651 - S: China AB194653 - S: China JX429965 - S: Australia HQ258895 - S: Australia AB636335 - S: Tokoro AB194646: S: Okinawa AB636326 - S: Okinawa AB636329 - S: Shinshinotsu NC_004011 - S: China DD462959 - S: China AB194647 - S: Korea AB636323 - S: Fukuoka SJU08 (KP236098) SJU11 (KP236099) GUA11 (KP236094) LYSV - MG (KP236096) SGO08 (KP236100) SJU07 (KP236097) SGO07 (KP236101) IPM08 (KP236095) BAN07 (KP236103) SP07 (KP236102) AB636327 - O: Okinawa AB636331 - Sp: Spain AB636332 - Sp: Spain AB636339- U: Uruguai JX429967 - N: Australia AB636319 - N: Fukuchi AB194650 - N: Aomori AB636334 - N: Tokoro AB194623 - N: Aomori AB194621 - N: Aomori AB194622 - N: Aomori AB194655 - N: Hokkaido AB636337 - N: Uruguai OYDV (AB219834) 100 79 100 100 99 96 99 100 72 82 53 94 99 77 97 100 98 78 63 90 100 60 0.2 Fig. 3 Phylogenetic relationships of garlic LYSV isolates based on the partial P1 amino acid sequence. The tree was constructed the Neighbor-joining method implemented in the MEGA 6.0 software. Bootstrap values of 1000 replicates are shown. OYDV (AB219834) was used as an outgroup. The Brazilian isolates originated from Bandeirantes (BAN), Ipameri (IPM), São Gotardo (SGO), Santa Juliana (SJU), São Paulo (SP), and Guarapuava (GUA). The fully-sequenced isolate LYSV-MG originated from Santa Juliana. Clades of isolates are shown as „S‟ (Southern Japan), „O‟ (Okinawa), „Sp‟ (Spain), „U‟ (Uruguay), and „N‟ (Northern Japan) as identified by Yoshida et al. (2012), with the Brazilian clade „B‟. S B Sp O U N 43 CAPÍTULO II Narcissus yellow stripe virus and different species of the genera Allexivirus, Carlavirus and Potyvirus are present in ornamental Allium species available in the United States2 2 Redigido conforme as normas da revista Plant Disease 44 Narcissus yellow stripe virus and different species of the genera Allexivirus, Carlavirus and Potyvirus are present in ornamental Allium species available in the United States Daiana Bampi1, Michael Reinsel2, Renate Krause-Sakate1, John Hammond2 1 Faculdade de Ciências Agronômicas, FCA-UNESP, Departamento Proteção Vegetal, Rua José Barbosa de Barros, 1780, CEP: 18610-307, Botucatu, SP, Brazil; 2USDA-ARS, USNA, Floral and Nursery Plants Research Unit, 10300 Baltimore Avenue, B-010A, Beltsville, Maryland, United States, 20705. Email:daiana@fca.unesp.br Abstract Plants of the genus Allium can be infected by several viruses of the genera Allexivirus, Carlavirus and Potyvirus, often in mixed infections. Bulbs of thirteen different species of Allium were purchased from retail nurseries and planted in the field in the fall of 2013; one additional species was sampled from plants established in a home garden. Leaf tissue from the flowering plants were collected in spring 2014, and tested by PCR using generic primers for the genus Allexivirus, Carlavirus and Potyvirus. PCR-positive samples were re-tested using specific primers for Allium-infecting virus species described for each genus. After flowering and foliage dieback, the bulbs were harvested and stored at 4º C for 60 days, before being planted in pots and maintained in a cooler until leaves emerged, and then transferred to a greenhouse. The sprouted plants were tested by ELISA using antibodies specific for the Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV-C), Shallot virus X (ShV- X), Garlic common latent virus (GarCLV), Shallot latent virus (SLV), Leek yellow stripe virus (LYSV), Onion yellow dwarf virus (OYDV) and Shallot yellow stripe virus (SYSV). RT-PCR detected the presence of GarV-B, GarV-C, Garlic virus D (GarV-D), and Garlic virus E (GarV-E) in Allium sphaerocephalon; GarV-B, GarV-C and ShV-X in Allium caeruleum; LYSV in Allium bulgaricum and Allium atropurpureum; SLV in Allium moly and Narcissus yellow stripe virus (NYSV) in Allium carinatum. ELISA testing with available antibodies confirmed the identity of viruses previously detected by PCR. Electron microscopy revealed the presence of flexuous particles of 650-800 nm, typical of Allexivirus, Carlavirus and Potyvirus. To our knowledge this is the first report of Narcissus yellow stripe virus in Allium spp. mailto:daiana@fca.unesp.br 45 Keywords: Allium spp., vírus detection, mechanical transmission. Introduction The genus Allium is one of the largest plant genera, including more than 750 species, and most species in the genus are bulb forming (Block, 2010). This genus includes important species used for human consumption, including onion (Allium cepa), chive (Allium schoenoprasum), garlic (Allium sativum), shallot (Allium ascalonicum), and leek (Allium ampeloprasum, syn. A. porrum) and many other species grown as ornamentals (Katis et al. 2012). The ornamental Allium species distinguish themselves by their great diversity in color, inflorescence and flowering height (Miglino et al. 2011). Most species belonging to this genus are vegetatively propagated. This feature allows the accumulation of viruses, which are perpetuated by bulbs from one growing cycle to the next (Katis et al. 2012). The species of flexuous viruses previously reported to infect Allium spp. include Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot yellow stripe virus (SYSV) and Turnip mosaic virus (TuMV) of the genus Potyvirus, Garlic common latent virus (GarCLV) and Shallot latent virus (SLV) of the genus Carlavirus, 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 virus (GarMbFV) and Shallot virus X (ShV-X) of the genus Allexivirus (Katis et al. 2012). Recently, a new Potexvirus infecting ornamental Allium was reported in Netherlands, for which the name Allium virus X was proposed (Miglino et al. 2011). In addition to the flexuous viruses listed above, Tobacco rattle virus (e.g. Miglino et al., 2006) and a few isometric viruses are also known to infect Allium spp., of which the most important worldwide are probably the tospoviruses Iris yellow spot virus (Katis et al. 2012), Impatiens necrotic spot virus (Hall et al. 1993), and Groundnut bud necrosis virus (Sujitha et al. 2012); some other isometric viruses are of regional importance (Katis et al. 2012; Miglino et al. 2006). Viruses of Allium spp. typically induce foliar mosaic and yellow stripe symptoms and may cause both yield losses and deterioration in crop quality (Lot et al. 1998). The occurrence of frequent mixed virus infections in the same plant makes it difficult to determine the symptoms and attribute yield loss caused by each virus (Ward et al. 2009). Members of the 46 genus Potyvirus are usually the most prevalent and most economically important, while carlaviruses and allexiviruses are typically latent (Katis et al. 2012). However, coinfection of carlaviruses or allexiviruses with the potyviruses can have synergistic effects, increasing the symptoms and losses (Conci et al. 2003; Lot et al. 1998). The viruses present in vegetatively propagated species may be transported long- distances with infected bulbs. The accurate knowledge of the viruses present within a country is important to ensure that phytosanitary controls for imported and exported plants are appropriate, and to enable disease control measures to be implemented (Pearson et al. 2009). As the information on the viruses affecting ornamental Allium grown in the United States (but largely imported as bulbs) is scarce, in this work we analyzed fourteen ornamental Allium species for the presence of viruses. We detected the same virus species commonly found in crops used for human consumption (garlic, onion, chive, shallot and leek) but also identified Narcissus yellow stripe virus (NYSV), a potyvirus in Allium carinatum as a first report of this species in the genus Allium. Material and methods Plant Material Bulbs of Allium amplectens “Graceful Beauty‟, A. carinatum ssp. pulchellum, A. flavum, A. ostrowskianum, A. triquetrum, A. caeruleum, A. sphaerocephalon, A. moly, A. atropurpureum, A. aflatuense „Purple Sensation‟, A. schubertii, A. karataviense „Ivory Queen‟, and A. christophii were purchased from retail nurseries and planted in the field in the fall of 2013; all of these bulbs were labeled as originating from the Netherlands. Leaves of A. bulgaricum were collected from an established planting in a private garden. Leaf tissue from the flowering symptomatic plants were collected in spring 2014 and tested by RT-PCR using generic and specific primers for viruses of the genera Allexivirus, Carlavirus and Potyvirus. After flowering and foliage dieback, the bulbs were harvested and stored at 4º C for 60 days, before being planted in pots and maintained in a cooler until leaves emerged, and then transferred to a greenhouse. The sprouted plants were tested by Enzyme-linked immunosorbent assay (ELISA) as described below. Electron microscopy assays were performed for positive samples. 47 Host Range Studies Various common indicator plants (Nicotiana benthamiana, N. glutinosa, N. tabacum, Chenopodium quinoa, C. amaranticolor, Tetragonia tetragonioides) and other Allium species, (Allium sativum, Allium cepa and Allium ampeloprasum) grown from seed were used to test mechanical transmission. Mechanical inoculations were typically made by grinding leaf tissue in 1% K2HPO4 in distilled water containing a small amount of Celite, and gently rubbing the sap extract to young leaves of the test plants. Plants were observed for symptom development in the greenhouse over at least four weeks after inoculation. Polymerase Chain Reaction (PCR) Assays The Total RNA of a