Incidence of Begomovirus and Crinivirus in tomato and potato crops in Paraná State, Brazil

ABSTRACT: In the last decades, the high incidence of viruses transmitted by whiteflies has become a problem in the tomato fields, threatening, more recently, the potato crops. The present study carried out a survey of begomoviruses and criniviruses in tomato and potato crops, from 2015 to 2018, in the municipalities of Araucária, Campo do Tenente, Campo Largo, Contenda, Lapa, Faxinal, Morretes, Reserva, Castro, Palmeira and São Mateus do Sul, in Paraná State, Brazil. Total DNA and RNA from leaves were extracted and used as templates to detect, respectively, begomoviruses by PCR and criniviruses by RT-PCR. Out of 215 tomato samples, 14 from Faxinal were infected by crinivirus. The other tomato samples and 243 potato samples showed negative results for begomovirus and crinivirus. Results indicated a low incidence (6.5%) of crinivirus infecting tomato crops in Paraná State, and the nucleotide sequence of one amplified fragment shared 99.71% identity with tomato chlorotic virus (ToCV)

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.Fundacsao de Apoio a Pesquisa do Estado de Sao Paulo - FAPESP [04/11799-0, 2008/52691-9]Fundacsao de Apoio a Pesquisa do Estado de Sao Paulo FAPES

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.Fundacsao de Apoio a Pesquisa do Estado de Sao Paulo - FAPESP [04/11799-0, 2008/52691-9]Fundacsao de Apoio a Pesquisa do Estado de Sao Paulo FAPES

Arabidopsis thaliana as a model host for Brevipalpus mite-transmitted viruses

ABSTRACT: Brevipalpus-transmitted viruses (BTV) are a taxonomically diverse group of plant viruses which severely affect a number of major crops. Members of the group can be sub-classified into cytoplasmic (BTV-C) or nuclear type (BTV-N) according to the accumulation sites of virions in the infected plant cells. Both types of BTV produce only local infections near the point of inoculation by viruliferous mites. Features of BTV-plant interactions such as the failure of systemic spread in their natural hosts are poorly understood. In this study we evaluated Arabidopsis thaliana, a model plant commonly used for the study of plant-virus interactions, as an alternative host for BTV. Infection of Arabidopsis with the BTV-N Coffee ringspot virus and Clerodendrum chlorotic spot virus, and the BTV-C Solanum violaefolium ringspot virus, were mediated by viruliferous Brevipalpus mites collected in the wild. Upon infestation, local lesions appeared in 7 to 10 days on leaves of, at least, 80 % of the assayed plants. Presence of viral particles and characteristic cytopathic effects were detected by transmission electron microscopy (TEM) and the viral identities confirmed by specific reverse-transcriptase polymerase chain reaction (RT-PCR) and further amplicon sequencing. The high infection rate and reproducibility of symptoms of the three different viruses assayed validate A. thaliana as a feasible alternative experimental host for BTV

Characterization of an isolate of the begomovirus Sida micrantha mosaic virus (SimMV)

A família Geminiviridae, caracterizada pela morfologia de partículas icosaédricas geminadas e genoma composto por DNA de fita simples circular, é dividida em quatro gêneros, de acordo com o tipo de inseto vetor, gama de hospedeiros, organização do genoma e relacionamento filogenético. Os begomovírus possuem dois componentes genômicos, são transmitidos por mosca-branca e infectam dicotiledôneas. A partir de 1994 houve relatos sucessivos de begomovírus em tomateiro em diversos estados brasileiros. O seqüenciamento parcial do genoma de alguns destes vírus revelou que há uma grande diversidade de espécies, provavelmente como resultado da disseminação de vírus nativos pelo biótipo B da mosca-branca Bemisia tabaci. O objetivo do presente trabalho foi a caracterização biológica e molecular de um isolado de begomovírus detectado em tomateiros do município de São Joaquim de Bicas, MG, denominado MG-Bi2. A caracterização biológica consistiu no teste de gama de hospedeiros, realizado por meio de inoculação via extrato foliar tamponado e biobalística. Os resultados indicaram que o isolado infecta plantas das famílias Solanaceae e Amaranthaceae e que a transmissão é altamente dependente do método de inoculação. Para a caracterização molecular, procedeu-se à amplificação do genoma viral (DNA-A e -B) utilizando a DNA polimerase do bacteriófago Φ29, seguida de clonagem e determinação de suas seqüências parciais de nucleotídeos. A comparação de seqüências e análise filogenética demonstraram relacionamento próximo com o genoma de Sida micrantha mosaic virus (SimMV), indicando que o isolado MG-Bi2 pertence a esta espécie, e reforçando a hipótese de que plantas daninhas e silvestres servem como reservatório natural para os geminivírus que infectam plantas cultivadas como o tomateiro.The Geminiviridae family, characterized by a particle morphology of twinned incomplete icosahedra and a single-stranded, circular DNA genome, is divided into four genera according to the type of insect vector, host range, genomic organization and phylogeny. Begomoviruses have two genomic components and are transmitted by whiteflies to dicotyledoneous plants. Since 1994 there have been successive reports of begomovirus infection in tomatoes in several states of Brazil. Partial genome sequencing of several isolates revealed a high degree of genetic diversity, probably due to the dissemination of indigenous viruses by the B biotype of the whitefly Bemisia tabaci. The objective of the present work was the biological and molecular characterization of a begomovirus isolate detected in tomatoes in the city of São Joaquim de Bicas, MG, named MG-Bi2. Biological characterization consisted on a host range study using sap inoculation and particle bombardment. Results indicated that the isolate infects species of Solanaceae and Amaranthaceae, and that transmition is highly dependent on the inoculation method. For the molecular characterization, the full-length genome (DNA-A and -B) was amplified using the DNA polimerase of bacteriophage Φ29, cloned and partially sequenced. Sequence comparisons and phylogenetic analyses demonstrated a close relationship with Sida micrantha mosaic virus (SimMV), indicating that the isolate MG-Bi2 is a member of this species, and reinforcing the hypothesis that weeds and wild plants could act as a natural reservoir of indigenous begomoviruses which can infect crop plants such as the tomato.Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorDissertação importada do Alexandri

Detection of Citrus leprosis virus C (CiLV-C) in the tissues of infected plants and of mite vector Brevipalpus phoenicis Geijskes (Acari: Tenuipalpidae)

A leprose é uma das principais doenças na citricultura brasileira devido à sua ocorrência difundida nos pomares e aos altos custos para o controle químico do ácaro vetor. A doença compromete a produção da planta e sua vida útil, manifestando-se através de lesões locais cloróticas ou necróticas em folhas, ramos e frutos, levando à queda prematura destes órgãos e à seca de ramos. O patógeno, Citrus leprosis virus C (CiLV-C), recentemente classificado como espécie tipo de um novo gênero de vírus de planta, Cilevirus, é transmitido pelo ácaro Brevipalpus phoenicis (Geijskes). Apesar de haver consenso de que a doença tem etiologia viral, ainda existem muitas questões pendentes sobre as interações vírus-planta-vetor, cujas soluções contribuirão para o controle integrado da doença. O presente trabalho teve como objetivo obter informações sobre a interação do vírus com células hospedeiras através de ensaios de imunolocalização das proteínas MP (putativa proteína de movimento), helicase (associada à replicação) e p29 (putativa proteína capsidial) do CiLV-C. Para tal, as sequências codificadoras das ORFs mp e hel foram amplificadas via RT-PCR e clonadas em vetor de expressão. Em seguida, promoveu-se a expressão in vitro das respectivas proteínas em células de E. coli e purificação por cromatografia de afinidade e troca iônica. A proteína MP pura foi utilizada para produção de antissoro policlonal específico que foi testado quanto à especificidade por métodos sorológicos. Os resultados do ELISA mostraram que o antissoro apresentou reação positiva com extratos foliares de lesões lepróticas em todos os estágios de desenvolvimento da doença, quando utilizado em altas concentrações. Além disso, lesões maduras reagiram mais intensamente que lesões mais novas. Por Western Blot, detectou-se somente a proteína pura, não sendo possível obter reação positiva em extrato de lesões foliares. Também não foi possível detectar a MP por imunolocalização in situ. Os resultados em conjunto sugerem que ocorre baixo nível de expressão da MP nos tecidos do hospedeiro. Empregando-se anticorpo policlonal contra proteína p29 do CiLV-C, foi possível detectar o CiLV-C em extratos de lesões foliares de leprose por ambos os métodos sorológicos testados e também por Tissue Blotting. Ensaios de imunolocalização in situ permitiram confirmar que as partículas baciliformes presentes em cisternas do retículo endoplasmático de tecidos de lesões lepróticas em plantas representam de fato vírions do CiLV-C. Além disso, também demonstrou-se que o viroplasma que ocorre no citoplasma representa o sítio de acúmulo da proteína p29. Este mesmo ensaio revelou que partículas baciliformes que ocorrem entre membranas de células adjacentes (intestino médio, glândulas prosomais, músculos e epiderme) de B. phoenicis virulíferos são de fato do CiLV-C. A ausência de viroplasma no ácaro sugere que a relação vírus/vetor seria do tipo circulativo, sem replicação. Baseado neste fato discutem-se alternativas para explicar como o vírus trafegaria do lúmen do intestino até o duto salivar para causar infecção numa planta sadia.Citrus leprosis is one of the most important diseases in the Brazilian citrus production due to the wide occurrence in orchards and also to the high costs involved in the chemical control of the mite vector. The disease affects the plant production and longevity and it is characterized by localized chlorotic and/or necrotic spots on the leaves, stems and fruits. Affected leaves and fruits may drop prematurely and dieback can be observed in stems. The pathogen, Citrus leprosis virus C (CiLV-C), recently considered as the type member of a new genus, Cilevirus, is transmitted by the mite Brevipalpus phoenicis Geijskes. Despite the consensus that citrus leprosis has viral etiology, there are many pending questions regarding the viral interactions with the infected plant and the viruliferous mites. The solution of these questions may contribute to a better disease integrated management. This work aimed to obtain a better understanding about the virus-plant-vector relationship with the host cell by immunolocalization assays of the putative movement protein (MP), helicase (protein involved in the viral replication) and p29 (putative coat protein) of the CiLV-C. ORFs coding sequences of mp and hel was amplified by RT-PCR and cloned in the expression vector. Afterwards, in vitro expression of these proteins in E. coli and its purification by affinity chromatography were realized. The purified MP was used to produce specific polyclonal antibody that was tested for specificity by serological methods. The ELISA results showed that high concentration antibody reacted with the leaves extracts from lesions in all the disease stage of development. Furthermore, the old lesions reacted more intensely than the younger. Western blot (which detected only the pure protein) and in situ immunolocalization assays failed to detect the native MP in lesioned leave extracts. The results as a hole suggest the occurrence of low expression of MP in host tissue. The polyclonal antibody against p29 was able to detect the virus in lesioned plant extracts by PTA-ELISA, Western Blot, and Tissue Blotting. The viral nature of the putative viral particles, present within endoplasmic reticulum cisternae of infected leaf tissue, was confirmed by immunogold label. The labeling also occurred intensely in the viroplasmas, indicating that these structures represent p29 protein accumulation site. Putative virus particles, visualized in viruliferous B. phoenicis, between membranes of adjacent cells (midgut, prosomal glands, epidermis, muscles), was also immunogold labeled indicating that they represent CiLV-C. The absence of viroplasma in the mite tissues suggests that CiLV-C / B. phoenicis relationship is of the circulative type, without replication. Based on this finding, we search for possible alternatives for the viral circulation in the mite body from the midgut lumen to the salivary duct for the infection of a healthy plant to occur

Arabidopsis thaliana as a model host for Brevipalpus mite-transmitted viruses

Brevipalpus-transmitted viruses (BTV) are a taxonomically diverse group of plant viruses which severely affect a number of major crops. Members of the group can be sub-classified into cytoplasmic (BTV-C) or nuclear type (BTV-N) according to the accumulation sites of virions in the infected plant cells. Both types of BTV produce only local infections near the point of inoculation by viruliferous mites. Features of BTV-plant interactions such as the failure of systemic spread in their natural hosts are poorly understood. In this study we evaluated Arabidopsis thaliana, a model plant commonly used for the study of plant-virus interactions, as an alternative host for BTV. Infection of Arabidopsis with the BTV-N Coffee ringspot virus and Clerodendrum chlorotic spot virus, and the BTV-C Solanum violaefolium ringspot virus, were mediated by viruliferous Brevipalpus mites collected in the wild. Upon infestation, local lesions appeared in 7 to 10 days on leaves of, at least, 80 % of the assayed plants. Presence of viral particles and characteristic cytopathic effects were detected by transmission electron microscopy (TEM) and the viral identities confirmed by specific reverse-transcriptase polymerase chain reaction (RT-PCR) and further amplicon sequencing. The high infection rate and reproducibility of symptoms of the three different viruses assayed validate A. thaliana as a feasible alternative experimental host for BTV

Arabidopsis thaliana as a model host for Brevipalpus mite-transmitted viruses

ABSTRACT: Brevipalpus-transmitted viruses (BTV) are a taxonomically diverse group of plant viruses which severely affect a number of major crops. Members of the group can be sub-classified into cytoplasmic (BTV-C) or nuclear type (BTV-N) according to the accumulation sites of virions in the infected plant cells. Both types of BTV produce only local infections near the point of inoculation by viruliferous mites. Features of BTV-plant interactions such as the failure of systemic spread in their natural hosts are poorly understood. In this study we evaluated Arabidopsis thaliana, a model plant commonly used for the study of plant-virus interactions, as an alternative host for BTV. Infection of Arabidopsis with the BTV-N Coffee ringspot virus and Clerodendrum chlorotic spot virus, and the BTV-C Solanum violaefolium ringspot virus, were mediated by viruliferous Brevipalpus mites collected in the wild. Upon infestation, local lesions appeared in 7 to 10 days on leaves of, at least, 80 % of the assayed plants. Presence of viral particles and characteristic cytopathic effects were detected by transmission electron microscopy (TEM) and the viral identities confirmed by specific reverse-transcriptase polymerase chain reaction (RT-PCR) and further amplicon sequencing. The high infection rate and reproducibility of symptoms of the three different viruses assayed validate A. thaliana as a feasible alternative experimental host for BTV

Characterisation of turnip mosaic virus isolates reveals high genetic variability and occurrence of pathotype 1 in Brazil

Turnip mosaic virus (TuMV) infects many plant species, being the only potyvirus able to infect brassicas. TuMV isolates have been classified into 12 pathotypes according to symptoms induced in lines of Brassica napus, and molecularly clustered into six lineages (basal-B, basal-BR, Asian-BR, world-B, Iranian and OMs). Despite being considered one of the most important viruses infecting brassicas worldwide, there is little information on this virus in the Neotropical region. Aiming to fill in this gap and advance knowledge on occurrence, genetic variability, and biological aspects of TuMV in Brazil, 40 isolates were identified and characterised. Five of these isolates were selected to determine their host range, sequence their genomes, and for phylogenetic, recombination and diversity analyses. Mechanical inoculations performed on plant species from 10 families showed differences in symptom expression among isolates. Inoculations of 13 TuMV isolates in B. napus lines revealed occurrence only of the pathotype 1. According to phylogenetic analyses of the coat protein, TuMV Brazilian isolates clustered into the groups: world-B (subgroups world-B2 and world-B3) and basal-BR. In the latter, there was a formation of a subclade named Brazilian subgroup composed by 31 Brazilian TuMV isolates. Intralineage and interlineage recombination events of world-B, basal-B and basal-BR suggest that Brazilian TuMV isolates had a European origin. Our diversity analysis suggest that a strong negative selection is acting on polyprotein coding region. We confirmed that Brazilian TuMV isolates showed high variability, which together with their ability to infect wild brassicas and to circumvent resistance genes highlight their genetic and epidemiological potential in causing damages in cultivated species of brassicas and other crops in Brazil

Characterization of Tomato yellow spot virus, a novel tomato-infecting begomovirus in Brazil Caracterização do Tomato yellow spot virus, um novo begomovírus isolado de tomateiro no Brasil

The objective of this work was the biological and molecular characterization of a begomovirus detected in São Joaquim de Bicas, Minas Gerais, Brazil, named TGV-[Bi2], by determining its host range, complete nucleotide sequence and phylogenetic relationships with other begomoviruses. Biological characterization consisted of a host range study using either sap inoculation or particle bombardment as inoculation methods. The yellow spot virus can infect plants in Solanaceae and Amaranthaceae, including economically importat crops as sweet pepper, and weeds as Datura stramonium and Nicotiana silvestris. For the molecular characterization, the full-length genome (DNA-A and DNA-B) was amplified, cloned and completely sequenced. Sequence comparisons and phylogenetic analyses indicated that TGV-[Bi2] constitutes a novel begomovirus species named Tomato yellow spot virus (ToYSV), closely related to Sida mottle virus (SiMoV).<br>O objetivo deste trabalho foi a caracterização biológica e molecular de um begomovírus detectado em tomateiros em São Joaquim de Bicas, Minas Gerais, denominado TGV-[Bi2]. A caracterização biológica consistiu em teste de gama de hospedeiros, realizado por meio de inoculação via extrato foliar tamponado ou bombardeamento de partículas. O isolado TGV-[Bi2] infecta plantas das famílias Solanaceae e Amaranthaceae, inclusive espécies economicamente importantes como o pimentão, e algumas plantas daninhas como Datura stramonium e Nicotiana silvestris. A caracterização molecular consistiu na clonagem e seqüenciamento de seu genoma completo (DNA-A e DNA-B). A comparação de seqüências e análise filogenética indicaram que o TGV-[Bi2] constitui uma nova espécie de begomovírus, denominada Tomato yellow spot virus (ToYSV), filogeneticamente relacionado ao Sida mottle virus (SiMoV)