Identification of a novel endornavirus in Geranium carolinianum and occurrence within three agroecosystems.

A putative endornavirus was detected in Carolina geranium (Geranium carolinianum) in Louisiana, USA. The virus was provisionally named Geranium carolinianum endornavirus 1 (GcEV1). The viral RNA was sequenced, and it consisted of 14,625 nt containing a single ORF coding a putative polyprotein of 4815 aa with conserved domains for a helicase 1, peptidase C97, glycosyl transferase GTB-type, and RNA-dependent RNA polymerase 2. The 5'end consisted of 130 nt while the 3'end consisted of 54 nt ending in nine cytosine residues. The closest relative to GcEV1 was Phaseolus vulgaris endornavirus 3. In phylogenetic analyses, GcEV1 clustered with members of the genus Alphaendornavirus. GcEV1 was detected in 57 of 60 G. carolinianum plants collected from three distinct agroecosystems. The virus was not detected in eight other species of the genus Geranium. There was no association of a particular phenotypic trait of the host with the presence or absence of the virus. GcEV1 was transmitted at a rate of 100% in seeds of a self-pollinated G. carolinianum plant.

Serological and Molecular Detection of Latent Viruses in the Apple Germplasm Bank of Santa Catarina

Abstract The Apple Germplasm Bank (AGB) of Santa Catarina Agricultural Research and Rural Extension Company - Epagri, AGB-Epagri, is the largest of the genus Malus in Brazil. Twenty-eight main accessions of this bank were virus screened through DAS-ELISA, RT-PCR and IC-RT-PCR during two consecutive reproductive cycles, and each accession showed latent mixed infection by at least two species, among ASGV, ASPV and ACLSV. The combined use of diagnostic methods helped overcome inconsistencies commonly found in apple virus detection and was shown essential for the AGB-Epagri can be safely used as a source of genetic variability and for the exchange of virus-free propagative material.

Occurrence of putative endornaviruses in non-cultivated plant species in South Louisiana.

Extraction and electrophoretic analysis of viral dsRNA from plants has been used successfully to detect infections by RNA viruses. We used this approach as an initial tool to test non-cultivated plant species for the presence of endornaviruses. Foliar samples were collected from symptomless plants in various locations within East Baton Rouge Parish, Louisiana, USA, and tested for viral dsRNA. After testing 208 plant species belonging to 74 families, five (Geranium carolinianum, Hydrocotyle umbellata, H. prolifera, Sorghum halepense, and Sisyrinchium atlanticum) yielded dsRNAs similar in size to the dsRNAs of members of the family Endornaviridae. The endornavirus nature of the dsRNAs was confirmed by reverse-transcription PCR (RT-PCR) and sequencing the RT-PCR products. Sequence data were used to determine relationships of the putative endornaviruses to members of the family Endornaviridae. The putative endornaviruses were detected in both native and introduced plants species. This is the first survey on the occurrence of endornaviruses in non-cultivated plant species.

Molecular characterization of a phytoplasma affiliated with the 16SrVII group representative of the novel 16SrVII-F subgroup.

Vernonia brasiliana is a wild perennial shrub frequently found in pasture areas. Plants of this species have been observed displaying typical symptoms induced by phytoplasmas, which were characterized by shoot proliferation, deformed leaves and leaf chlorosis. The present study confirmed the presence of phytoplasmas in association with affected plants. Sequencing of the 16S rRNA gene, computer-simulated RFLP analysis and phylogenetic analysis revealed that one of the phytoplasmas identified was representative of novel subgroup. The sequence identity scores between the novel strain and those of previously described 'CandidatusPhytoplasma fraxini'-related strains was 99 %, while similarity coefficient values were lower than 0.97. These findings provide support to delineate the phytoplasma found in vernonia plants as a reference phytoplasma for a novel subgroup designated 16SrVII-F. This representative of the novel subgroup was denominated VbSP phytoplasma (Vernonia brasiliana Shoot Proliferation; GenBank KX342018). The results of the present study revealed V. brasiliana to be a host of phytoplasmas, evidenced a novel phytoplasma associated with phytoplasmal disease in Brazil and extended the knowledge of the genetic diversity existing within the 16SrVII group.

Complete genome sequence of bean rugose mosaic virus, genus Comovirus.

Since the first report in Costa Rica in 1971, bean rugose mosaic virus (BRMV) has been found in Colombia, El Salvador, Guatemala and Brazil. In this study, the complete genome sequence of a soybean isolate of BRMV from Paraná State, Brazil, was determined. The BRMV genome consists of two polyadenylated RNAs. RNA1 is 5909 nucleotides long and encodes a single polypeptide of 1856 amino acids (aa), with an estimated molecular weight of 210 kDa. The RNA1 polyprotein contains the polypeptides for viral replication and proteolytic processing. RNA2 is 3644 nucleotides long and codes for a single polypeptide of 1097 aa, containing the movement and coat proteins. This is the first complete genome sequence of BRMV. When compared with available aa sequences of comoviruses, the highest identities of BRMV coat proteins and proteinase polymerase were 57.5 and 58 %, respectively. These were below the 75 and 80 % identity limits, respectively, established for species demarcation in the genus. This confirms that BRMV is a member of a distinct species in the genus Comovirus.

Evaluation of limonoid production in suspension cell culture of Citrus sinensis

ABSTRACTThe use of cell and plant tissue culture techniques to produce economically important active metabolites has been growing. Among these substances are total limonoid aglycones, which are produced by "pera" orange (Citrus sinensis (L.) Osbeck, Rutaceae) and have received considerable attention because of their anticancer actions. The main objective of the present study was to analyze and compare the levels of limonoid aglycones in seeds, callus cultures (originating from seeds), callus cultures (originating from hypocotyls), cell suspensions from hypocotyls cells, and cell suspensions from cotyledons. The cell cultures or C. sinensis were obtained by inoculating two strains of callus in MS medium supplemented with 2.0 µM 2,4-dichlorophenoxyacetic acid, 7.0 µM benzyl aminopurine, and 3% (w/v) sucrose in the dark. The highest concentrations of limonoid aglycone that were obtained were observed in cotyledon cell lines (240 mg/100 g dry weight) that were produced on day 21 of culture and hypocotyl cell lines on day 7 (210 mg/100 g dry weight). Explants of different origins under the same culture conditions had different limonoid aglycone content. The present results may suggest strategies for enhancing the productivity of biologically important limonoid aglycones and investigating the complex pathways of these secondary metabolites in plant tissue cultures.

Detection and partial molecular characterization of Grapevine fleck virus, Grapevine virus D, Grapevine leafroll-associated virus -5 and -6 infecting grapevines in Brazil / Detecção e caracterização molecular parcial de Grapevine fleck virus, Grapevine virus D, Grapevine leafroll-associated virus -5 e -6 que infectam videiras no Brasil

Grapevine fleck, rugose wood and leafroll are three grapevine viral diseases whose causal agents (or associated viruses) respectively are Grapevine fleck virus (GFkV), Grapevine virus D (GVD) and Grapevine leafroll-associated virus 5 and 6 (GLRaV-5 and -6). The objective of this work was to perform a partial molecular characterization of local isolates of these four viral species that infect grapevines. The nucleotide and deduced amino acid sequences of complete genes of the coat protein (CP) (of GFkV), the CP and the RNA binding protein (of GVD), the CP and the partial hHSP70 gene (of GLRaV-5) and the partial hHSP70 gene (of GLRaV-6) were aligned and compared in silico with other isolates. These data extend the available information about Brazilian isolates of GFkV, GLRaV-5 and -6, and reports for the first time the GVD occurrence in Brazil.

Mancha das nervuras, lenho rugoso e enrolamento das folhas são três doenças virais da videira, cujos agentes causais (ou vírus associados) são o Grapevine fleck virus (GFkV), o Grapevine virus D (GVD) e os Grapevine leafroll-associated virus 5 e 6 (GLRaV-5 e -6), respectivamente. O objetivo deste trabalho foi realizar a caracterização molecular parcial de isolados locais dessas quatro espécies virais que infectam videira. As sequências de nucleotídeos e de aminoácidos deduzidos dos genes completos da proteína capsidial (CP) (do GFkV), da CP e da RNA binding protein (do GVD), da CP do GLRaV-5 e parte do gene codificador da hHSP70 do GLRaV-5 e -6 foram alinhadas e comparadas in silico com sequências de outros isolados. Os dados obtidos expandem a informação existente sobre isolados brasileiros de GFkV, GLRaV-5 e - 6 e relatam pela primeira vez a ocorrência do GVD no Brasil.

Levantamento da incidência de Leifsonia xyli subsp. xyli em plantios de cana-de-açúcar do noroeste do Paraná / Survey of Leifsonia xyli subsp. xyli incidence in sugarcane plantations of the northwest region of Paraná State

O raquitismo-da-soqueira, causado por Leifsonia xyli subsp. xyli é considerado uma das mais importantes doenças bacterianas da cana-de-açúcar (Saccharum spp.). Para verificar a possível ocorrência de Leifsonia xyli subsp. xyli na região noroeste do Paraná, foi realizado um levantamento em 32 áreas de plantio de duas usinas, utilizando-se o método sorológico "Dot blot" e um protocolo baseado na reação em cadeia da polimerase. As amostras foram coletadas em áreas cultivadas com 8 variedades e 2 clones, a partir de plantas em diferentes épocas de corte, perfazendo um total de 1395 amostras. Constatou-se a presença da bactéria no fluído fibrovascular de plantas da variedade SP77-5181 coletado em uma das propriedades, com índice de incidência de 23 por cento. Esses resultados indicam a incidência de Leifsonia xyli subsp. xyli no noroeste do Paraná.

Ratoon stunting disease caused by Leifsonia xyli subsp. xyli is one of the most important bacterial diseases of sugarcane (Saccharum spp.). A ratoon stunting desease survey was carried in 32 sugarcane producing areas from two mills in the northwest region of Paraná State through a dot blot serological protocol and a polymerase chain reaction-based assay. A total of 1395 samples were collected in areas planted with 8 varieties and 2 sugarcane clones collected from plants at different harvest periods. The bacterium was detected in the fibrovascular fluid of the SP77-5181 variety from a producing area in a 23 percent rate of incidence. These data indicated that Leifsonia xyli subsp. xyli is present in the northwest region of Paraná.

Detection and identification of TMV infecting tomato under protected cultivation in Paraná State

During an inspection in plastic houses in Sapopema, Paraná, 90 percent of tomato plants showed leaf abnormalities, probably associated with herbicide toxity. However, virus like symptoms developed in selected hosts after mechanical inoculatation. RT-PCR reactions using primers for an internal region within the movement protein gene of TMV and ToMV resulted in the amplification of a 409 bp cDNA fragment only by TMV primers. Deduced amino acids showed 100 percent identity when compared to TMV movement protein and 94 percent with ToMV. The RT-PCR protocol was efficient for quick and conclusive determination of virus species. The virus was purified and a polyclonal antiserum was raised for future surveys in tomato crops of Paraná. The partial genomic sequence obtained for TMV-Sapopema has been deposited under the accession number DQ173945, which is the first partial genomic sequence of an isolate of TMV from Brazil in the GenBank, and the first tomato virus isolate from Paraná to have some of its biological and molecular properties determined.

Durante uma inspeção em cultivos protegidos de tomate em Sapopema, Paraná, foram observadas anormalidades foliares em 90 por cento das plantas, indicando possivelmente a existência de um problema de fitotoxidade causada por herbicidas. Todavia, os sintomas manifestados nas hospedeiras após os ensaios de inoculação mecânica revelaram que os sintomas estariam relacionados a uma infecção por Tobamovirus. As reações de RT-PCR com oligonucleotídeos específicos para uma região interna da proteína de movimento de dois vírus comuns em tomate, TMV e ToMV, resultaram na amplificação de um fragmento de 409 pares de bases, apenas com os oligonucleotídeos específicos para o TMV. Após o sequenciamento, os aminoácidos deduzidos apresentaram identidade de 100 por cento quando comparados com as seqüências das proteínas de movimento de outros isolados do TMV, e 94 por cento de identidade com seqüências do ToMV. A RT-PCR demonstrou ser um método eficiente para a rápida e conclusiva determinação da espécie viral envolvida na infecção do tomateiro. A seqüência parcial do genoma do isolado de TMV de Sapopema, está depositada no GenBank sob o número de acesso DQ173945, sendo esta a primeira seqüência genômica parcial de um isolado de TMV do Brasil, e conforme o nosso conhecimento, o primeiro isolado de TMV do Paraná a ter algumas de suas propriedades biológicas e moleculares determinadas. Um anti-soro policlonal foi produzido, o que permitirá futuros levantamentos da ocorrência de Tobamovirus nas principais áreas de cultivo de tomate do Paraná.

Comparação de diferentes metodologias para obtenção de cultura de calos de Stevia rebaudiana (Bertoni) Bertoni / Comparison of different methodologies for obtaining callus cultures from Stevia rebaudiana (Bertoni) Bertoni

As folhas de Stevia rebaudiana (Bertoni) Bertoni (Asteraceae) contêm glicosídeos diterpenóides (GDS), que são cerca de 300 vezes mais doce que a sacarose a 4%. O objetivo deste estudo foi avaliar a formação de calos, a partir de folhas obtidas in vivo e in vitro de S. rebaudiana em dois meios já descritos na literatura: Murashige e Skoog (MS), suplementado com 3 mg L-1 de ácido 2,4-diclorofenóxiacético (2,4-D), e o MS suplementado com 1 mg L-1 de ácido naftalenoacético (ANA) e 0,5 mg L-1 de 6-benzilaminopurina 6-BAP) e um desenvolvido em nosso laboratório o Woody Plant Medium (WPM), suplementado com 6 mg L-1 de ANA e 4 mg L-1 de cinetina (CIN). Os explantes obtidos in vitro iniciaram a formação de calos um pouco mais rapidamente que os das folhas de plantas advindas da natureza. A utilização dos nutrientes do meio WPM, associada a uma combinação de fitorreguladores adequada, proporcionou velocidade de indução e multiplicação de calos bem maiores que as apresentadas nos meios que empregaram os nutrientes do MS. Novos experimentos serão realizados, depois de alcançada a estabilidade genética dos calos, visando avaliar a capacidade destes em biossintetizar os GDS

The leaves from Stevia rebaudiana (Bertoni) Bertoni (Asteraceae) contain diterpenoid glycosides (GDS), which are almost 300 times sweeter than sucrose at 4%. The subject of this study was to evaluate the callus-formation from in vivo and in vitro leaves of Stevia rebaudiana in two already described in literature: Murashige and Skoog (MS) supplemented with 3 mg L-1 of 2,4-dichlorophenoxyacetic acid (2,4-D); MS supplemented with 1 mg L-1 of naphthaleneacetic acid (NAA) and 0.5 mg L-1 of 6-benzylaminopurine (6-BAP) and other developed in our laboratory the Woody Plant Medium (WPM) with 6 mg L-1 of NAA and 4 mg L-1 of Kinetin (KIN). The explants obtained in vitro initiated callus formation faster than leaves from natural plants. The utilization of WPM nutrients, associated with an adequate combination of phytoregulators, provided greater callus induction velocity and multiplication than the media that using MS nutrients. New experiments will be conducted after reaching genetic stability of the calluses, seeking to evaluate the capacity of these calluses to biosynthesize GDS

Multiplicação in vitro de Aspidosperma ramiflorum Muell. Arg. (Apocynaceae) / In vitro multiplication of Aspidosperma ramiflorum Muell. Arg. (Apocynaceae)

O presente estudo relata um método simples e promissor para multiplicação in vitro de Aspidosperma ramiflorum, uma espécie encontrada no sudeste do Brasil e seriamente ameaçada de extinção, utilizada com propósitos medicinais e como fonte de compostos que podem ser usados para desenvolver novos fármacos sintéticos. O trabalho teve como objetivo o estabelecimento de um protocolo de multiplicação in vitro de Aspidosperma ramiflorum (guatambu), a partir de segmentos apicais de material juvenil originários de plântulas obtidos a partir de sementes. A avaliação da multiplicação in vitro foi realizada em meio de cultura Woody Plant Médium (WPM), suplementado com concentrações variadas de ácido naftalenoacético (ANA) e 6-Benzilaminopurina (6-BAP). A multiplicação de A. ramiflorum foi positivamente influenciada principalmente nas combinações aonde as concentrações de 6-BAP foram relativamente maiores do que as do ANA, nessas concentrações houve a indução de múltiplas brotações.

The present study described a simple and promissory method for in vitro multiplication of Aspidosperma ramiflorum, a species found in the South of Brazil and seriously extinction menaced. The method was used for medicinal proposes and as a source of compounds to develop new synthetic drugs. The objective of this work was to establish an in vitro multiplication protocol of Aspidosperma ramiflorum (guatambu), from apical segments of juvenile material of plantlets obtained from seeds. The in vitro multiplication evaluation was done in WPM medium, supplemented with variable concentrations of Naphthalene acetic acid (NAA) and 6- Benzyl aminopurine (6-BAP). The multiplication of A. ramiflorum was positively influenced mainly in the combinations when 6-BAP concentrations were relatively higher than NAA. In these concentrations multiple shoots were induced.