Characterization of Soybean yellow shoot virus, a New Member of the Family Potyviridae Infecting Soybean Plants in Brazil.

A new virus species, belonging to the family Potyviridae and capable of infecting most of the soybean cultivars grown in Brazil, was collected in Lavras, Minas Gerais, Brazil, and named Soybean yellow shoot virus (SoyYSV). In this study, the complete 9,052-nucleotide genome of SoyYSV was determined and the structural, biological, and molecular properties of the virus were investigated. The SoyYSV genome encoded a single polyprotein that could be subsequently cleaved, generating 11 proteins. The SoyYSV genome shared 49% nucleotide and 36% amino acid sequence identity with Blackberry virus Y. However, the P1 protein of SoyYSV was much smaller and lacked the ALK1 domain characteristic of the genus Brambyvirus. Electron microscopy revealed flexuous filamentous virus particles, 760 to 780 nm in length, and cytoplasmic inclusions typical of those found in plant cells infected with Potyviridae species. In addition to soybean, SoyYSV infected species in the Amaranthaceae, Caricaceae, Fabaceae, and Solanaceae families. Among the most common potyviruses present in Brazil, only SoyYSV induced local necrotic lesions in Carica papaya L. SoyYSV was transmissible by Myzus persicae and Aphis gossypii but lacked the HC-Pro domain required for aphid transmission in other potyviruses. No seed transmission in soybean was observed.

First report and molecular characterization of an isolate of papaya ringspot virus (PRSV-W) detected in pumpkin in Cuba / Primeiro relato e caracterização molecular de um isolado de papaya ringspot virus (PRSV-W) detectado em planta de abóbora proveniente de Cuba

In this work, a virus isolate collected from pumpkin plants (Cucurbita pepo L.), showing severe symptoms of mosaic and leaf deformation, grown in Cuba, was analyzed using indicator plants, electron microscopy, and phylogenetic analysis. Plants of pumpkin, cv. Caserta, inoculated with this virus isolate showed mosaic, leaf distortion and blistering symptoms, whereas papaya plants were immune and did not show any symptoms. A transmission electron microscopic examination of leaf dip preparations made from infected pumpkin leaves revealed the presence of elongated and flexuous particles, approximately 780-800 x 12 nm in size. Genomic fragments containing the coat protein (CP) and HC-Pro genes, amplified by specific primers for Papaya ringspot virus, W strain (PRSV-W), showed amino acid identities of both genes higher than 94% when compared to other PRSV-W isolates from America. In the phylogenetic tree, this virus isolate has grouped with other virus isolates from America, Australia, and India and was more distant from the Asian isolates. Taken together, the analyses allow the conclusion that this virus isolate is a W strain of PRSV, detected for the first time in Cuba.

Neste trabalho um isolado viral coletado em planta de abóbora (Cucurbita pepo L), apresentando sintomas severos de mosaico e deformação foliar, proveniente de uma lavoura localizada em Cuba, foi analisado utilizando plantas indicadoras, microscopia eletrônica de transmissão e análise filogenética. Plantas de abóbora cv. Caserta, inoculadas com este isolado do vírus mostrou mosaico, distorção foliar e bolhas, enquanto que as plantas de mamão foram imunes e não apresentaram sintoma. Exame ao microscópio eletrônico de tranmissão de telas preparadas com a técnica leaf dip, empregando o extrato de folhas de abóbora infectadas revelou a presença de partículas alongadas e flexuosas, medindo cerca de 780-800 x 12 nm. A análise de fragmentos genômicos contendo os genes da proteína capsidial (CP) e HC-Pro, amplificados por primers específicos para Papaya ringspot virus, estirpe W (PRSV -W), mostrou identidades de aminoácidos superiores a 94 % quando ambos os genes foram comparados a outros isolados americanos de PRSV -W. Na árvore filogenética, este isolado estudado se agrupou com os isolados de PRSV-W da América, Austrália e Índia, ficando mais distante dos isolados asiáticos. Tomadas em conjunto, as análises permitem concluir que este isolado viral pertence à estirpe W do PRSV, detectada pela primeira vez em Cuba.

Variability of seed-borne Colletotrichum strains in cotton based on ITS1 and ITS2 ribossomal genes analysis / Variabilidade de estirpes de Colletotrichum provenientes de sementes de algodão com base na análise dos genes ribossomais ITS1 e ITS2

The use of DNA sequences analysis has been an important mean to distinguish and to identify populations of organisms at different levels. By molecular markers several complex organisms have been successful detected in plants for distinct aims. Ribosomal DNA (rDNA) has been used to evaluate genetic variability, microorganism phylogeny and to develop specific primers for detection of plant pathogens in plant tissues. In this study, the objective was to characterize isolates of Colletotrichum gossypii var. cephalosporioides and Colletotrichum gossypii, collected in different regions of Brazil, by analyzing the nucleotide sequence of rDNA regions. ITS1, ITS2, and the intervening 5.8S gene were amplified by PCR and their sequences compared to each other and to those from other species registered in the GenBank. The rDNA of isolates associated with Gossypium spp. showed sequence identities ranging from 96 to 100% in the ITS1 region, 98 to 100% in the 5.8S gene, and 97 to 100% in the ITS2 region. The sequences were submitted to UPGMA analysis, and according to the phylogenetic trees, the C. gossypii var. cephalosporioides and C. gossypii species clustered together along with isolates of Glomerella cingulata from mango and papaya, and thus no distinction could be made between isolates of those organisms.

O uso de sequências de fragmentos de DNA tem sido importante ferramenta para distinguir e identificar populações de organismos em diferentes níveis de variação. Por meio de marcadores moleculares alguns organismos com variações taxonômicas complexas têm sido detectados com sucesso em tecidos vegetais. O DNA ribossomal tem sido utilizado para avaliar variabilidade genética, filogenia de micro-organismo e para desenvolver oligonucleotídeos específicos visando à detecção de fitopatógenos. Nesse estudo o objetivo foi comparar isolados do complexo Colletotrichum, incluindo C. gosssypii var. cephalosporioides e C. gossypii, coletados em diferentes regiões do Brasil, todos associados às sementes de algodão, pela análise de sequências de nucleotídeos de regiões de rDNA. ITS1, ITS2 e o gene 5.8S que foram amplificados por PCR e suas sequências comparadas entre si com outras sequências depositadas no GenBank. O rDNA de isolados associados com Gossypium spp. mostraram identidades de sequências na faixa de 96 to 100% na região ITS1, 98 to 100% na região de 5.8S, e 97 a 100% na região ITS2. As sequências foram submetidas a análise UPGMA, e de acordo com as árvores filogenéticas , C. gossypii var. cephalosporioides e C. gossypii fizeram parte de um mesmo cluster junto com isolados de Glomerella cingulata de manga e mamão, e assim nenhuma distinção pode ser feita entre os isolados destes organismos.

Caracterização morfológica, genética e patogenicidade de isolados de Rhizoctonia solani provenientes de algodoeiros no Brasil / Morphological, genetic characterization and pathogenicity of Rhizoctonia solani isolates from cotton in Brazil

A espécie Rhizoctonia solani é um patógeno importante na cultura do algodão associada a doenças conhecidas como tombamento e mela. A variabilidade entre os isolados são extremamente importantes, porque existem diferenças entre os grupos de anastomose, tomadas como aqueles isolados capazes de trocar informações genéticas entre si. A caracterização morfológica, quando confirmada pela a caracterização genética fornece informações concretas sobre a distribuição do isolados quando agem como um agente patogênico. O objetivo foi identificar e caracterizar os grupos de anastomose no Brasil e confirmá-los pela caracterização genética. Foram consideradas 51 isolados de Rhizoctonia solani com o objetivo de caracterizar os grupos de anastomose e determinar a patogenicidade. A caracterização morfológica foi realizada observando-se o número de núcleos, morfologia da colônia e identificação de grupos de anastomose (AG). Na caracterização genética foram sequenciados e analisados os fragmentos genômicos contendo as regiões 5.8 S, ITS1 e ITS2, comparando-se os isolados listados no GenBank. A patogenicidade foi avaliada utilizando a escala de infecção com graus que variam de 1 a 4. Considerando-se o AG foram identificados 36 de 51 isolados como AG-4 e dois isolados como AG-7, 13 isolados não tiveram classificação. Em análises moleculares foram confirmados os 36 isolados identificados pela caracterização morfológica e mais 10 isolados como AG-4. Todos os isolados AG-7 foram confirmados e encontrado mais um nas análises moleculares. Para a patogenicidade verificou-se que cinco isolados não diferem do controle. A virulência intermediária e alta virulência foram observadas em 16 e 24 isolados, respectivamente, com médias 2,52-3,3 e 3,4-3,9.

The Rhizoctonia solani is an important pathogen in cotton crop associated with damping-off disease. The variability among isolates are extremely important, because differences exist between anastomosis groups, taken as those isolates capable of exchanging genetic information with each other. Morphological characterization, when confirmed by genetic characterization provides concrete information about the isolates distribution when it acts like a pathogen. Our study was to identify and characterize the anastomosis groups in Brazil and confirm them by genetic characterization. We considered 51 Rhizoctonia solani isolates aiming to characterize the anastomose group and determine their pathogenicity. The morphological characterization was done observing the number of cores, colony morphology and identification of anastomosis groups (AG). In genetic characterization were sequenced and analyzed the genomic fragments containing the 5.8 S, ITS1 and ITS2 regions and compared them to Rhizoctonia isolates listed in the GenBank. The pathogenicity was evaluated for the disease severity, using the scale of infection with grades ranging from 1 to 4. Considering the AG we identified 36 of 51 isolates as AG-4 and two isolates as AG-7 and 13 isolates were listed as unrated. In molecular analyses were confirmed those 36 isolates and were identified more 10 isolates as AG-4. All the AG- 7 isolates were confirmed and we found one more considering the molecular analyses. For the pathogenicity was found that five strains did not differ from the control. Intermediate virulence and high virulence were observed in 16 and 24 isolates, respectively with averages from 2.52 to 3.3 and from 3.4 to 3.9.

Genetic transformation with the gfp gene of Colletotrichum gloeosporioides isolates from coffee with blister spot

Blister spot (Colletotrichum gloeosporioides) is now widespread in most coffee producing states of Brazil, becoming a limiting factor for production. The lack of data relating to the reproduction of typical symptoms (light green, oily patches) leaves a gap within the pathosystem, forcing the search for new methodologies for monitoring the disease. Monitoring of genetically modified organisms has proven to be an effective tool in understanding the host x pathogen interactions. Thus, the present study was carried out to evaluate the effectiveness of two systems of genetic transformation in obtaining mutants using the gfp reporter gene. Using the two transformation systems (PEG and electroporation) revealed the efficiency of both, confirmed by fluorescence microscopy and resistance to the antibiotic hygromycin-B, when incorporated into the culture medium. The fungus maintained its cultural and morphological characteristics when compared to wild strains. When inoculated on coffee seedlings, it was found that the pathogenicity of the processed isolates had not changed.

Complete genome sequence of the first Andean strain of potato virus S from Brazil and evidence of recombination between PVS strains.

An isolate of the Andean strain of potato virus S (PVS), named BB-AND, was detected for the first time in a Brazilian potato crop, fully sequenced and analyzed. A comparison of BB-AND with other PVS isolates (Andean and Ordinary) showed that BB-AND is quite distinct. The lowest amino acid sequence identity to the only other fully sequenced Andean isolate was found in ORF 1 (82%) and ORF 6 (87%). Recombination analysis showed that the isolate Vltava (AJ863510), from Germany, is a recombinant between PVS(O) and PVS(A) isolates, with the recombination event located between nucleotides 6125 and 8324.

Reação fenotípica de genótipos de abóboras ao vírus da mancha anelar do mamoeiro, estirpe melancia (Pappaya ringspot virus, strain watermelon ­ PRSV-W) / Phenotypic reaction of Cucurbita spp. genotypes to Pappaya ringspot virus, watermelon strain ­ PRSV-W

O Pappaya ringspot vírus, estirpe melancia - PRSV-W, é a principal doença virótica das abóboras em condições tropicais. O trabalho objetivou avaliar a resistência fenotípica e o padrão de sintomas apresentados por genótipos de abóboras (Cucurbita spp.) ao PRSV-W. O experimento foi conduzido no delineamento inteiramente casualizado com quatro repetições e 10 plantas por parcela. Foram realizadas duas inoculações, a primeira na fase cotiledonar e a segunda cinco dias após a primeira. As plantas foram avaliadas quanto ao aparecimento de sintomas durante 30 dias, começando 10 dias após a segunda inoculação, através de uma escala de notas, em que: nota ­ 1: plantas com folhas sem sintomas de mosaico e; nota ­ 5: plantas com folhas apresentando mosaico intenso, com bolhosidade e presença de deformações foliares mais severas. Foram avaliados nove genótipos, dos quais três são regionais [ABTO#01 e ABTO#02 (C. maxima); ABTO#03 (C. moschata)]; e seis são cultivares comerciais [cv. Caserta (C. pepo); cvs. Menina Brasileira; Paulista; Baianinha; Jacarezinho (C. moschata); e o híbrido interespecífico Tetsukabuto ou Kabutiá (C. máxima x C. moschata)]. Também foram avaliadas progênies endogâmicas do acesso ABTO#01. Apenas o genótipo regional ABTO#01 não apresentou sintomas durante o período de avaliação, sendo considerado resistente. Todos os genótipos comerciais foram suscetíveis e os sintomas apresentados foram bolhosidades, mosaicos, estreitamento foliar e subdesenvolvimento. Todas as progênies endogâmicas oriundas do acesso ABTO#01 avaliadas foram resistentes ao PRSV-W e, portanto, constituem progênies promissoras para serem utilizadas em programas de melhoramento genético da abóbora para a região do Tocantins.

The Pappaya ringspot virus, watermelon strain - PRSV-W is the most important viral disease of Cucurbita spp. in the tropical conditions. The study evaluated the phenotypic resistance and the pattern of symptoms presented by the genotypes of Cucurbita spp. to PRSV-W. The experiment was conducted in completely randomized design with four replications. The plants were evaluated for symptoms 30 days after inoculation, using a scale where: Note - 1: plants with no symptoms of mosaic and; note - 5: plants with leaves showing mosaic intense with blistering and the presence of more severe leaf deformation. Nine genotypes were evaluated, three landraces [ABTO#01; ABTO#02 (C. maxima) e ABTO#03 (C. moschata) and six commercial cultivars [cv.Caserta (C. pepo); cvs. Menina Brasileira; Paulista; Baianinha; Jacarezinho (C. moschata) and the interspecific hybrid Tetsukabuto or Kabutiá (C. moschata x C. maximum)]. Inbred progenies from ABTO#01 were also evaluated. All commercial genotypes were susceptible and symptoms were blistering, mosaics, leaf narrowing and underdevelopment. The genotype ABTO#01 and their inbred progeny presented no virus symptoms during the evaluation period, being considered resistant and a promising genotype to be used in breeding programs of the Cucurbita spp. aiming resistance to PRSV-W.

Genetic transformation with the gfp gene of Colletotrichum gloeosporioides isolates from coffee with blister spot.

Blister spot (Colletotrichum gloeosporioides) is now widespread in most coffee producing states of Brazil, becoming a limiting factor for production. The lack of data relating to the reproduction of typical symptoms (light green, oily patches) leaves a gap within the pathosystem, forcing the search for new methodologies for monitoring the disease. Monitoring of genetically modified organisms has proven to be an effective tool in understanding the host × pathogen interactions. Thus, the present study was carried out to evaluate the effectiveness of two systems of genetic transformation in obtaining mutants using the gfp reporter gene. Using the two transformation systems (PEG and electroporation) revealed the efficiency of both, confirmed by fluorescence microscopy and resistance to the antibiotic hygromycin-B, when incorporated into the culture medium. The fungus maintained its cultural and morphological characteristics when compared to wild strains. When inoculated on coffee seedlings, it was found that the pathogenicity of the processed isolates had not changed.

Combining ability of summer-squash lines with different degrees of parthenocarpy and PRSV-W resistance

The aim was to assess heterosis in a set of 16 summer-squash hybrids, and evaluate the combining capacity of the respective parental lines, which differed as to the degree of parthenocarpy and resistance to PRSV-W (Papaya Ringspot Virus-Watermelon strain). The hybrids were obtained using a partial diallel cross design (4 x 4). The lines of parental group I were 1 = ABX-037G-77-03-05-01-01-bulk, 2 = ABX-037G-77-03-05-03-10-bulk, 3 = ABX-037G77-03-05-01-04-bulk and 4 = ABX-037G-77-03-05-05-01-bulk, and of group II, 1' = ABX-037G-77-03-05-04-08-bulk, 2' = ABX-037G-77-03-05-02-11-bulk, 3' = Clarice and 4' = Caserta. The 16 hybrids and eight parental lines were evaluated for PRSV-W resistance, parthenocarpic expression and yield in randomized complete-block designs, with three replications. Parthenocarpy and the resistance to PRSV-W were rated by means of a scale from 1 to 5, where 1 = non-parthenocarpic or high resistance to PRSV-W, and 5 = parthenocarpic or high susceptibility to PRSV-W. Both additive and non-additive gene effects were important in the expression of parthenocarpy and resistance to PRSV-W. Whereas estimates of heterosis in parthenocarpy usually tended towards a higher degree, resistance to PRSV-W was towards higher susceptibility. At least one F1 hybrid was identified with a satisfactory degree of parthenocarpy, resistance to PRSV-W and high fruit-yield.

Seleção de linhagens de melancia resistentes ao Watermelon mosaic virus e ao Papaya ringspot virus / Selection of resistant watermelon lines to Watermelon mosaic virus and Papaya ringspot virus

Foram avaliadas 20 linhagens de melancia, provenientes do cruzamento da cultivar comercial suscetível Crimson Sweet e da introdução PI 595201 resistente ao Watermelon mosaic virus (WMV) e Papaya ringspot virus (PRSV-W). As linhagens, e os parentais foram inoculados com o WMV ou com o PRSV-W em casa-de-vegetação distintas. Aos 35 e 49 dias após a primeira inoculação (DAI), as plantas foram avaliadas por meio de uma escala de notas, em que 1 (ausência de sintomas) a 5 (intenso mosaico e deformações foliares). Pelos resultados infere-se que, aos 35 DAI, as linhagens 1, 2 e 20 apresentaram resistência tanto para o WMV como para o PRSV-W, com médias de 1,95, 1,80 e 2,25 para o WMV, e de 2,50, 2,30 e 2,50 para o PRSV-W, respectivamente. As linhagens 5, 7 e 13 foram resistentes somente ao WMV e as plantas das linhagens 3, 10 e 18 para o PRSV-W. A reação das linhagens permaneceu em geral pouco alterada aos 49 DAI. A existência de linhagens resistentes somente ao WMV e somente ao PRSV-W, ao lado de linhagens resistentes a ambos os vírus, é indicativo de que as resistências ao WMV e ao PRSV-W não são controladas pelos mesmos genes.

Twenty advanced watermelon breeding lines, derived from the cross between cv. Crimson Sweet (susceptible) and PI 595201 (resistant to WMV and PRSV-W), were screened for resistance to both potyviruses. The twenty lines, among with Crimson Sweet and PI 595201, were inoculated with either WMV or PRSV-W, in two different greenhouse trials. Plants were evaluated for symptoms 35 and 49 days after the first inoculation (DAI), using a scale from 1 (no symptoms) to 5 (severe mosaic and foliar distortion). Evaluations at 35 DAI indicated that lines 1, 2 and 20 had good levels of resistance to both WMV and PRSV-W, with ratings of 1,95, 1,80 and 2,25 for WMV, and of 2,50, 2,30 and 2,50 for PRSV-W, respectively. Lines 5, 7 and 13 were resistant to WMV only, whereas lines 3, 10 and 18 were resistant to PRSV-W only. The reaction of the lines 49 DAI remained essentially unchanged. The existence of lines with resistance to WMV only and to PRSV-W only, along with lines with resistance to both viruses, indicates that resistance to WMV and PRSV-W are under control of different genes.