Marcelo Gravina de Moraes

Biochemical characterization of a new cysteine endopeptidase from Carica candamarcensis L.

This work describes a novel cysteine endopeptidase from C. candamarcensis L., which displays high esterase activity. The enzyme was isolated from latex collected from the unripe fruit and maintained at −20°C until processed. Purification was done by chromatography on Sephadex G-10, CM-Sephadex, SP-Sephadex, and thiol-Sepharose. The enzyme is a monomer with an optimal pH of 7.0, an estimated Mr 28 600, a pI above 10.5 and a cysteine residue that, upon reaction with E-64, abolished enzyme activity. The 25 NH2-terminal sequence of protein C reveals partial homology when compared with the sequences for chymopapain (89%), papaya proteinase IV (85%), proteinase Ω (85%) and papain (82%). The electrophoretic behavior of endopeptidase C in denaturing gels reveals significant size differences when compared to chymopapain. Also, mobility of endopeptidase C in acid non-denaturing gels is slower than chymopapain. The esterase activity of endopeptidase C toward BAPNA is approximately 7-fold higher than the values recorded for chymopapain and endopeptidase Ω.

Incidence of pectolytic erwinias associated with blackleg of potato in Rio Grande do Sul

Erwinia carotovora subsp. atroseptica (Eca), E. carotovora subsp. carotovora (Ecc) and E. chrysanthemi (Ech) may cause potato (Solanum tuberosum) blackleg. To determine the occurrence of these pathogens in the conditions found in the State of Rio Grande do Sul (RS), potato plants showing blackleg symptoms were harvested from 22 fields in nine counties in Serra do Nordeste, Planalto, Depressao Central, and Grandes Lagoas, from September to December of 1999 (Spring-Summer season). Green pepper (Capsicum annuum) fruits were used as a host to enrich for pectolytic erwinia from potato stems with blackleg symptoms. Bacteria were subsequently isolated on non-selective medium. Isolates that were Gram-negative, facultatively anaerobic, and pitted crystal-violet-pectate medium were tested for biochemical traits to identify the species and subspecies. Four hundred strains were identified as either Eca, Ecc or Ech. Although the three erwinias were found in RS potato fields, only three strains of Ech were found in one field. Frequencies of Eca and Ecc were 55 and 42%, respectively. Eight strains could not be assigned based on the biochemical characterization.

Caracterização de estirpes de Ralstonia solanacearum isoladas de plantas de batata com murcha bacteriana, por PCR-Rep e RAPD

Considered one of the most important potato (Solanum tuberosum) pathogens in tropical and subtropical regions, as well as in temperate regions with warmer climate, Ralstonia solanacearum is a species with significant genetic diversity. It has been characterized in a binary system of races and biovars based on the range of host species and on the ability to use carbon sources. The attempts to use genetic resistance as a strategy to control R. solanacearum were shown to be unstable due to climatic changes in different regions and due to the variability of the pathogenic strains. Due to different epidemiological characteristics of the biovars, strains of biovar 2 are more likely to be eradicated in an integrated control system. In a survey performed in four regions where potato is produced in Rio Grande do Sul, isolates of R. solanacearum were obtained from 25 crop fields in ten municipalities. The biochemical analyses of the isolates revealed the occurrence of biovars 1 and 2, the latter being predominant. The isolates obtained were evaluated for their genetic variability by PCR, using repetitive sequences ERIC and BOX and random primers (RAPD). The PCR-ERIC and BOX were able to clearly differentiate the biovars 1 and 2. However, neither analysis was able to demonstrate variability among isolates of biovar 2 and only PCR-BOX showed some degree of variability among isolates of biovar 1. The amplification by RAPD demonstrates the distinction between the biovars and revealed that they show characteristic profiles that are closely related to the region where they were obtained.

Ocorrência das biovares 1 e 2 de Ralstonia solanacearum em lavouras de batata no estado do Rio Grande do Sul

Occurrence and distribution of biovars of Ralstonia solanacearum in the State of Rio Grande do Sul were assessed by harvesting potato (Solanum tuberosum) plants showing bacterial wilt symptoms in the spring of 1999. Plants were collected from 25 potato fields in ten counties in the four main production regions: Serra do Nordeste, Planalto Superior, Depressao Central and Grandes Lagoas. Species and biovar determinations were based on serological test with ELISA and the oxidative metabolism of sugars and alcohols. Ninety-four percent of 490 strains were identified as biovar 2. Occurrence of biovar 1 was recorded in Serra do Nordeste and Depressao Central. The incidence of biovars showed no correlation with local temperature or potato cultivars.

Cadmium Accumulation in Tomato Cultivars and Its Effect on Expression of Metal Transport-Related Genes

Cadmium (Cd) content was quantified in the shoot of six tomato cultivars and the Cd effect on the expression of LeNRAMP3, LeFER, LeIRT1 and LeNRAMP1 was evaluated. The six tomato cultivars accumulated high Cd concentrations and were able to transport Cd to the fruits. Among the evaluated genes, the Cd-induced level of LeFER expression appeared to provide an evidence regarding the capacity of foliar Cd accumulation in tomato. The results indicate that tomato represents a possible pathway for Cd entry into the food chain and an attractive model organism for the elucidation of the mechanisms involved in Cd accumulation.

Epidemiological analysis of clones and cultivars of potato in soil naturally infested with Ralstonia solanacearum biovar 2

Os objetivos deste estudo foram avaliar o progresso da murcha bacteriana, a resistencia do clone MB 03, selecionado para a raca 1 em Brasilia, e detectar a presenca do patogeno em tuberculos colhidos de plantas assintomaticas. Durante a primavera (setembro a final de novembro no hemisferio sul) de 1999 e 2000, 14 cultivares ou clones foram cultivados num campo naturalmente infestado com R. solanacearum biovar 2, em Caxias do Sul, RS. O numero de plantas com sintomas de murcha foi registrado semanalmente para a obtencao da curva de progresso da doenca. Para comparar a resistencia dos genotipos de batata a murcha bacteriana foi calculada a area sob a curva de progresso da doenca. Dentre os modelos avaliados para ajuste das curvas, o modelo logistico foi o que melhor se ajustou. Ao final das avaliacoes, em cada periodo de cultivo, os tuberculos produzidos por plantas assintomaticas foram coletados e armazenados ate o inicio da brotacao, quando foram submetidos a testes para detectar a presenca de R. solanacearum. A cultivar Cruza 148 e o clone MB 03 foram os genotipos mais resistentes, mas ambos apresentaram tuberculos com infeccoes latentes. As implicacoes epidemiologicas da incidencia de R. solanacearum raca 3 em lavouras de batata, bem como da resistencia de determinados genotipos que podem propiciar tuberculos com infeccoes latentes, sao aspectos de importância para o manejo integrado da murcha bacteriana no RS.

Ocurrence of Rice stripe necrosis virus in rice crops from Rio Grande do Sul, Brazil

Ocurrence of Rice stripe necrosis virus in rice crops from Rio Grande do Sul, Brazil A viral disease in rice (Orysa sativa), caused by Rice stripe necrosis virus (RSNV), is reported for the first time in Rio Grande do Sul, Brazil. Na safra agricola 2001/02, constatou-se sintomas de plântulas mortas, listras cloroticas em folhas e retorcimento de folhas e paniculas (Figura 1A) em lavouras de arroz (Oryza sativa L.) irrigado, localizadas proximas ou no municipio de Dona Francisca, Regiao da Depressao Central do Rio Grande do Sul. Como hipotese, sugeriu-se como agente causal dos sintomas o Benyvirus denominado Rice stripe necrosis virus (RSNV) transmitido pelo protista Polymyxa graminis Ledingham, causador do “entorchamiento” ou “crinkling”. Para confirmar a hipotese, plantas sintomaticas dessa regiao foram analisadas para deteccao do virus e do vetor. Amostras dessas plantas com sintoma de virose foram enviadas ao Centro Internacional de Agricultura Tropical (CIAT), na Colombia, sendo, as raizes submetidas a coloracao com fucsina e analisadas em microscopio otico para visualizacao das estruturas do vetor, e os extratos foliares analisados em microscopio eletronico (JEOL 100SX) para visualizacao das particulas virais. No Brasil, apos purificacao parcial segundo o protocolo de Fauquet & Thouvenel, exceto as etapas de ultracentrifugacao (C. R. Acad. Sci. Paris 296:575. 1983), o virus foi inoculado em folhas de Nicotiana benthamiana Domin, especie indicadora e, como controle foi inoculado tampao fosfato de potassio (0,01 M) pH 7,2. Tambem realizou-se Double Antibody Sandwich–Enzyme-Linked Immunosorbent Assay (DASELISA) com anticorpo policlonal reativo contra RSNV (Morales et al., European Journal of Plant Pathology 5:643. 1999). Alem disso, 160 plantas da cultivar IRGA 417 foram cultivadas ate 42 dias apos a emergencia, 80 em vasos contendo solo coletado em areas com plantas sintomaticas e as outras 80 em solo de areas com plantas sem sintomas. Na analise dos extratos foliares observaram-se particulas virais caracteristicas de RSNV (Kanyuka et al., Molecular Plant Pathology 4:393. 2003) e nas raizes verificaram-se cistosoros de P. graminis (Figura 1B). Somente as plantas de N. benthamiana inoculadas com RSNV apresentaram necrose (Figura 1C). No DAS-ELISA todas as amostras de plantas com sintomas foram positivas para RSNV. Os sintomas foram observados apenas nas plantas cultivadas em solo de areas com plantas sintomaticas. Assim, conclui-se que o agente causal dos sintomas descritos e Rice stripe necrosis virus. FIG. 1 A. Planta de arroz (Oryza sativa) com sintoma de Rice stripe necrosis virus (RSNV); B. cistosoro de Polymyxa graminis em aumento de 100x; C. Folhas de Nicotiana benthamiana, a da esquerda inoculada com tampao fosfato de potassio (controle) e da direita inoculada com RSNV, parcialmente purificado.

Padrão molecular e de virulência de isolados de Pyricularia grisea do Estado do Rio Grande do Sul

Selection for rice blast resistance is a major problem in any rice (Oryza sativa) breeding program due to the variability of the fungus Pyricularia grisea. This diversity has been characterized by DNA molecular markers and by virulence of the isolates that has been demonstrated on rice genotypes with different blast resistance genes. Molecular markers allow the identification of groups of isolates, so called lines, genetically related to each other, and the isogenic lines (NILs) in groups of virulence with the same or similar virulence pattern. The objectives of this work were to verify the molecular and virulence patterns from isolates of P. grisea from the State of Rio Grande do Sul. The DNA from 51 monosporic isolates obtained in Rio Grande do Sul were used in PCR reactions with primers based on the repetitive sequence Pot2. The same isolates were also used to inoculate six NILs. The statistical analysis indicates the presence of six lines and seven virulence groups. The frequency of virulent isolates is higher in the NIL that contains the Pi-1 allele and lower in the NIL containing the Pi-2 allele. The compatible reaction on the NIL that carries Pi- 2 is very weak, exhibiting, in general, a type 4 reaction. No obvious relationship between phenotypic virulence and line determination has been found.

Análise da variabilidade genética de duas cultivares Raminad Str. 3 utilizadas como diferenciadoras de raças de Pyricularia grisea

O conhecimento sobre a ocorrencia e distribuicao de racas do fungo Pyricularia grisea e um importante aspecto a ser considerado em programas de melhoramento genetico de arroz (Oryza sativa). No entanto, para que a identificacao das racas seja correta e passivel de comparacoes com outros levantamentos e necessario que os genotipos utilizados apresentem as mesmas caracteristicas geneticas. O presente trabalho foi realizado com os objetivos de comparar a variacao genetica entre dois genotipos identificados como pertencentes a cultivar Raminad Str. 3, que e uma das oito cultivares utilizadas como direnciadoras de racas de P. grisea, e examinar a composicao de racas deste fungo no estado do Rio Grande do Sul. Constatou-se a amplificacao de fragmentos polimorficos em 20 dos 39 marcadores microssatelites utilizados para verificar a pureza genetica de dois genotipos de arroz identificados como sendo a cultivar Raminad Str. 3. A identificacao das racas baseou-se na reacao das oito cultivares diferenciadoras de racas submetidas a inoculacao com 85 isolados monosporicos de P. grisea, provenientes de 14 municipios produtores de arroz no Rio Grande do Sul. A avaliacao do grau de infeccao foi realizada de acordo com a escala preconizada pelo sistema internacional de avaliacao de doencas do arroz, 14 dias apos a inoculacao.

Incidence of Biovars 1 and 2 of Ralstonia solanacearum in Potato Fields at Rio Grande do Sul State, Brazil

Bacterial wilt caused by Ralstonia solanacearum is one of the most serious limitations to potato (Solanum tuberosum L.) crop at tropical, sub-tropical, and warmer areas of temperate zones of the world. R. solanacearum strains are grouped in races by host range and biovars according to their oxidative metabolism of sugars and alcohols. Strains of biovars 1 and 2 are able to infect potato. The first group has a wide host range, survives in the soil for long periods, and predominates in high temperature regions. The second one basically infects potato, occurs in temperate regions and presents higher incidence of latent infections. These different pathogen traits determine different measures on disease management. Biovar 2 is considered endemic in the South and Southeast regions of Brazil. Rio Grande do Sul State is located at South, latitude 28 to 34 and humid subtropical climate. Potato is grown yearly in two seasons, comprising an area larger than 48,000 ha, represented by at least four distinct production regions. The purpose of this research was to study the incidence and distribution of biovars 1 and 2 of R. solanacearum in those potato production regions.