Valmir Duarte

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.

Identificação de Pectobacterium carotovorum subsp. brasiliensis através de PCR-RFLP do gene recA

Pectobacterium carotovorum subsp. brasiliensis has been proposed to be the major causal agent of blackleg of potato (Solanum tuberosum) in Brazil. In order to identify this subspecies, primers were selected from heterologous regions of recA genes present in P. carotovorum subsp. brasiliensis ATCC BAA-41, P. carotovorum subsp. carotovorum BAB1, and other DNA sequences of pectobacteria available in the GenBank. These primers, however, showed low specificity. The recA gene PCR product, a DNA fragment of ± 730 bp, from 38 strains of P. chrysanthemi, subspecies of P. carotovorum, was digested with TasI and HhaI restriction enzymes. These enzymes were selected based on the sequences of recA gene from strains of P. carotovorum subsp. brasiliensis ATCC BAA-416 (581 pb) and P. carotovorum subsp. carotovorum BAB1 (626 pb). The restriction fragment length polymorphism (PCR-RFLP) analysis with TasI and HhaI enzymes generated seven and 12 patterns, respectively. Analysis of the combined results allowed for the separation of 13 distinct groups and differentiation of P. carotovorum subsp.brasiliensis.

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.

Detecção e epidemiologia da podridão branca da maçã

Although white rot caused by Botryosphaeria dothidea is one of the most important diseases of apple (Malus domestica) in Brazil during rainy summer seasons, local information on the disease and pathogen is scarce. The aim of this research was to define methods of inoculating B. dothidea on apple fruits without wounding them, to detect latent infection and to analyze the temporal and spatial patterns of the disease in a commercial orchard. The best results of inoculum production were obtained on PDA covered with filter paper. The higheest apple infection rates were obtained after two days in a humid chamber before inoculation with a conidial suspension carried by a piece of paper. Latent infection was better detected when infected fruits were disinfested with NaOCl, 1.25% of active chlorine and 9.6 oGL of ethylic alcohol for 2 min. Production of B. dothidea on pruning stems occurred in different periods of each season. The disease distribution in a fungicide treated orchard showed clear aggregation of diseased plants but slight aggregation of fruits inside the plant.

Ocorrência de pectobactérias em tubérculos de batata-semente no estado do Rio Grande do Sul

Tuberculos de batata (Solanum tuberosum)-semente, pre-basica, basica, registrada e certificada, de oito cultivares, oriundos de 21 lavouras localizadas nos municipios de Vacaria, Cangucu, Piratini e Ibiraiaras, no Rio Grande do Sul, foram coletados nos meses de maio a agosto de 2002. Cada tuberculo foi lavado em agua corrente, deixado secar a temperatura ambiente, perfurado com palitos em dez lenticelas, coberto com fina camada de oleo de soja, colocado individualmente em cima de folha de papel toalha umedecida dentro de saco plastico transparente e incubado a 23 oC por quatro dias. A incidencia de podridao mole a partir das lenticelas variou de 20_100% entre as cultivares. Pectobacterium sp. foi constatada em tuberculos das 21 lavouras. Duzentos e vinte e tres isolados de Pectobacterium sp. foram obtidos em meio CPG, a partir das lenticelas com podridao mole, e identificados por testes bioquimicos, fisiologicos e PCR em nivel de subespecie. Cento e dezenove isolados foram identificados como P. carotovorum subsp. brasiliensis e e 96 com o P. carotovorum subsp. carotovorum. Oito isolados nao se enquadraram na classificacao bioquimica. Pectobacterium carotovorum subspp. estavam presentes em tuberculos de batata-semente, independente da cultivar, classe ou municipio de origem. Pectobacterium carotovorum subsp. atrosepticum, a principal responsavel por causar canela preta em batata em outros paises, nao foi detectada.

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.

Determinação da presença da raça 1 de Venturia inaequalis no Sul do Brasil

This study aims to identify races of V. inaequalis present in the apple-growing regions of Southern Brazil. Apple plants and detached leaves were inoculated by spraying and also by using four drops of inoculum (2.5 x 105 conidia.mL-1) on the surface of each leaf. Four fungal isolates from Rio Grande do Sul and five from Santa Catarina State, 2005 season, were used. Following inoculation, the plants were incubated in a controlled environment (RH 90-100%, 16-20 oC, darkness) for 48 h and then moved to the greenhouse. Sprayed leaves were maintained in agarized water medium and incubated for 48 h in the dark at 17oC. Afterwards, leaves were dried off and incubated together with the single-drop-inoculated detached leaves, under conditions of 19oC and 18 h of light. Evaluations were made 20 days after inoculation. The pattern of symptoms led to a conclusion that the nine isolates were of race 1. The same was observed on the spray-inoculated detached leaves but not with the single-drop inoculation method. This is the first report of the presence of race 1 of V. inaequalis in South America.

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.

Detecção de bactérias associadas a gemas de pereiras europeias no Rio Grande do Sul

The death of flowers in European pear (Pyrus communis L.) orchards was observed in Vacaria, State of Rio Grande do Sul, Brazil. Two bacterial isolates, obtained from symptomatic flowers, reproduced these symptoms on flowers of detached branches and were identified as Pseudomonas sp. Considering the lack of reports of this disease in Brazil, the record of P. syringae pv. syrigae and Erwinia amylovora associated with similar symptoms in other countries and the possible presence of these bacteria in buds, a method to detect the three bacteria was developed. The method comprised: bud maceration with liquid nitrogen, addition of PBS and 2% PVP, transfer of juice to FTA cards (Whatman), treatment of card circles with 70% ethanol, TE buffer and FTA reagent, elution of DNA followed by PCR with specific primers for each bacterium. The method was further used to monitor these bacteria in buds collected bimonthly during 2008. E. amylovora was not detected. The average frequency of P. syringae pv. syringae and Pseudomonas sp. was 0.7 and 16.3%, respectively, the latter showing a frequency peak of 30% in October. The species of the causal agent was not made clear, but the association with the genus Pseudomonas was demonstrated, which already provides a basis for the development of methods for disease control.

Caracterização de populações de Ralstonia solanacearum Smith em tabaco (Nicotiana tabacum L.) no Brasil

Ralstonia solanacearum, causal agent of bacterial wilt on different hosts, including members of Solanaceae, causes severe losses in tobacco crop, if not managed. It is a complex species and classified in several ways. Lately, the species was divided into three taxonomic levels: phylotype, sequevar and clone. Due to the lack of studies on populations of R. solanacearum in tobacco, this research aimed to characterize 120 isolates from soil and wilted tobacco plants, from 13 counties from Parana, 24 from Santa Catarina, 13 from Rio Grande do Sul, one from Paraiba and two from Pernambuco, by biovar, phylotype, and by using repetitive polymerase chain reaction (rep-PCR) element (BOX, ERIC and REP) primers. The results indicated that all isolates belong to the biovar 1 and phylotype II. Although there was homogeneity regarding biovar and phylotype, the results of rep-PCR allowed the formation of six groups (A, B, C, D, E and F) from 61% of similarity, regardless of sample type, geographical origin, collecting date or cultivar. Groups A and B were those that had the greatest number of isolates, 47 and 20 clonal lines, respectively. Knowledge regarding R. solanacearum population variability as well as the virulence of different isolates may help in the success of breeding programs and integrated disease management.