Evaluation of a PCR kit for the detection ofErwinia carotovora subsp.atroseptica on potato tubers

Abstract

SummaryA PCR-based kit, Probelia TM, for the detection of Erwinia carotovora subsp, atroseptica (Eca) on potatoes was evaluated at five laboratories in four countries. The kit is based on DNAspecific PCR amplification followed by detection of amplicons by hybridization to a peroxidase-labelled DNA probe in a microplate. Specificity of the PCR primers for Eca, regardless of serogroups, was confirmed by testing against 246 bacterial, fungal and plant species. Detection limits of the assay varied little between six Eca strains in pure cultures (1.3• to 1.5x103 cells ml-l). When Eca-free tuber peel extract from four cultivars was inoculated with known numbers of 15 Eca strains, detection limits were more variable (1.0xl0 l to 6.2x103 cells ml -I peel extract), attributed probably to inconsistency in the recovery of DNA during extraction. When the PCR assay was compared with three current commercial Eca detection methods, using naturally contaminated tubers, results matched most closely those from viable counts on a selective medium, the most sensitive method (88%), followed by enrichment ELISA (72%) and last ELISA (30%), the least sensitive method.

Elevation of three subspecies of Pectobacterium carotovorum to species level: Pectobacterium atrosepticum sp. nov., Pectobacterium betavasculorum sp. nov. and Pectobacterium wasabiae sp. nov.

“…Several DNA probes were found to be specific for P. carotovorum subsp. atrosepticum (Darrasse et al, 1994a;Ward & De Boer, 1994) and PCR primers were proposed for the detection of the same subspecies on potatoes (De Boer & Ward, 1995;Frechon et al, 1998). P. carotovorum subsp.…”

“…P. carotovorum subsp. atrosepticum was also consistently considered rather uniform in terms of PCR-RFLP fingerprints using a pel gene (Darrasse et al, 1994b;Helias et al, 1998) and comparison of the recA gene (Waleron et al, 2001). By contrast, P. carotovorum subsp.…”

Assessment of recent outbreaks of Dickeya sp. (syn. Erwinia chrysanthemi) slow wilt in potato crops in Israel

“…The DNA was purified using the silica beads method (Frechon et al 1998). The PCR conditions were used as described by Rademaker et al (1997), with few modifications as described below.…”

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

“…Os isolados obtidos foram identificados através dos seguintes testes: gram, atividade pectolítica em batata, oxidase, catalase, fermentação de glicose (oxidação/ fermentação), crescimento a 37°C, utilização de α-metil glicosídeo (estirpes positivas formam colônias com centro vermelho), sensibilidade à eritromicina, produção de substâncias redutoras de sacarose, produção de ácidos a partir de maltose D(+), lactose e sorbitol (Hyman et al, 1998;De Boer & Kelman, 2001 A capacidade dos isolados em utilizar diferentes açúcares foi testada em placas de microtitulação (96 amostras/ placa) (Silveira et al, 2002;Oliveira et al, 2003) contendo 150 µl de meio de cultura (peptona, 10 g; púrpura de bromocresol 1,5%, 0,7 ml; ágar, 0,3 g; açúcar 10%, 100ml/l, pH 6,8) (Dickey & Kelman, 1988). Culturas com 24-48 h de crescimento foram transferidas para o meio com o auxílio de um palito de madeira esterilizado, e incubadas a 28 °C por 48 h. A mudança de cor de púrpura para amarela foi considerada positiva.…”

“…No Rio Grande do Sul, a coleta de plantas com sintomas de canela preta resultou na obtenção de 408 isolados (Oliveira et al, Fitopatol. bras (De Boer & Ward, 1995;Fréchon et al, 1998) amplificaram o DNA da estirpe controle de P. carotovorum subsp. atrosepticum, mas não das estirpes identificadas como P. carotovorum subsp.…”