Evaluation of procedures for reliable PCR detection of Ralstonia solanacearum in common natural substrates

Poussier, Stéphane; Cheron, Jean-Jacques; Couteau, Annie; Luisetti, Jacques

doi:10.1016/s0167-7012(02)00111-2

Cited by 49 publications

(26 citation statements)

References 31 publications

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“…Os resultados positivos determinados pela técnica IFAS são confirmados por bioensaio com plantas de tomate (indicadoras) e pela detecção de seqüências de DNA específicas por PCR a partir da cultura pura da bactéria isolada em meio seletivo de cultura (OEPP/EPPO, 2004). Testes diagnósticos utilizando PCR a partir de amostras de água, sementes, solo e de material vegetal têm mostrado rapidez e especificidade de detecção (Seal et al, 1999;Lee & Wang, 2000;Poussier et al, 2002).…”

Section: Discussionunclassified

Ralstonia solanacearum em viveiros clonais de eucalipto no Brasil

Alfenas

Máfia

Sartório³

et al. 2006

Fitopatol. bras.

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A incidência da murcha bacteriana, causada por Ralstonia solanacearum, em viveiros clonais de eucalipto, no período de abril a setembro de 2005, resultou no descarte de cerca de 553.991 minicepas, 6.837.691 propágulos na fase de enraizamento e 11.266.819 mudas, nos Estados da Bahia, do Espírito Santo, do Maranhão, de Minas Gerais e do Pará, totalizando um prejuízo estimado em, no mínimo, seis milhões de reais (US$ 2,7 milhões). Em minijardim clonal, a doença caracteriza-se por necrose foliar, escurecimento anelar ou completo do lenho, murcha e morte de minicepas. Os sintomas na parte aérea são similares à morte gradual de minicepas submetidas a podas drásticas ou com sistema radicular malformado. Na fase de enraizamento, miniestacas infectadas podem apresentar arroxeamento das nervuras do limbo foliar e podridão. No campo, a doença caracteriza-se por bronzeamento e necrose foliar, desfolha basal, ascendente escurecimento interno do lenho e morte da planta, geralmente a partir do quarto mês após o transplantio. Os sintomas geralmente se agravam em árvores com enovelamento de raízes e afogamento de coleto. A etiologia da doença foi confirmada por meio de testes de exsudação, microscopia de varredura, isolamento da bactéria, análises de PCR/RFLP, reação de hipersensibilidade (HR) em mudas de fumo, testes de patogenicidade em plântulas de eucalipto e tomate e re-isolamento da bactéria. Como o sistema de produção de mudas clonais de eucalipto é altamente favorável à multiplicação bacteriana e na falta de conhecimento sobre a resistência genética e de outras estratégias de controle da doença, é essencial evitar a introdução da bactéria em viveiros.

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Section: Discussionunclassified

Ralstonia solanacearum em viveiros clonais de eucalipto no Brasil

Alfenas

Máfia

Sartório³

et al. 2006

Fitopatol. bras.

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“…Series of 1-ml aliquots were sampled and diluted down to 10 Ϫ4 . Afterwards, macerates (50 l) were streaked either on Imazaki medium (rhizosphere and soil) (66) or on modified Sequeira medium (67). Plates were then incubated at 28°C for 2 to 3 days.…”

Section: Methodsmentioning

confidence: 99%

New Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Scheme for Fine-Scale Monitoring and Microevolution-Related Study of Ralstonia pseudosolanacearum Phylotype I Populations

Guinard

Latreille

Guérin

et al. 2017

Appl Environ Microbiol

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Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is considered one of the most harmful plant diseases in the world. Special attention should be paid to R. pseudosolanacearum phylotype I due to its large host range, its worldwide distribution, and its high evolutionary potential. So far, the molecular epidemiology and population genetics of this bacterium are poorly understood. Until now, the genetic structure of the RSSC has been analyzed on the worldwide and regional scales. Emerging questions regarding evolutionary forces in RSSC adaptation to hosts now require genetic markers that are able to monitor RSSC field populations. In this study, we aimed to evaluate the multilocus variable-number tandem-repeat analysis (MLVA) approach for its ability to discriminate genetically close phylotype I strains and for population genetics studies. We developed a new MLVA scheme (MLVA-7) allowing us to genotype 580 R. pseudosolanacearum phylotype I strains extracted from susceptible and resistant hosts and from different habitats (stem, soil, and rhizosphere). Based on specificity, polymorphism, and the amplification success rate, we selected seven fast-evolving variable-number tandem-repeat (VNTR) markers. The newly developed MLVA-7 scheme showed higher discriminatory power than the previously published MLVA-13 scheme when applied to collections sampled from the same location on different dates and to collections from different locations on very small scales. Our study provides a valuable tool for fine-scale monitoring and microevolution-related study of R. pseudosolanacearum phylotype I populations.IMPORTANCE Understanding the evolutionary dynamics of adaptation of plant pathogens to new hosts or ecological niches has become a key point for the development of innovative disease management strategies, including durable resistance. Whereas the molecular mechanisms underlying virulence or pathogenicity changes have been studied thoroughly, the population genetics of plant pathogen adaptation remains an open, unexplored field, especially for plant-pathogenic bacteria. MLVA has become increasingly popular for epidemiosurveillance and molecular epidemiology studies of plant pathogens. However, this method has been used mostly for genotyping and identification on a regional or global scale. In this study, we developed a new MLVA scheme, targeting phylotype I of the soilborne Ralstonia solanacearum species complex (RSSC), specifically to address the bacterial population genetics on the field scale. Such a MLVA scheme, based on fast-

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“…Plant material is particularly notorious for PCR inhibitor compounds, many of which often are co-precipitated during DNA extraction (45). Strategies to overcome PCR inhibition and increase confidence in negative results include using a range of DNA template dilutions (40), spiking of negative samples (40), buffer additions during the DNA extraction process (4,34,63) magnetic DNA capture (4,45,63), post-extraction purification (44), and the use of alternative polymerases (3). In the interests of speed, economy, and wide applicability of the tests, it is desirable to avoid repetitious assays or lengthy and involved DNA purification techniques.…”

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confidence: 99%

Development of Nested Polymerase Chain Reaction Detection ofMycosphaerellaspp. and Its Application to the Study of Leaf Disease inEucalyptusPlantations

Glen¹,

Smith²,

Langrell³

et al. 2007

Phytopathology®

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Glen, M., Smith, A. H., Langrell, S. R. H., and Mohammed, C. L. 2007. Development of nested polymerase chain reaction detection of Mycosphaerella spp. and its application to the study of leaf disease in Eucalyptus plantations. Phytopathology 97:132-144.Mycosphaerella leaf disease (MLD) is a serious disease of two of the major eucalypt species grown in temperate regions worldwide, Eucalyptus globulus and E. nitens. More than 30 species of Mycosphaerella have been reported on eucalypts worldwide. Accurate, rapid, and early discrimination of Mycosphaerella spp. causing crown damage to E. globulus and E. nitens will assist the development of sustainable management strategies. This study describes the development, and incorporation in a nested polymerase chain reaction (PCR) approach, of specific primers for the detection and identification of Mycosphaerella spp. commonly reported from leaf lesions of E. globulus and E. nitens in Australia. Primer design was assisted by sequence alignment and phylogenetic analysis of 165 nonredundant sequences from the nuclear ribosomal DNA internal transcribed spacer regions of Mycosphaerella and related species. Phylogenetic analysis revealed very high sequence similarity for two taxon groups, Mycosphaerella grandis and M. parva, and M. vespa, M. ambiphylla, and M. molleriana, and primers were designed to differentiate each of the two groups. Three other species, M. cryptica, M. nubilosa, and M. tasmaniensis, were distinct and distinguished by species-specific primers. In double-blind trials, the detection test accurately and rapidly identified Mycosphaerella spp. in cultures and discriminated against other pathogens that co-occur in or on Eucalyptus leaves, thereby verifying its reliability. The detection test has an internal amplification control in the first-round PCR with fungal-specific primers to raise confidence in test results, particularly to highlight negative results due to PCR inhibition. When applied to DNA extracted from leaf or stem samples either as multiple or single lesions, it detected and identified up to five Mycosphaerella spp. or taxon groups in both positively identified and in young (putative) MLD lesions. The samples were 20 mm 2 or larger in surface area and were collected while undertaking disease rating assessments in an experimental investigation of Eucalyptus plantations and regrowth forest. Using nested PCR detection, Mycosphaerella spp. were positively identified in 2 days, 1 to 5 months earlier than by classical methods, demonstrating the potential application of this detection test to the early discrimination of MLD components in ecological, epidemiological, and genetic investigations.

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Evaluation of procedures for reliable PCR detection of Ralstonia solanacearum in common natural substrates

Cited by 49 publications

References 31 publications

Ralstonia solanacearum em viveiros clonais de eucalipto no Brasil

Ralstonia solanacearum em viveiros clonais de eucalipto no Brasil

New Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Scheme for Fine-Scale Monitoring and Microevolution-Related Study of Ralstonia pseudosolanacearum Phylotype I Populations

Development of Nested Polymerase Chain Reaction Detection ofMycosphaerellaspp. and Its Application to the Study of Leaf Disease inEucalyptusPlantations

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