A competitive PCR-based method for the detection and quantification of Erwinia carotovora subsp. atroseptica on potato tubers

Hyman, L. J.; Birch, Paul R. J.; Dellagi, Alia; Avrova, Anna O.; Toth, Ian K.

doi:10.1046/j.1472-765x.2000.00736.x

Cited by 23 publications

(12 citation statements)

References 21 publications

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“…The choice between the two PCR systems will be dictated by cost as the ABI Prism 7700 sequence detector system (Applied Biosystems) is more expensive than the equipment used for conventional PCR, even following the further development of this latter system to allow quantification, for example, based on the use of competitor template fragments (Bell et al. , 1999; Hyman et al. , 2000).…”

Section: Discussionmentioning

confidence: 99%

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real‐time PCR

Cullen¹,

Lees²,

Toth³

et al. 2002

Plant Pathology

144

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Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genusspecific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .

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Section: Discussionmentioning

confidence: 99%

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real‐time PCR

Cullen¹,

Lees²,

Toth³

et al. 2002

Plant Pathology

144

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“…'Adesoto 101' hosts and P. tomentosa scions were additionally evaluated by reverse transcription-polymerase chain reaction (RT-PCR) as described by Rosales et al (1998), which detects a 220 bp region corresponding to the 3'-non coding region (3'-NCR) of the PPV RNA. Total RNA from leaf samples from 'Adesoto 101' rootstocks and P. tomentosa scions was performed according to Hyman et al (1998). Evaluations of the grafted populations and 'Adesoto101' hosts were carried out during three consecutive springsummer seasons (September to January 2006January -2007January , 2007January -2008January , and 2008January -2009.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of the Resistance of Transgenic C5 Plum (Prunus domestica L.) against Four Chilean Plum Pox Virus Isolates through Micro-Grafting

Wong

Barba

Álvarez

et al. 2010

Chilean J. Agric. Res.

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The transgenic plum (Prunus domestica L.) C5, in which the coat protein (CP) gene of the Plum pox virus (PPV) is inserted, represents a unique example of the use of genetic engineering for fruit crop improvement in Prunus spp. Field trials in Poland, Romania, and Spain have demonstrated resistance of C5 to several D and M strain PPV isolates. In Chile, the quarantine regulations for PPV and for genetically modified (GM) plants require that the testing of C5 for resistance to Chilean PPV isolates be done under controlled isolated conditions. To carry out these tests C5 shoots were multiplied in vitro and micro-grafted onto four Adesoto101 (Prunus insititia L.) rootstock populations that had been previously infected each with one of four Chilean PPV-Ds. Tests were carried out under controlled conditions in a biosafety greenhouse. Symptoms appearance, virus detection, and viral mRNA levels for the cylindrical inclusion (CI) and CP genes were determined during three consecutive growing seasons. Complete resistance to all PPV isolates was demonstrated during the first 2 yr in all of the C5 plants. In the third season, four of 10 C5 plants showed mild symptoms on leaves close to the graft union and low but detectable CI mRNA levels in the C5 scions. These results support the effectiveness of using of micro-grafting on P. insititia for PPV resistance studies, especially in the limited space of a quarantine facility; whereas resistance levels in C5 after 3 yr indicate the importance of long term and field scale evaluations.

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“…All serogroups of Eca were detected, leading to formulation of a key relating product ratios to the likelihood of incidence of blackleg disease. The competitive PCR assay has the potential for use in quarantine and certification programs (Hyman et al 2000).…”

Section: Polymerase Chain Reaction-based Assaysmentioning

confidence: 99%

Detection of Bacterial and Phytoplasmal Pathogens

Narayanasamy¹

2010

Microbial Plant Pathogens-Detection and Disease Diagnosis:

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Bacterial pathogens classified as prokaryotes are much simpler in structure and smaller in size and they have less complex structural features compared to fungal plant pathogens. As the morphological characteristics of bacterial cells are less variable, biological, biochemical, physiological, immunological and genomic characteristics have to be determined for reliable identification and meaningful classification of bacterial pathogens. Detection and identification of bacterial plant pathogens present in whole plants and in propagative plant materials have been possible by employing isolation on cultural media and metabolic fingerprinting methods. But they are labor-intensive and require long time. Results often are inconclusive. Isozyme analysis, direct colony thin layer chromatography and gel electrophoresis techniques have been successfully applied for the detection of some bacterial pathogens. Immunoassays and nucleic acid-based assays have become widely accepted techniques, providing more sensitive and specific detection and quantification of bacterial pathogens affecting a wide range of host plant species. However, the major limitation of the molecular techniques is their inability to discriminate living cells from the dead ones. Attempts have been made to overcome this limiting factor by using DNA binding dyes and estimating pathogen RNAs as an indicator of cell viability. The diagnostic methods have both merits and demerits and hence, appropriate method has to be selected based on cost-effectiveness and possibility of obtaining reliable results rapidly to suit the requirements of the investigation concerned.Phytoplasmas are cell wall-less, nonculturable bacteria belonging to Mollicutes. Lack of cultural characteristics has made obligatory to study the morphological characteristics of phytoplasmas present in the phloem cells of infected plant hosts by observing under electron microscopes. As most of the phytoplasmas look alike in the ultrathin sections, the morphological characters have no diagnostic value. Histochemical methods using DNA binding dyes have been employed to localize the phytoplasmas in the host cells. Application of immunoassays and nucleic acidbased techniques has been effective in providing information for the identification and differentiation of phytoplasams. Polyclonal and monoclonal antibodies have been generated for detection of phytoplasmas infecting several plant species. Universal and species-specific primers and probes have been designed based on

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A competitive PCR-based method for the detection and quantification of Erwinia carotovora subsp. atroseptica on potato tubers

Cited by 23 publications

References 21 publications

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real‐time PCR

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real‐time PCR

Evaluation of the Resistance of Transgenic C5 Plum (Prunus domestica L.) against Four Chilean Plum Pox Virus Isolates through Micro-Grafting

Detection of Bacterial and Phytoplasmal Pathogens

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