A simple and rapid protocol of crude DNA extraction from apple trees for PCR and real-time PCR detection of ‘Candidatus Phytoplasma mali’

Aldaghi, M.; Massart, Sébastien; Dutrecq, O.; Bertaccini, Assunta; Jijakli, M. Haïssam; Lepoivre, Philippe

doi:10.1016/j.jviromet.2008.10.011

Cited by 13 publications

(8 citation statements)

References 36 publications

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“…They are absolute parasites of plant phloem and transmitted by insect vectors, especially by leafhoppers [5,19]. Fortunately, specific primers designed for highly conserved genes, such as the 16S ribosomal gene, together with the use of molecular probes randomly cloned from the phytoplasma genome, allow these pathogens to be discriminated and molecularly classified [2,4,6,17,21,27,33].…”

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confidence: 99%

Evaluation of Anti-Phytoplasma Properties of Surfactin and Tetracycline Towards Lime Witches’ Broom Disease Using Real-Time PCR

Askari¹

2011

J. Microbiol. Biotechnol.

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The anti-phytoplasma activities of surfactin (derived from Iranian native Bacillus subtilis isolates) and tetracycline towards Candidatus "Phytoplasma aurantifolia", the agent of lime Witches' broom disease, were investigated. HPLC was used to quantify the surfactin production in four previously characterized native surfactin-producing strains, and the one producing the highest amount of surfactin (about 1,500 mg/l) was selected and cultivated following optimized production and extraction protocols. Different combinations of purified surfactin and commercial tetracycline were injected into artificially phytoplasmainfected Mexican lime seedlings using a syringe injection system. An absolute quantitative real-time PCR system was developed to monitor the phytoplasma population shifts in the lime phloem during 3 months following the injections. The results revealed that the injections of surfactin or tetracycline had a significant inhibitory effect on Candidatus "P. aurantifolia". However, the combined treatment with both surfactin and tetracycline (1:1) resulted in the highest inhibition due to a synergic effect, which suppressed the phytoplasma population from about 2×10 5 to less than 10 phytoplasma units/g plant tissue.

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confidence: 99%

Evaluation of Anti-Phytoplasma Properties of Surfactin and Tetracycline Towards Lime Witches’ Broom Disease Using Real-Time PCR

Askari¹

2011

J. Microbiol. Biotechnol.

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“…This new procedure was useful by providing suitable DNA samples for accurate detection of 'Ca. P. mali' in different types of apple and periwinkle samples (Aldaghi et al 2009). As the PCR-based assays are more sensitive, precise and rapid, detection of a large number of phytoplasmal diseases has been possible and some of them are listed in Table 2.10.…”

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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“…Phytoplasmas are non-helical obligate parasites that belong to the prokaryotic class Mollicutes, and are transmitted by sap-feeding insects and vegetative plant propagation materials [ 15 – 18 ]. Diagnosis of phytoplasma pathogens has proven difficult because they cannot be cultured in vitro [ 19 – 21 ].…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, the high sensitivity of this method allows for quantitative description of all phases of phytoplasma infection [ 31 ] when labeling systems are used for the DNA amplicons [ 25 ]. Real-time qPCR assays have been developed for individual or group specific detection and quantification of many phytoplasmas [ 18 , 25 , 32 – 35 ] based on the 16S rRNA [ 36 ] and 23S rRNA gene sequences [ 37 ].…”

Section: Introductionmentioning

confidence: 99%

Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors

et al. 2016

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Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

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A simple and rapid protocol of crude DNA extraction from apple trees for PCR and real-time PCR detection of ‘Candidatus Phytoplasma mali’

Cited by 13 publications

References 36 publications

Evaluation of Anti-Phytoplasma Properties of Surfactin and Tetracycline Towards Lime Witches’ Broom Disease Using Real-Time PCR

Evaluation of Anti-Phytoplasma Properties of Surfactin and Tetracycline Towards Lime Witches’ Broom Disease Using Real-Time PCR

Detection of Bacterial and Phytoplasmal Pathogens

Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors

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