Development  of RT-PCR tests for the routine detection of latent and ILAR viruses in fruit trees

Marbot, Sophie; Kummert, J.; Salmon, Michel; Vendrame, Marina; Huwaert, Astrid; Dutrecq, O.; Lepoivre, Philippe

doi:10.17221/10312-pps

Cited by 4 publications

(3 citation statements)

References 5 publications

(6 reference statements)

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“…The position of a mismatch is crucial to the specificity of an MGB probe. MGB probes have been successfully employed to detect single‐nucleotide mutations in the central region (Marbot et al , 2002; Salmon et al , 2002 b ) or the 3′ terminal region (Kutyavin et al , 2000; Salmon et al , 2002 a ; Massart et al , 2005) of the probe. Yao et al (2006) observed that two mismatches in the centre of the probe‐annealing region generated a signal in their MGB probe assay.…”

Section: Discussionmentioning

confidence: 99%

“…TaqMan MGB probes display higher specificity than classical TaqMan probes (Yao et al , 2006). Successful discrimination of a single mismatch has been achieved by various authors (Marbot et al , 2002; Salmon et al , 2002 a ; Yoshitomi et al , 2003; Van Hoeyveld et al , 2004; Massart et al , 2005). Yet, recent studies (Yao et al , 2006) have shown that a TaqMan MGB probe may tolerate one or two mismatches with its template and cause a nonspecific fluorescence emission.…”

Section: Introductionmentioning

confidence: 99%

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Development of a new probe for specific and sensitive detection of ‘Candidatus Phytoplasma mali’ in inoculated apple trees

Aldaghi

Massart

Roussel

et al. 2007

Annals of Applied Biology

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A new TaqMan minor groove binding (MGB) probe and new PCR conditions were designed for quick, specific and sensitive detection of 'Candidatus Phytoplasma mali'. The new probe can distinguish a single mismatch between 'Ca. P. mali' and 'Candidatus Phytoplasma prunorum', this constituting an improvement over a previously published method. In our study, the relative position of the mismatch in the MGB probe influenced greatly the specificity of detection. Our new real-time PCR protocol was able to detect one plasmid copy in water and 100 copies in healthy plant DNA extracts. The sensitivity of this new real-time PCR method, three other real-time PCR protocols and a conventional PCR with fU5/rU3 primers was compared. Periwinkles and MM106 rootstocks were grafted with infected material and surveyed over time by symptom observation, conventional PCR and real-time PCR. Phytoplasma infection was detected by symptom observation in all periwinkles within 4 months and in 75% of the MM106 rootstocks by the end of 7 months. Conventional PCR detected phytoplasma infection in all periwinkles within 4 months and in all MM106 rootstocks within 7 months. Best results were obtained by our realtime PCR, which detected phytoplasma infection in all grafted plants within 3 months after inoculation.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development of a new probe for specific and sensitive detection of ‘Candidatus Phytoplasma mali’ in inoculated apple trees

Aldaghi

Massart

Roussel

et al. 2007

Annals of Applied Biology

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“…The DNA extraction efficiency of this crude preparation method was further assessed by semi-quantitative evaluation of phytoplasma DNA using real-time PCR. The application of crude preparation of infected plants has been previously tested for virus detection by PCR amplification (Marinho et al, 1998;Marbot et al, 2002;Delanoy et al, 2003); furthermore a simple plant tissue preparation without homogenization suitable for detection of viruses by PCR was described by Thomson and Dietzgen (1995).…”

Section: Introductionmentioning

confidence: 99%

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

Aldaghi

Massart

Dutrecq

et al. 2009

Journal of Virological Methods

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a b s t r a c tDifferent PCR protocols have been established for detection of European fruit trees phytoplasmas; however the majority of the procedures for extracting phytoplasma DNA are complex, time consuming, and expensive, with a risk of contamination or loss of target DNA. In present study, a crude extract preparation method previously used to detect other plant pathogens was adapted to samples from apple trees infected by 'Candidatus Phytoplasma mali'. End-point and real-time PCR detection of 'Ca. P. mali' were used to compare this extraction procedure with an established method for efficient extraction of purified DNA. The crude extract proved fully adequate for phytoplasma detection in samples from 86 in vitro and 35 in vivo apple shoots or plants and 10 periwinkle plants. High inter-and intra-run reproducibility was obtained for phytoplasma detection with different TaqMan MGB-or SYBR Green-based real-time PCR protocols applied to the crude extracts. Real-time PCR applied to serially diluted crude and purified extracts revealed the same phytoplasma detection limit (dilution up to 10 5 ). All results confirm the suitability of this simple, quick, efficient extraction technique for accurate detection of 'Ca. P. mali' in different types of apple and periwinkle samples.

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Durable resistance to stripe rust is due to three specific resistance genes in French bread wheat cultivar Apache

Paillard

Trotoux-Verplancke

Perretant³

et al. 2012

Theor Appl Genet

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Quantitative resistance is postulated to be more durable than qualitative (R-gene mediated) resistance, which is usually quickly overcome by the pathogen population. Despite its wide use for nearly 10 years in France, the French bread wheat cultivar Apache remains resistant to stripe rust. Here, we investigated the genetic architecture of cv. Apache resistance to examine whether its durability could be explained by quantitative characteristics. We identified quantitative trait loci (QTL) by composite interval mapping of disease progress data recorded throughout 4 years of field assays. These assays included inoculation with three different pathotypes on a segregating population originating from a cross between cv. Apache and cv. Taldor, a French cultivar susceptible to stripe rust. Three QTLs derived from Apache, QYr.inra-2AS, QYr.inra-2BL and QYr.inra-4B, were detected. Each of these QTLs contributed between approximately 15 and 69 % of the phenotypic variance and corresponds to a race-specific resistance gene. We showed that QYr.inra-2AS and QYr.inra-2BS map to the positions of Yr17 and Yr7, respectively, whereas QYr.inra-4B corresponds to an adult plant resistance gene. Our results demonstrate that a combination of two or more race-specific resistance genes can confer durable resistance provided that it is properly managed at a continental level. Race-specific resistance genes should not be removed from breeding programs provided that they are properly managed.

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Development of RT-PCR tests for the routine detection of latent and ILAR viruses in fruit trees

Cited by 4 publications

References 5 publications

Development of a new probe for specific and sensitive detection of ‘Candidatus Phytoplasma mali’ in inoculated apple trees

Development of a new probe for specific and sensitive detection of ‘Candidatus Phytoplasma mali’ in inoculated apple trees

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

Durable resistance to stripe rust is due to three specific resistance genes in French bread wheat cultivar Apache

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