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A comparison of real-time PCR protocols for the quantitative monitoring of asymptomatic olive infections by Verticillium dahliae pathotypes

AuthorsGramaje, David ; Pérez Serrano, V.; Montes Borrego, Miguel ; Navas Cortés, Juan Antonio ; Jiménez-Díaz, Rafael M. ; Landa, Blanca B.
Issue DateMay-2013
PublisherDeutsche Phytomedizinische Gesellschaft
Citation11th International Verticillium Symposium (2013)
AbstractEarly, specific and accurate in planta detection and quantification of Verticillium dahliae are essential to prevent the spread of Verticillium wilt in olive using certified pathogen-free planting material and development of resistance. We comparatively assessed the accuracy, specificity and efficiency of eight real-time quantitative PCR (qPCR) protocols published since 2002 for the specific detection and quantification of V. dahliae in various host plant species and in soil, using a background of DNAs extracted from olive roots, stems and leaves. Results showed that some of those protocols were not specific for V. dahliae and/or were inhibited when using backgrounds other than water. Ranking of protocols according to a weighted score system placed protocol TAQ (based on IGS rDNA target gene; Bilodeau et al . 2012) and SYBR-4 (based on β -tubulin 2 target gene; Atallah et al . 211) first in sensitivity and efficiency for the quantification of V. dahliae DNA in small amounts and different types of olive tissues (root and st em) tested. Use of TAQ and SYBR-4 protocol allowed accurate quantification of V. dahliae DNA regardless of the background DNA with a detection limit being fixed at a C T of 36 (approximately 18 fg for SYBR-4 and 15 fg for TAQ) of V. dahliae ). The amount of DNA from defoliating (D) and nondefoliating (ND) V. dahliae pathotypes was monitored in Verticillium wilt-resistant 'Frantoio' olive using the TAQ and SYBR-4 protocols. In the infection bioassay, higher amounts of D- V. dahliae DNA were measured in olive stems, whereas the aver age amount of fungal DNA in roots was higher for ND-infected plants than D-infected ones. Overall, V. dahliae DNA amount in all olive tissues tested tended to slightly decrease or remain stable by the end of the experiment (35 days). The SYBR-4 and TAQ protocols further enabled detection of V. dahliae in tissues of symptomless plants, suggesting that both techniques can be useful for implementing certification schemes of pathogen-free planting material as well as a helpful tool in breeding resistance to V. dahliae in olive.
DescriptionPóster presentado en el 11 th International Verticillium Symposium, celebrado en Göttingen (Alemania) del 5 al 8 de mayo de 2013.
Appears in Collections:(IAS) Comunicaciones congresos
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