A Comparison of Real-Time PCR Protocols for the Quantitative Monitoring of Asymptomatic Olive Infections by Verticillium dahliae Pathotypes

Abstract

Early, 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 polymerase chain reaction 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 or were inhibited when using backgrounds other than water. Ranking of protocols according to a weighted score system placed protocols TAQ (based on intergenic spacer ribosomal DNA target gene) and SYBR-4 (based on the β-tubulin 2 target gene) first in sensitivity and efficiency for the quantification of V. dahliae DNA in small amounts and different types of olive tissues (root and stem) tested. Use of TAQ and SYBR-4 protocols allowed accurate quantification of V. dahliae DNA regardless of the background DNA, with a detection limit being fixed at a cycle threshold of 36 (≈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 average amount of fungal DNA in roots was higher for ND-infected plants than D-infected ones. Overall, V. dahliae DNA amounts in all olive tissues tested tended to slightly decrease or remain stable by the end of the experiment (35 days after inoculation). 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 helpful tools in breeding resistance to V. dahliae in olive.