Development and validation of a new real-time assay for the quantification of Verticillium dahliae in the soil: a comparison with conventional soil plating

Abstract

Verticillium wilt of olive, caused by the soil-borne fungus Verticillium dahliae, is one of the most serious diseases of olive tree. In this study, a SYBR Green-based quantitative polymerase chain reaction (Q-PCR) assay targeting the intergenic spacer (IGS) region of the ribosomal DNA (rDNA) was developed to quantify V. dahliae microsclerotia (MS) in soils cropped with olive tree. In order to make the assay quantitative, the number of rDNA units in the genome was estimated using Q-PCR and fixed at 25 copies/genome. The assay was highly specific for V. dahliae, with no cross-amplification with other soil-borne pathogens. The sensitivity analysis showed similar slopes and efficiency, from both fungal DNA (slope = −3.405, r2 = 0.976, E = 96.64 %) and the positive recombinant plasmid (y = −3.36, r2 = 0.989, E = 98.43 %), thus indicating a high accuracy of the assay. The assay exhibits a high intra- and inter-run reproducibility at a very low concentration of 102 copies/μL (CV% ≈ 1 %). When the real-time PCR assay was applied to quantify MS in five naturally infested soil samples, it was able to detect V. dahliae in as few as two MS g−1 of soil. Q-PCR estimates of pathogen DNA were significantly correlated with disease severity (r2 = 0.944) and with the soil plating method (r2 = 0.845). This new assay will be a valuable tool and can be applied for disease risk prediction before installing new plantations, and provides a more complete and rapid examination for soils subjected to such a treatment program.