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A Newly Developed Real-Time PCR Assay for Detection and Quantification of Fusarium oxysporum and Its Use in Compatible and Incompatible Interactions with Grafted Melon Genotypes

First, second, third, fourth, and seventh authors: Consiglio per la ricerca e la sperimentazione in agricoltura (CRA)–Centro di Ricerca per la Patologia Vegetale, Via C. G. Bertero 22, 00156 Rome; fifth author: Department of Agronomy Foods Natural Resources Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro, Padova, Italy; and sixth author: CRA–Unità di Ricerca per l'Orticoltura Via Salaria 1, 63077 Monsampolo del Tronto (AP), Italy.

A reliable and species-specific real-time quantitative polymerase chain reaction (qPCR) assay was developed for detection of the complex soilborne anamorphic fungus Fusarium oxysporum. The new primer pair, designed on the translation elongation factor 1-α gene with an amplicon of 142 bp, was highly specific to F. oxysporum without cross reactions with other Fusarium spp. The protocol was applied to grafted melon plants for the detection and quantification of F. oxysporum f. sp. melonis, a devastating pathogen of this cucurbit. Grafting technologies are widely used in melon to confer resistance against new virulent races of F. oxysporum f. sp. melonis, while maintaining the properties of valuable commercial varieties. However, the effects on the vascular pathogen colonization have not been fully investigated. Analyses were performed on ‘Charentais-T’ (susceptible) and ‘Nad-1’ (resistant) melon cultivars, both used either as rootstock and scion, and inoculated with F. oxysporum f. sp. melonis race 1 and race 1,2. Pathogen development was compared using qPCR and isolations from stem tissues. Early asymptomatic melon infections were detected with a quantification limit of 1 pg of fungal DNA. The qPCR protocol clearly showed that fungal development was highly affected by host–pathogen interaction (compatible or incompatible) and time (days postinoculation). The principal significant effect (P ≤ 0.01) on fungal development was due to the melon genotype used as rootstock, and this effect had a significant interaction with time and F. oxysporum f. sp. melonis race. In particular, the amount of race 1,2 DNA was significantly higher compared with that estimated for race 1 in the incompatible interaction at 18 days postinoculation. The two fungal races were always present in both the rootstock and scion of grafted plants in either the compatible or incompatible interaction.

Additional keywords: Cucumis melo, Fusarium wilt, SYBR green.