3 years ago

Comparative genomics-based markers: discrimination of host-specificity in Fusarium oxysporum.

Anneliek Ter Horst, Michelle van der Gragt, Martijn Rep, Peter van Dam, Mara de Sain
The polyphyletic nature of many formae speciales of Fusarium oxysporum (Fo) prevents molecular identification of newly encountered strains based on conserved, vertically inherited genes. Alternative molecular detection methods that could replace labour- and time-intensive disease assays are therefore highly desired. Effectors are functional elements in the pathogen-host interaction and have been found to show very limited sequence diversity between strains of the same forma specialis, which makes them potential markers for host-specific pathogenicty. We therefore compared candidate effector genes extracted from 60 existing and 22 newly generated genome assemblies, specifically targeting strains affecting cucurbit plant species. Based on these candidate effector genes, in total eighteen PCR primer pairs were designed to discriminate between each of the seven Cucurbitaceae-affecting formae speciales. When tested on a collection of strains encompassing different clonal lineages of these formae speciales, non-pathogenic strains and strains of other formae speciales, they allowed clear recognition of the host range of each evaluated strain. Within Fo f. sp. melonis more genetic variability exists than anticipated, resulting in three melonis marker patterns that partially overlapped with the cucurbit-infecting formae speciales cucumerinum, niveum, momordicae and/or lagenariae. For Fo f. sp. niveum, a multiplex TaqMan assay was evaluated, which allowed quantitative and specific detection of template DNA quantities as low as 2.5 pg. These results provide ready-to-use marker sequences for the mentioned Fo pathogens. Additionally, the method can be applied to find markers distinguishing other host-specific forms of F. oxysporumImportance Pathogenic strains of Fusarium oxysporum are differentiated into formae speciales based on their host range, that is normally restricted to only one or a few plant species. However, horizontal gene transfer between strains in the species complex has resulted in a polyphyletic origin of host specificity in many of these formae speciales This hinders accurate and rapid pathogen detection through molecular methods. In our research, we compared the genomes of 88 strains of F. oxysporum with each other, specifically targeting virulence-related genes that are typically highly similar within each forma specialis Using this approach, we identified marker sequences that allow the discrimination of F. oxysporum strains affecting various cucurbit plant species through different Polymerase Chain Reaction-based methods.

Publisher URL: http://doi.org/10.1128/AEM.01868-17

DOI: 10.1128/AEM.01868-17

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