Genetic Variation, qPCR-Based Detection and Virulence of Berkeleyomyces Species in Ilex crenata

Abstract

Berkeleyomyces, a globally spread genus of soil-borne fungi comprising B. basicola and B. rouxiae, causes black root rot in over 170 plant species including the ornamental shrub Ilex crenata. These two species are morphologically indistinguishable and were previously grouped under Thielaviopsis basicola. Historical records did not account for hidden species diversity, including pathogenic diversity on specific host plant species such as lettuce. The first objective of this study was to determine whether strains of both Berkeleyomyces species affect I. crenata, as this knowledge could influence the selection of isolates for resistance breeding. We collected 33 isolates from I. crenata plants in gardens, parks and nurseries in Flanders (Belgium) and the Netherlands, and characterised this Berkeleyomyces collection to species and subspecies level using Genotyping-by-Sequencing (GBS). Most I. crenata isolates belonged to two near-clonal groups within B. basicola. Virulence testing revealed that B. basicola and B. rouxiae could incite similar disease levels in I. crenata, with smaller differences in virulence related to the host of origin. Our second objective was to develop novel qPCR assays, allowing fast species identification and quantification of both Berkeleyomyces species. We developed two sets of species-specific qPCR assays: one set on the 60S locus (with sensitivity down to 100 fg DNA), and another set on the rDNA ITS locus (with sensitivity down to 10 fg DNA), which can be run in duplex format. The qPCR assays were successfully used to quantify the pathogen. They show potential for use in other hosts as well as in epidemiological studies.