Species-Specific Detection of Monilinia fructicola from California Stone Fruits and Flowers

Abstract

ABSTRACT A set of molecular diagnostics was developed for Monilinia fructicola, causal agent of brown rot of stone fruits, capable of sensitive detection of the pathogen in planta. Species-specific repetitive sequences were identified from a partial library of 312 recombinant clones hybridized with total DNA, followed by subsequent screening for specificity. One hundred isolates, comprising 12 fungal species common to California stone fruits, were surveyed for specificity. Three clones hybridized to 60 geographically diverse M. fructicola isolates (California, Michigan, Georgia, Oregon, and Australia) to the exclusion of all other fungi surveyed, including the closely related M. laxa (n = 12). Two clones were identical and of extrachromosomal origin (pMF73 and pMF150), whereas the third (pMF210) migrated with uncut DNA. The sensitivity of all three was comparable and capable of detecting 50 pg of fungal DNA in dot blot hybridizations. Six species-specific primer pair sets were designed. They maintained the same specificity patterns observed in the initial hybridization surveys and were sensitive enough to detect 50 fg of fungal DNA template, approximately equivalent to 10 spores. The species-specific clones were capable of detecting the pathogen in planta, specifically from infected plum flowers and nectarine fruit tissue, using both hybridization- and polymerase chain reaction-based methodologies.