Characterization of molecular markers for specific and sensitive detection of Botrytis cinerea Pers.: Fr. in strawberry ( Fragaria × ananassa Duch.) using PCR

Abstract

Random amplified polymorphic DNA (RAPD) assays were applied on 34 fungal strains isolated from strawberry and other host plants, in order to detect polymorphism to consequently identify and isolate molecular markers specific to Botrytis cinerea. Among the 26 10-mer primers tested, one primer mainly amplified a 750-bp product present in all the B. cinerea strains and absent in the other species and genera examined. This product was cloned and sequenced in order to design a specific 20-mer primer pair, which was tested on the 34 fungal isolates by polymerase chain reaction (PCR). A single 0.7-kb band was amplified in all the 13 strains of B. cinerea isolated from different host plants. Moreover, a 0.6-kb band was amplified in both Botrytis fabae strains. No band was observed for the nine other Botrytis species and 12 fungal genera isolated from strawberry plants. A comparison between the 0.7-kb B. cinerea sequence and the 0.6-kb B. fabae sequence revealed 98% homology and one 122-bp deletion for B. fabae, including an EcoRI site. Hybridization of Southern blots with RAPD and EcoRI-digested DNA confirmed the specificity of the marker. The limit of detection of B. cinerea genomic DNA was approximately 0.2 pg. The PCR procedure was able to amplify the 0.7-kb B. cinerea fragment form mixed samples of DNA as low as 2 pg B. cinerea genomic DNA and 1 microg plant DNA. Thus this PCR-based detection procedure is a powerful tool for diagnosis of B. cinerea in symptomless strawberry plants, and should allow infection and latency sites to be localized in order to improve knowledge of the epidemiology of the pathogen under field conditions.