Strain-specific detection of two Aureobasidium pullulans strains, fungal biocontrol agents of fire blight by new, developed multiplex-PCR

Abstract

The aim of this study was to develop a specific and sensitive identification method for two Aureobasidium pullulans biocontrol strains, CF10 and CF40, based on a sequence-characterized amplified region (SCAR) derived from RAPD - and multiplex-RAPD PCR analysis. The random amplified polymorphic DNA (RAPD) and multiplex RAPD-PCR techniques were used for a preliminary screening of A. pullulans genetic variability among 200 isolates. This approach allowed the selection of ten fragments present solely in strains CF10 and CF40. The RAPD fragments were cloned, sequenced and used to design two SCAR primers. Two primer pairs obtained from SCH3RAPD fragment of CF 40 and 6RAPD of CF10 were highly specific and sensitive. In this study, we developed strain-specific multiplex-PCR based on sequence-characterized amplified region (SCAR) markers to simultaneously detect both strains in a single PCR. This new multiplex-PCR provides a valuable tool for specific and sensitive identification of CF10 and CF40, and could be used in studies on the efficacy and persistence of introduced strains of A. pullulans for fire blight control.