Two New Primers Highly Specific for the Detection of Botrytis cinerea Pers. Fr.

Abstract

Of thirty-nine Botrytis cinerea isolates originating in different host-plants and grown in pure cultures, twenty-six produced abundant grey aerial mycelium and sporulated intensely, whilst thirteen produced a thin mycelial layer, abundant sclerotia and secreted an unidentified yellow pigment in PDA culture media. The commonly used C729 +/– primers (5’-AGCTCGAGAGAGATCTCTGA-3’; 5’-CTGCAATGTTCTGCGTGGAA-3’) designed to detect B. cinerea did not amplify the DNA fragment of 0.73 kb in this smaller group of strains under standard conditions, whereas a shorter DNA fragment (0.60 kb) was amplified at a lower annealing temperature (50°C). This fragment was sequenced and two new internal primers were designed, BC108 + (5’-ACCCGCACCTAATTCGTCAAC-3’) and BC563 – (5’-GGGTCTTCGATACGGGAGAA-3’). These new primers were used to amplify a DNA fragment of 0.48 kb for the main group of 26 B. cinerea strains and a shorter fragment (0.36 kb) for the smaller group of 13 strains due to a deletion of 0.12 kb, which was not detected with the primers C729 +/–. All the strains were amplified to detect the presence or absence of Boty and Flipper transposable elements. No correlation was found between strains possessing the deletion and those belonging to either the vacuma or the transposa sibling species. Other closely related Botrytis species such as B. allii and B. fabae were not amplified with these primers, confirming their specificity for B. cinerea and enhancing the sensitivity of the molecular tools available to detect this fungus in host-plants.