Amplification of Polyketide Synthase gene fragments in ochratoxigenic and nonochratoxigenic black aspergilli in grapevine

Abstract

Members of the Aspergillus section Nigri, also known as black aspergilli produce Ochratoxin A (OTA), a contaminant of wine. Despite potentially severe health effects and economic losses caused by OTA in wine, almost nothing is known about the genetics that lies behind its biosynthesis in black aspergilli in grapevine. In this work, degenerate primer sets were used to amplify 49 Polyketide Synthase (PKS) gene fragments in reference strains of A. carbonarius, A. niger and A. tubingensis. Deduced amino acid sequences were then compared with those of aolc35-12 and aoks1, two PKS genes involved in OTA biosynthesis in A. westerdijkiae. A putative homologue of aolc35-12 was found in A. carbonarius (63% amino acid identity), the main OTA producer on grapes and in an ochratoxigenic A. niger strain (58%). In A. niger this fragment corresponded to an15g07920, a PKS already annotated in the sequenced A. niger CBS 513.88 genome as putatively involved in OTA biosynthesis. No aolc35-12 candidates were found in atoxigenic A. tubingensis isolates and no putative homologues of aoks1 were found in any of the screened strains. A screening of A. niger field isolates using specific primers for an15g07920 indicated that the absence of this gene is apparently related to a failure to produce OTA. The present work gives a first insight into the genetics of OTA biosynthesis in black aspergilli in grapevine and represents a starting point for further investigation of the OTA biosynthesis pathway and the development of molecular methods to detect the producers in vineyards.