Comprehensive molecular system to study the presence, growth and ochratoxin A biosynthesis ofPenicillium verrucosumin wheat

Abstract

Based on the sequence of the ochratoxin A polyketide synthase gene (otapksPV), a polymerase chain reaction (PCR) system for the specific detection of Penicillium verrucosum in wheat has been developed. In a further approach, a real-time PCR system has been applied to determine the growth kinetics of P. verrucosum in wheat at cell numbers above 10(3) colony-forming units (cfu) ml(-1). The data obtained by real-time PCR correlated well with the data obtained by the plate count technique. For this purpose, the DNA was isolated directly from contaminated wheat without any further enrichment step. In a reverse transcriptase real-time PCR, the expression of the otapksPV gene in wheat was detected 22 days after inoculation and storage at ambient temperature. Reasonable amounts of ochratoxin A, however, could not be detected before day 30. This early activation of ochratoxin A related genes was confirmed by microarray analysis.

Aspergillus westerdijkiae polyketide synthase gene “aoks1” is involved in the biosynthesis of ochratoxin A

“…These results suggest that production pattern of OTA seems to be associated with expression pattern of aoks1 gene. On the basis of expression studies several other PKS genes producing reduced type polyketides were found to be important for OTA biosynthesis in other organisms like P. nordicum (Fär-ber and Geisen, 2004;Karolewiez and Geisen, 2005) and P. verrucosum (Schmidt-Heydt et al, 2008).…”

Recent advances in ochratoxin A-producing fungi detection based on PCR methods and ochratoxin A analysis in food matrices

“…By using qPCR, a positive correlation between OTA content and gene expression has been indicated for P. nordicum, P. verrucosum, A. ochraceus and A. carbonarius (Atoui et al, 2007;Geisen et al, 2004;Mulè et al, 2006;Schmidt-Heydt et al, 2008). These studies demonstrated that the production of OTA in relation to the DNA content is dependent either on environmental conditions or the physiological activity of the species analyzed.…”

“…Fungal strains were grown on appropriate medium and mycelium was collected after 2e7 days. Then, the mycelium was frozen in liquid nitrogen, ground to a powder with mortar and pestle, and incubated with lysis buffer containing EDTA and SDS during 15e60 min at 65 C (Bogs et al, 2006;Geisen, Mayer, Karolewiez, & Färber, 2004;Schmidt-Heydt, Richter, Michulec, Buttinger, & Geisen, 2008). This treatment with alkaline chemicals was used as a non-mechanical disruption method.…”

“…Some pks genes have also been identified in P. nordicum (Geisen et al, 2004) and P. verrucosum (Schmidt-Heydt et al, 2008).…”

Development of a Real Time PCR system for detection of ochratoxin A-producing strains of the Aspergillus niger aggregate

“…Among the ochratoxigenic fungi, different PKS genes involved in OTA biosynthesis have been identified in Aspergillus ochraceus (O'Callaghan, Caddick, & Dobson, 2003), A. westerdijkiae (Atoui, Dao, Mathieu, & Lebrihi, 2006;Bacha, Atoui, Mathieu, Liboz, & Lebrihi, 2009), A. carbonarius (Atoui et al, 2006Gallo et al, 2009), Penicillium nordicum (Geisen, Mayer, Karolewiez, &Färber, 2004) andP. verrucosum (Schmidt-Heydt, Richter, Michulec, Buttinger, &Geisen, 2008). Some of these genes have been used as a target to develop accurate methods which allow a rapid, sensitive and specific identification and quantitation of OTA-producing strains.…”