Development of a generic PCR detection of deoxynivalenol- and nivalenol-chemotypes ofFusarium graminearum

Abstract

Based on the intergenic sequences of Tri5-Tri6 genes involved in the mycotoxin pathways of Fusarium species, a generic PCR assay was developed to detect a 300 bp fragment of deoxynivalenol (DON)-chemotypes and a 360 bp sequence of nivalenol (NIV)- chemotypes of Fusarium graminearum. Mycotoxin chemotypes identified by the PCR assays were confirmed by the chemical analyses of HPLC or GC/MS. Further analysis of 364 F. graminearum isolates from 12 provinces of China showed that 310 were DON-chemotypes and 54 were NIV-chemotypes. Sequence analyses revealed that DON-chemotypes display more variations than NIV-chemotypes. This PCR assay could be used to detect mycotoxin-producing Fusarium-species and may thus help to develop strategies to avoid or reduce mycotoxin contamination of cereals. Also this assay may provide useful alternatives to antibody-based mycotoxin tests.

Assessment ofFusariuminfection in wheat heads using a quantitative polymerase chain reaction (qPCR) assay

“…In particular, the molecular characterization of the Tri-5 gene cluster, which contains most of the genes involved in trichothecene biosynthesis, has been exploited in the development of several PCR-based assays to target Fusarium species and chemotypes (Lee et al 2001;Ward et al 2002;Kimura et al 2003). On these sequences, both generic assays that target all tricothecene producers (Li et al 2005) as well as species-specific assays have been developed (Demeke et al 2005). Generic primers designed on Tri5 gene sequence that amplify all thricothecene producers have been developed by Doohan et al (1999), Edwards et al (2001) and Schnerr et al (2001).…”

“…In a recent work (Terzi et al 2007), a qPCR-based analytical method was developed targeted at the quantification of the biomass of thricothecene-producing F. graminearum and F. culmorum strains using a pair of primers designed on the Tri5-Tri6 intergenic sequence which is involved in DON synthesis. This intergenic sequence has been already used (Bakan et al 2002;Li et al 2005), but the analytical approach developed by Terzi et al (2007) is quantitative and can be used also in samples characterized by a high level of DNA fragmentation, as those derived from food and feed, because of the small size of the amplicon. For this reason, it can be potentially used along the entire production chain.…”

Novel Methods for the Quantification of Pathogenic Fungi in Crop Plants: Quantitative PCR and ELISA Accurately Determine Fusarium Biomass

“…Based on this knowledge three genes were chosen in all studies to identify the chemotypes: tri3, tri5, tri7 and tri13 or regions between these genes. The developed assays were applied to Fusarium monitoring programs in numerous countries of Europe and Nortehrn America (Chandler et al, 2003;Jennings et al 2004;Lia et al, 2005;Quarta et al, 2006;Stepien et al, 2008;).…”

Microbial Diversity in Milled Rice as Revealed by Riosomal Intergenic Spacer Analysis

“…For the Tri13NIV/ Tri13R primer set 8) , the program consisted of 2 min at 94°C, followed by 40 cycles of 30 sec at 94°C, 30 sec at 65°C, and 15 sec at 72°C, and a final extension step of 7 min at 72°C. For the ToxP1/ToxP2 primer set 30) , the program consisted of 2 min at 94°C, followed by 40 cycles of 30 sec at 94°C, 30 sec at 62.5°C, and a final extension step of 7 min at 72°C. PCR amplifications were carried out using a Gene Amp ® PCR System (Applied Biosystems), except for the UBC85F 410 /UBC85R 410 primers for which we used TAKARA 480 (Takarabio).…”

“…This gene encodes C-4 monooxygenase, and is thought to convert deoxynivalenol (DON) to nivalenol (NIV) 29) . The ToxP1/ToxP2 primer set was developed based on the intergenic sequence between the Tri5 and Tri6 genes 30) .…”