cerealis (Chandler et al., 2003) were published. Recently, Pasquali et al. (2011), comparing three PCR genotyping methods, were not able to identify NIV genotypes of F. poae based on the tri7, tri12 and tri13 genes, using primers previously designed for other species (Ward et al., 2002; Quarta et al., 2006; Wang et al., 2008). Diagnostic assays based on the PCR allow researchers to analyse the potential contamination of cereal-based selleck chemicals llc food with certain mycotoxins and to determine the potential risk for human and animal health. Therefore, the aim of this study was to develop a PCR method for the detection of potential NIV-producing F. poae isolates. A total of 125
F. poae isolates from different countries and hosts previously identified by a species-specific PCR (Parry & Nicholson, 1996), four F. cerealis (NIV producers), two F. culmorum (NIV producers), one F. langsethiae (NIV producer), one F. sporotrichioides (NIV producer) and seven F. graminearum (NIV and DON producers) were analysed (Table S1, Supporting information). Moreover, NIV producers F. austroamericanum NRRL 2903, F. meridionale NRRL 28436, F. graminearum sensu stricto
NRRL 31084 and F. cortaderiae NRRL 29297, from the ARS Culture Collection, and Fusarium species isolated from seed samples (F. graminearum, F. oxysporum, BKM120 F. chlamydosporum, F. sporotrichioides, F. equiseti and F. acuminatum) were also evaluated. Twelve barley/wheat seed samples (2 kg) were provided by farmers from Buenos Aires province, Argentina. Seeds (400 per sample) were surface sterilized by immersing them for 3 min in 50% ethanol, 3 min in sodium hypochlorite (commercial 55 g Cl L−1), washed three times with sterilized distilled water and deposited
in Petri dishes (9 cm diameter) with potato dextrose agar (PDA) with chloramphenicol (50 μg mL−1) and incubated for 7 days at 25 ± 2 °C under 12-h light/dark conditions. Potential Fusarium isolates were placed in tubes with PDA and in Petri dishes containing Spezieller Nährstoffarmer Agar (SNA) and incubated for 7 days at 25 ± 2 °C under 12-h light/dark conditions for the identification according to Leslie & Summerell (2006). Monosporic genomic DNA from Fusarium isolates were extracted using a cetyltrimethylammonium bromide (CTAB) method described by Stenglein & Balatti (2006). From cereal samples, 20 g of seeds per sample were ground to a fine powder for Interleukin-2 receptor 1 min in a coffee-grinder and the DNA was extracted using the CTAB method described by Nicholson et al. (1996). The quality of seed and fungal DNA was examined by electrophoresis in 0.8% (w/v) agarose gels containing GelRed™ (Biotium; Hayward) at 80 V in 1× Trisborate-EDTA buffer for 3 h at room temperature. The DNA was visualized under UV light. DNA concentrations were calculated using a fluorometer (Qubit Fluorometer; Invitrogen). Different set of primers (data not shown) derived from the tri13 and the tri7 genes of the F. graminearum 88-1 NIV producer (Lee et al.