Quantification of Fusarium culmorum in Wheat and Barley Tissues Using Real‐Time PCR in Comparison with DON Content

Abstract

A real-time polymerase chain reaction (PCR) assay was developed for the specific detection of Fusarium culmorum in infected seeds. Primers and TaqMan minor groove binder probe were derived from the sequences of a F. culmorum specific PCR product. The specificity of the assay was confirmed by test in seven Fusarium species and 21 non-Fusarium fungal species. With serial dilutions of purified genomic DNA from F. culmorum isolate B as the template, the detection limit of the assay was found to be 0.9 pg of fungal genomic DNA per reaction. A significant correlation (r 2 average ¼ 0.982) and collinearity was found between DNA concentration and Ct (cycle threshold) values of real-time PCR assay with serial diluted DNAs extracted from three seed samples with different deoxynivalenol (DON) content. Eight barley and nine wheat varieties infected by F. culmorum isolate B were evaluated in 1 (barley samples) and in 4 years (wheat samples). The results of real-time PCR analysis and enzyme-linked immunosorbent assay testing for DON content were compared and a significant correlation was found for barley samples (r 2 ¼ 0.935). Concerning wheat we found rather complicated relationship between Ct values and DON contents influenced by environmental conditions of field trials. The real-time PCR assay was found to be highly specific and sensitive. It could be used in phytopathological studies and praxis.

Evaluation of Slovak Wheat Cultivars for Fusarium Culmorum Infection by Real-Time PCR and By Conventional Assays

“…The standard curve constructed using dilution series of pure F. culmorum DNA showed very high correlation between Ct values and the logarithm of the amount of input DNA (r 2 = 0.998) comparable with the results of Leišová et al (2006) and Moradi et al (2010). The contents of F. culmorum DNA in analysed samples expressed in nanograms of pathogen DNA per 1 g of wheat seeds and the infection per cent calculated as (Fusarium DNA/ total DNA) × 100 as given in Table 1.…”

“…The grains of modern cultivars were infected Agriculture (Poľnohospodárstvo), 58, 2012 (1): 18−24 17.8% more than the grains of old cultivars. The average contamination of grains in the tested group with the mycotoxin DON was 24.9 mg/kg, what is similar to DON content in wheat samples without a fungicide treatment found by Leišová et al (2006). The results show that old cultivars had average DON content in grain 35.9% lower than modern cultivars.…”

“…For the quantification of F. culmorum DNA we successfully used the real-time PCR assay with Taq-Man probe developed by Leišová et al (2006). The standard curve constructed using dilution series of pure F. culmorum DNA showed very high correlation between Ct values and the logarithm of the amount of input DNA (r 2 = 0.998) comparable with the results of Leišová et al (2006) and Moradi et al (2010).…”

“…SYBR-Green real-time PCR was successfully used for the specific detection of trichothedene-producing Fusarium spp. (Schnerr et al 2001;Moradi et al 2010) and also more specific Taq-Man real-time PCR was used to quantify main Fusarium species (Reischer et al 2004;Waalwijk et al 2004;Leišová et al 2006;Yli-Mattila et al 2008). Leišová et al (2006) found a significant correlation between the amounts of F. culmorum DNA (expressed by transformed threshold cycle (Ct) values) detected by Taq-Man real-time PCR and the content of DON detected by ELISA in barley, while in wheat the relationship was rather complicated and influenced by environmental conditions.…”

“…The Leišová et al (2006). Reaction was carried out in 25 µl reaction volume consisting of 12.5 µl TaqMan Universal PCR Master Mix (Applied Biosystems), 300 nM each primers, 200 nM TaqMan MGB probe (labelled with FAM fluorescent dye) and 25 ng of DNA in 1 µl.…”