Quantification of Fusarium culmorum in Wheat and Barley Tissues Using Real-Time PCR in Comparison with DON Content
2006
Leišová, L. | Kučera, L. | Chrpová, J. | Sýkorová, S. | Šíp, V. | Ovesná, J.
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 ( [graphic removed] = 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 (r² = 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.
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