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Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer
Institute of Phytopathology, Christian-Albrechts-University Kiel, Hermann-Rodewald-Str. 9, D-24118, Kiel, Germany
Botany and Microbiology Department, College of Science, King Saud University, P.O. Box: 2455, Riyadh 1145, Saudi Arabia
Plant Pathology Research Institute, Agricultural Research Centre, Giza, Egypt
Laboratory of Phytopathology, Wageningen University, Droevendaalsesteeg 1, NL-6708 PB Wageningen, The Netherlands
* Author to whom correspondence should be addressed.
Received: 2 December 2010; in revised form: 4 January 2011 / Accepted: 4 January 2011 / Published: 19 January 2011
Abstract: Early detection of infection is very important for efficient management of Mycosphaerella graminicola leaf blotch. To monitor and quantify the occurrence of this fungus during the growing season, a diagnostic method based on real-time PCR was developed. Standard and real-time PCR assays were developed using SYBR Green chemistry to quantify M. graminicola in vitro or in wheat samples. Microsatellite dinucleotide specific-primers were designed based on microsatellite repeats of sequences present in the genome of M. graminicola. Specificity was checked by analyzing DNA of 55 M. graminicola isolates obtained from different geographical origins. The method appears to be highly specific for detecting M. graminicola; no fluorescent signals were observed from 14 other closely related taxa. Primer (CT) 7 G amplified a specific amplicon of 570 bp from all M. graminicola isolates. The primers did not amplify DNA extracted from 14 other fungal species. The approximate melting temperature (Tm) of the (CT) 7 G primer was 84.2 °C. The detection limit of the real-time PCR assay with the primer sets (CT) 7 G is 10 fg/25 µL, as compared to 10 pg/25 µL using conventional PCR technology. From symptomless leaves, a PCR fragment could be generated two days after inoculation. Both conventional and real-time PCR could successfully detect the fungus from artificially inoculated wheat leaves. However, real-time PCR appeared much more sensitive than conventional PCR. The developed quantitative real-time PCR method proved to be rapid, sensitive, speciﬁc, cost-effective and reliable for the identiﬁcation and quantiﬁcation of M. graminicola in wheat.
Keywords: Septoria tritici blotch; microsatellite; wheat; Dothidiomycete; molecular diagnostics
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Abd-Elsalam, K.; Bahkali, A.H.; Moslem, M.; De Wit, P.J.G.M.; Verreet, J.-A. Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer. Int. J. Mol. Sci. 2011, 12, 682-693.
Abd-Elsalam K, Bahkali AH, Moslem M, De Wit PJGM, Verreet J-A. Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer. International Journal of Molecular Sciences. 2011; 12(1):682-693.
Abd-Elsalam, Kamel; Bahkali, Ali H.; Moslem, Mohamed; De Wit, Pierre J. G. M.; Verreet, Joseph-Alexander. 2011. "Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer." Int. J. Mol. Sci. 12, no. 1: 682-693.