Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer

Abd-Elsalam, Kamel, Bahkali, A. H., Moslem, M., Wit, P. J. De and Verreet, Joseph Alexander (2011) Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer International Journal of Molecular Sciences, 12 (1). pp. 682-93. DOI 10.3390/ijms12010682.

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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 degrees C. The detection limit of the real-time PCR assay with the primer sets (CT) 7 G is 10 fg/25 muL, as compared to 10 pg/25 muL 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.

Document Type: Article
Additional Information: Abd-Elsalam, Kamel Bahkali, Ali H Moslem, Mohamed De Wit, Pierre J G M Verreet, Joseph-Alexander Switzerland International journal of molecular sciences Int J Mol Sci. 2011 Jan 19;12(1):682-93.
Research affiliation: Kiel University > Faculty of Agricultural and Nutritional Sciences > Institute of Phytopathology
Refereed: Yes
DOI etc.: 10.3390/ijms12010682
ISSN: 1422-0067
Date Deposited: 01 Nov 2012 04:59
Last Modified: 15 Jan 2014 13:07
URI: http://eprints.uni-kiel.de/id/eprint/16590

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