Next Article in Journal
In Silico Theoretical Molecular Modeling for Alzheimer’s Disease: The Nicotine-Curcumin Paradigm in Neuroprotection and Neurotherapy
Previous Article in Journal
Combined Effects of Cyclooxygenase-1 and Cyclooxygenase-2 Selective Inhibitors on Ovarian Carcinoma in Vivo
Int. J. Mol. Sci. 2011, 12(1), 682-693; doi:10.3390/ijms12010682
Article

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

1,2,3,* , 2
, 2
, 2,4
 and 1
Received: 2 December 2010; in revised form: 4 January 2011 / Accepted: 4 January 2011 / Published: 19 January 2011
(This article belongs to the Section Molecular Pathology)
View Full-Text   |   Download PDF [674 KB, uploaded 19 June 2014]   |   Browse Figures
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 Septoria tritici blotch; microsatellite; wheat; Dothidiomycete; molecular diagnostics
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Export to BibTeX |
EndNote


MDPI and ACS Style

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.

AMA Style

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.

Chicago/Turabian Style

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.


Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert