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

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.

Species-Specific Detection of Mycosphaerella polygoni-cuspidati as a Biological Control Agent for Fallopia japonica by PCR Assay

“…Furthermore, touchdown PCR used in the present study resulted in higher sensitivity with the primer compared to normal PCR with consistent annealing temperature (data not shown). This is more sensitive than the previous reports including that the detection limit of M. graminicola or M. parva is as low as 10 pg [ 19 , 28 ]. In addition, the primers could detect M. polygoni - cuspidati from 1 pg of genomic DNA extracted from the lesions (data not shown), which showed sufficient and higher sensitivity than that shown in other report [ 19 ].…”

Assessing the Consequences of Microbial Infection in Field Trials: Seen, Unseen, Beneficial, Parasitic and Pathogenic

“…This can be achieved by specifically testing asymptomatic leaves e.g., [49,50] but a more general approach is to examine levels of symptom expression relative to total infection by accounting for the relationship between the two traits. Such an approach has been used to identify genetic loci that have differential effects on overall infection relative to symptom development [27].…”

Simple and rapid protocol for the isolation of PCR-amplifiable DNA from medicinal plants

“…MP-PCR patterns were produced following the protocol of Abd-Elsalam et al (2011). PCR products were obtained in a total volume of 25 mL with 20 ng template DNA, 10 pmol of the primer T3B (5ꞌ-AGGTCGCGGGTTCGAATCC-3ꞌ) and M13 (5ꞌ-GAGGGTGGCGGTTCT-3ꞌ) (MWG, Germany), 200 mM of each dNTP, 1 U Taq polymerase (JenaBioscience, Germany) and 1X polymerase reaction buffer.…”