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.

Keywords: Dothidiomycete; Septoria tritici blotch; microsatellite; molecular diagnostics; wheat.

Figure 1

Polymerase chain reaction (PCR) amplification of genomic DNA isolated from 55 isolates of M. graminicola using primer pair (CT) C9 showing a amplification product of 570 bp. Lane M, 100 bp DNA marker; arrows indicate the 1000 and 500 bp DNA marker.

Figure 2

Polymerase chain reaction (PCR) amplification performed to assess specificity of the primer pair (CT) C9. A PCR fragment of 570 bp could only be amplified from Lanes 1–8 of genomic DNA from different isolates of Mycosphaerella graminicola, lane 9, DNA from Stagonospora nodorum (SN), lane 10, DNA from Pseudocercosporella herpotrichoides (PT), lane 11, healthy plant (HP), and lane 12–13, plants infected by M. graminicola (IP1 and IP2). Lane M, 100 bp DNA marker.

Figure 3

Comparison of the sensitivity of conventional (A) and real-time PCR (B), using different concentrations of Mycosphaerella graminicola DNA. For both (A) and (B), Lane 1, 10 ng; lane 2, 1 ng; lane 3, 100 pg; lane 4, 10 pg; lane 5,1 pg; lane 6, 100 fg; lane 7, 50 fg. Lane M, 100 bp DNA marker.

Figure 4

Amplification plot of fungal DNA from Mycosphaerella graminicola and other fungal species, as well as in symptomless wheat tissue and a non-template negative control by using a LightCycler instrument and (CT) C9 primer pair.

Figure 5

Polymerase chain reaction amplification performed to diagnose Mycosphaerella graminicola in artificially inoculated wheat leaves by (CT) C9 primer pair (product 570 bp). Lanes 1 to 10, DNA from inoculated leaves sampled at 0, 2, 6, 8, 10, 12, 14, 16, 18 and 20 days post inoculation with M. graminicola; lanes 11–12, DNA from healthy plants; lane 13, DNA from leaves inoculated with Pseudocercosporella herpotrichoides; lane 14, leaves inoculated d with Fusarium graminearum. Lane M, 100 bp DNA marker.