8th INTERNATIONAL WHEAT CONFERENCE

quANTITATIVe PCR AS A TooL foR TILLeTIA SPP.
deTeCTIoN ANd quANTIfICATIoN IN TImBeRINg WheAT
PLANTS
Evženie Prokinová 1 , Jana Mazáková 1 ,
Miloslav Zouhar 1 , Pavel Ryšánek 1 and Marie Váňová 2
1 Department of Plant Protection, Faculty of Agrobiology, Food and Natural Resources,
Czech University of Life Sciences Prague, Prague-Suchdol, Czech Republic
2 Agricultural Research Institute Kroměříž, Ltd., Kroměříž, Czech Republic
e-mail: mazakova@af.czu.cz
Common and dwarf bunt caused by Tilletia spp. belong to the most important diseases
of the wheat plants across the world. In the Czech Republic three closely related species
of this genus (Tilletia caries (DC.) Tul. & C. Tul. [1847], Tilletia controversa J.G. Kühn
[1874], Tilletia foetida (Wallr.) Liro [1920]) cause significant losses of wheat crops by
replacing grains with a mass of teliospores. The smut balls full of millions of teliospores
are crashed during wheat harvesting and seeds are contaminated by released spores. The
effective control measures against the bunts consist of seed surface treatment and tolerant
or resistant wheat varieties growing in particular. The objective of this present study was
to screen collection of Czech wheat varieties for smut fungi infection during first stages
of the wheat plant growth in order to predict sensitive/tolerant (resistant) reaction of the
wheat variety. Real-time qPCR is more suitable technique then conventional PCR for the
specific, accurate and sensitive detection of a wide range of plant pathogens. Therefore we
used real-time qPCR SYBER Green I assay with target-specific primers pairs for detection
and quantification of mycelium in wheat varieties naturally infected in a field trial.
The usability and specificity of designed primers based on intergenic spacers I region
sequence was firstly carried out by conventional PCR on isolates of Tilletia spp., healthy
plants, infected plants and other possible wheat pathogens. Quantitative PCR was verified
by analyzing different DNA isolation methods from various wheat plant parts of naturally
infected wheat plants, mycelium and spores of Tilletia spp.. The detection limit of qPCR
for the specific identification of fungal DNA was 0.22 ng of mycelium and the negative
correlation between target DNA quantity and cycle threshold (Ct) was high with coefficient
of determination R 2 = 0.992. This work was supported by project of Ministry of
Agriculture of the Czech Republic, grant No. QH71105 and Ministry of Education of the
Czech Republic, grant No. MSM6046070901.
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