Quantification of Tilletia caries and Tilletia controversa mycelium in wheat apical meristem by real-time PCR

• Published in: Plant protection science Vol. 46; no. 3; pp. 107 - 115
• Main Authors: Zouhar, M.,Ceska Zemedelska Univ., Prague (Czech Republic). Katedra Ochrany Rostlin, Mazakova, J.,Ceska Zemedelska Univ., Prague (Czech Republic). Katedra Ochrany Rostlin, Prokinova, E.,Ceska Zemedelska Univ., Prague (Czech Republic). Katedra Ochrany Rostlin, Vanova, M.,Zemedelsky Vyzkumny Ustav, Kromeriz (Czech Republic), Rysanek, P.,Ceska Zemedelska Univ., Prague (Czech Republic). Katedra Ochrany Rostlin
• Format: Journal Article
• Language: English
• Published: Prague Czech Academy of Agricultural Sciences (CAAS) 01.09.2010; Czech Academy of Agricultural Sciences
• Subjects: absolute quantification; ADN; AGENT PATHOGENE; apical meristem; APICAL MERISTEMS; BLE; Cereal crops; Deoxyribonucleic acid; DNA; EXTRACCION; EXTRACTION; FUSION; http://www.fao.org/aos/agrovoc#c_2347; http://www.fao.org/aos/agrovoc#c_24524; http://www.fao.org/aos/agrovoc#c_34079; http://www.fao.org/aos/agrovoc#c_36910; http://www.fao.org/aos/agrovoc#c_4731; http://www.fao.org/aos/agrovoc#c_5017; http://www.fao.org/aos/agrovoc#c_528; http://www.fao.org/aos/agrovoc#c_5630; http://www.fao.org/aos/agrovoc#c_7657; http://www.fao.org/aos/agrovoc#c_7951; http://www.fao.org/aos/agrovoc#c_8373; MELTING; MERISTEMAS APICALES; MERISTEME APICAL; Meristems; MICELIO; MYCELIUM; ORGANISMOS PATOGENOS; PATHOGENS; PCR; Polymerase chain reaction; Real time; real-time pcr; Teliospores; TEMPERATURA; TEMPERATURE; TILLETIA CARIES; TILLETIA CONTROVERSA; tilletia spp; TILLETIA TRITICI; TRIGO; TRITICUM AESTIVUM; Wheat; WHEATS
• ISSN: 1212-2580; 1805-9341
• DOI: 10.17221/50/2009-pps

In the Czech Republic, three closely related species of the genus Tilletia belong to pathogens that cause significant losses of wheat crops by replacing grains with a mass of teliospores. A quantitative real-time qPCR assay using SYBR Green I has been developed to quantify the amount of T. caries and T. controversa mycelium in apical meristems of different wheat varieties. The real-time PCR reaction was validated by evaluating selected extraction methods, examining the specificity of designed target-specific IGS primers and verifying the optimised amplification reaction on naturally infected wheat plants. The PCR detection limit for the specific identification of fungal DNA was 0.22 ng of mycelium, and the negative correlation between the target DNA quantity and cycle threshold (Ct) was high with a coefficient of determination of R2 = 0.992. The developed method was used to quantify pathogens mycelium in five wheat varieties in the range from 0.34 ng to 15 microg per one growing tip.