Abstract
Common bunt, caused by Tilletia laevis Kühn [syn. T. foetida (Wallr) Liro] and Tilletia tritici (Bjerk.) Wint. [syn. T. caries (DC) Tul.], is an important wheat disease worldwide. To quickly differentiate the closely related fungi T. laevis, T. tritici and Tilletia controversa (a pathogen that causes dwarf bunt of wheat and has been requested as a quarantined pathogen in many countries), a rapid diagnostic and detection method for an ISSR molecular marker was developed for the first time in this study. Based on the T. laevis-specific band (1300 bp) amplified by the primer ISSR860, a pair of SCAR primers (L60F/L60R) was designed to amplify a specific 660-bp DNA fragment from the isolates of T. laevis but not other related pathogens. The detection limit of the SCAR marker was 0.4 ng/μl of DNA from T. laevis; moreover, a SYBR Green I real-time PCR method was also successfully developed based on the SCAR marker with the detection limit of 10 fg/μl T. laevis DNA. This is the first report of a rapid, specific and highly sensitive SCAR marker and SYBR Green I real-time PCR method for detection of the teliospores of T. laevis based on ISSR technology. This method allows highly efficient, rapid and accurate differentiation of the pathogen from related pathogens, especially from the very similar pathogens T. tritici and T. controversa.
Highlights
Contamination of wheat with common bunt spores has resulted in considerable loss of yield and seed quality accompanied by a fish-like odor[1,2]
There has been no report on molecular markers that could distinguish T. laevis from similar species of pathogens obtained by ISSR analysis, such as T. tritici and T. controversa
The main aim and objectives of this study were to develop a detection method that could be rapid, reliable and sensitive for distinguishing the teliospores of T. laevis from those of similar species by ISSR technology, which will contribute to a diagnostic method based on a SCAR marker and SYBR Green I real-time PCR
Summary
Contamination of wheat with common bunt spores has resulted in considerable loss of yield and seed quality accompanied by a fish-like odor[1,2]. T. caries (DC) Tul.], both could be variants of the same species together with the pathogen that causes dwarf bunt, Tilletia controversa Kühn, as proposed by several genetic, biochemical, and molecular studies[5]. There has been no report on molecular markers that could distinguish T. laevis from similar species of pathogens obtained by ISSR analysis, such as T. tritici and T. controversa. The main aim and objectives of this study were to develop a detection method that could be rapid, reliable and sensitive for distinguishing the teliospores of T. laevis from those of similar species (especially T. tritici and T. controversa) by ISSR technology, which will contribute to a diagnostic method based on a SCAR marker and SYBR Green I real-time PCR
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