J H Liu1, L Gao, T G Liu, W Q Chen. 1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, CAAS, Beijing 100193, China.
Abstract
AIMS: Dwarf bunt of wheat, caused by Tilletia controversa Kühn, is a destructive disease on wheat as well as an important international quarantined disease in many countries. The objective of this investigation was to develop a diagnostic molecular marker generated from amplified fragment length polymorphism (AFLP) for rapid identification of T. controversa. METHODS AND RESULTS: A total of 30 primer combinations were tested by AFLP to detect DNA polymorphisms between T. controversa and related species. The primer combination E08/M02 generated a polymorphic pattern displaying a 451-bp DNA fragment specific for T. controversa. The marker was converted into a sequence-characterized amplified region (SCAR), and specific primers (SC-01(49)/SC-02(415)), designed for use in PCR detection assays, amplified a unique DNA fragment in all isolates of T. controversa, but not in the related pathogens. The detection limit with the primer set SC-01(49)/SC-02(415) was 10 ng of DNA which could be obtained from 11 microg of teliospores in a 25-microl PCR reaction. CONCLUSIONS: An approach to distinguish T. controversa from similar pathogenic fungi has been developed based on the use of a SCAR marker. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of the simple, high throughput assay kit for the rapid diagnosis of dwarf bunt of wheat and detection of T. controversa is anticipated in further studies.
AIMS: Dwarf bunt of wheat, caused by Tilletia controversa Kühn, is a destructive disease on wheat as well as an important international quarantined disease in many countries. The objective of this investigation was to develop a diagnostic molecular marker generated from amplified fragment length polymorphism (AFLP) for rapid identification of T. controversa. METHODS AND RESULTS: A total of 30 primer combinations were tested by AFLP to detect DNA polymorphisms between T. controversa and related species. The primer combination E08/M02 generated a polymorphic pattern displaying a 451-bp DNA fragment specific for T. controversa. The marker was converted into a sequence-characterized amplified region (SCAR), and specific primers (SC-01(49)/SC-02(415)), designed for use in PCR detection assays, amplified a unique DNA fragment in all isolates of T. controversa, but not in the related pathogens. The detection limit with the primer set SC-01(49)/SC-02(415) was 10 ng of DNA which could be obtained from 11 microg of teliospores in a 25-microl PCR reaction. CONCLUSIONS: An approach to distinguish T. controversa from similar pathogenic fungi has been developed based on the use of a SCAR marker. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of the simple, high throughput assay kit for the rapid diagnosis of dwarf bunt of wheat and detection of T. controversa is anticipated in further studies.