A rapid and sensitive polymerase chain reaction (PCR) assay for detection of Ascochyta rabiei, the cause of ascochyta blight of chickpea

Abstract

Ascochyta blight, caused by Ascochyta rabiei, is a major constraint to chickpea production in Australia. A diagnostic test based on restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) amplified DNA was developed to detect A. rabiei in infected leaves and seeds of chickpea. The test was developed by using primers designed to conserved sequences of the 18–25S ribosomal genes to amplify the internal transcribed spacer (ITS) regions of A. rabiei and other closely related Ascochyta species commonly found in pulses (A. lentis, A. pinodes and A. fabae) and identifying restriction enzymes that specifically cut the A. rabiei ITS region. The test was so sensitive that it detected DNA of A. rabiei at a quantity of 0.1 pg. The test also detected DNA extracted from infected leaf or seed in dilutions to 1×10−4 and 1 × 10−5, respectively, thus indicating that fungal genomic DNA can be specifically amplified by PCR from a plant genomic DNA background. This study also showed that the best method to detect chickpea seeds infected with A. rabiei was based on incubating surfacesterilised chickpea seeds in a liquid, fungal-growth medium (Czapek-Dox) for 18 h prior to the PCR and RFLP methods. This test was sensitive enough to detect a 1% infection.