Early detection of Ascochyta blight (Ascochyta rabiei) of chickpea by traditional PCR

Abstract

Ascochyta blight is the major disease affecting chickpea (Cicer arietinum) around the world. Since the first report of Ascochyta rabiei's isolation in Argentina in 2012, the pathogen has caused severe economic losses in crop production; so, the detection and rapid identification of the pathogen in early stages is key for the management of the disease. In this work, a traditional PCR procedure for detection of A. rabiei directly from plant tissues has been described based on beta-tubulin gene. The TP-6/TP-9 specific primers designed, amplified only a single PCR band of 770 bp from A. rabiei. The specificity of the primers was checked using 12 isolates of A. rabiei and DNA from 10 other different fungi including common pathogens of chickpea as Alternaria alternata, Botrytis cinerea, Sclerotinia sclerotiorum and Phoma medicaginis that cause similar symptoms. The detection sensitivity with primers was 2 × 104 ng μl−1 genomic DNA. In inoculated plant material, PCR amplification gave a band of the expected size and no amplification was observed when DNA was from healthy and uninoculated plants. The results suggested that the assay detected the pathogen more rapidly and accurately than standard isolation methods. The PCR-based method developed here can simplify both plant disease diagnosis, and pathogen monitoring in an early phase, as well as aid in effective management practices that avoid the disease advance and minimize losses.