Determination of Ascochyta blight disease in chickpea using real-time PCR
MetadataShow full item record
Ascochyta blight is a devastating disease of chickpea caused by Ascochyta rabiei. In this article, we described a real-time PCR assay for the determination and quantification of A. rabiei infection in chickpea tissues and accurate monitoring of disease progression in plant materials inoculated with different inoculation methods. The primer pairs HEF1/HEF2 were designed to anneal to conserved regions of translation elongation factor 1 alpha (EF) gene for specific amplification of 82-bp fragment of A. rabiei based on SYBR Green I technology. The detection limit of assay was determined as 0.1 pg DNA. PCR specificity was confirmed by testing against uninfected chickpea tissues and another fungal species associated with chickpea. The chickpea plants were inoculated by the methods of whole-plant and detached leaflet inoculation. Disease progression in resistant and susceptible cultivars was evaluated at certain time intervals after pathogen inoculation by real-time PCR. The results revealed a good correlation between visual assessments of disease reaction and pathogen quantification in infected chickpea tissues. The target DNA sequence was also amplified from the samples of DNA extracts from artificially infested seed. This technique could provide a useful approach for efficient selection of resistant breeding material in an early stage of infection as an alternative to the visual disease assessment and will be also used for the determination and quantification of A. rabiei infection.