Development of a simple and reliable species-specific detection of Phomopsis azadirachtae, using the translation elongation factor 1-alpha gene

Abstract

Phomopsis azadirachtae (PA), a deuteromycetous fungus, is responsible for die-back disease in neem. The conventional method of identification of the causative fungi has been based on morphological and microscopic studies which are inconclusive and laborious. To assist in a reliable and faster detection of the infection, molecular methods are widely used in various studies of characterizing different species of Phomopsis. Lack of adequate polymorphism at the ITS region in most Phomopsis spp. have led to identification of other gene sequences which assist in accurately resolving the species. The translation elongation factor 1-alpha (EF1-α) locus has assisted in accurate delineation of various fungal species as this is highly consistent and also exhibits sufficient polymorphism. In the present study, EF1-α sequences of 25 PA isolates were considered, along with some of the Phomopsis and Diaporthe spp. obtained using BLAST analyses. Subsequently, a pair of specific primers was designed to amplify a 240-bp product. The results illustrated that the primers were specific to all the PA isolates and could detect 50 ng/μl DNA template. To enhance the sensitivity of detection, a nested PCR protocol was designed which raised the detection limit to 10 fg/μl. In addition, the primers also proved efficient in detection of the pathogen in neem twigs obtained by artificial inoculation one day after infection, with the nested PCR method. The results provide a highly sensitive method in early and rapid detection of PA and facilitate in primary screening of the infection.