Species-Specific PCR-Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die-Back Disease in Azadirachta indica

摘要

Die-back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P.azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species-specific PCR-based assay was developed for rapid and reliable identification of P.azadirachtae by designing a species-specific primer-targeting ITS region of P.azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313-bp product from all the isolates of P.azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100fg when genomic DNA of all isolates was analysed. The PCR-based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species-specific PCR assay for identification and detection of P.azadirachtae. Thus, PCR-based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P.azadirachtae at early stages.