Nested PCR assay for specific and sensitive detection of Alternaria carthami

Alternaria leaf spot (ALS) caused by Alternaria carthami Chowdhary is one of the major threats to the cultivation of safflower in the world. The pathogen is seed borne and requires early detection for restricting its transmission and proliferation. A PCR-based diagnostic assay was developed for easy, quick and reliable detection of A. carthami in infected seeds and leaf samples of safflower. A primer set, AcSPF and AcSPR was designed using ribosomal internal transcribed spacer regions of A. carthami that consistently produced a distinct amplicon of 340 bp with DNA extracted from thirty A. carthami isolates. The specificity of the primer was confirmed using strains of 26 other strains of Alternaria and four other fungal pathogens of safflower. The sensitivity of detection was further enhanced from concentration of 100 pg by simple PCR to as low as 10 pg fungal DNA by a nested PCR assay using ITS and AcSPF and AcSPR primers. The primer pair also facilitated detection of A. carthami in infected seeds and leaf samples. The study provides an accurate and sensitive diagnostic tool for detection of A. carthami.