Development of Conventional and Real-Time PCR Assays to Detect Alternaria burnsii in Cumin Seed

Abstract

Alternaria burnsii is the causal agent of cumin blight, a seed-borne disease of economic concern for all cumin growing areas. Current detection and identification methods for the pathogen are based on visual examination of morphological features, which are time-consuming and laborious. The present study describes conventional and real-time PCR assays for rapid and accurate detection of A. burnsii in cumin seeds. Based on sequence differences in Alternaria allergen a1 (Alt a1) gene, two primer pairs, Ab35/326 and AB177/403, were designed for conventional and real-time PCR assays, respectively. Both primer pairs amplified the expected target PCR fragment from A. burnsii genomic DNA. The sensitivity of conventional PCR with primer pairs Ab35/326 was 1 pg of genomic DNA and allowed the detection of pathogen in cumin seeds samples with 0.2% infestation rate. Real-time PCR assay was highly sensitive and allowed the quantification of 0.1 pg pathogen DNA. Also, this assay confirmed the presence of pathogen in cumin seeds up to 0.1% infestation level. The standard curve (r2 = 0.99) showed a good correlation between fungal DNA quantities and Cq values. The specificity of primer pairs was confirmed by the absence of amplified product with DNA of related fungi species and healthy plant tissue. The assays developed in this study provide a rapid and sensitive tool for the detection and quantification of Alternaria burnsii in cumin seed.