Detection and Quantification of Alternaria solani in Tomato by Real Time PCR

Abstract

A conventional and real-time PCR assays using SYBR Green for the detection and quantification of A. solanihave been developed and validated. A primer set (ALP and ITS4) designed from the ITS region of A. linicola/ A. solani complex, yielded a 536 bp product when DNA from 38 isolates of A. solani were amplified. No product was amplified from A. alternata, A. brassicae, A. brassicicola, A.helianthi, A. porri, A. sesami, A.carthami, A.ricini, Colletotrichum gloeosporioides, C. capsici, C. falcatum, Cercospora canescens, C. capsici, Phytophthora infestans, Sclerotium rolfsii, Fusarium equiseti, F. oxysporum, Rhizoctonia solani, Phoma exigua, Curvularia spp and Drechslera. In addition, ALP/ITS4 primers were successfully utilized in real-time PCR assays of A. solani. The efficiency of conventional and real-time PCR assays was compared. The conventional PCR was able to detect the pathogen on symptomatic artificially infected tomato plants 5 days after pathogen inoculation. The detection limit was 100 conidia and 10 pg of DNA in the case of conventional PCR. Real-time PCR exhibited a detection limit 10 times lower (10 conidia, 10fg of DNA). The application of real time PCR assay for rapid detection of A.solani in infected tomato plant material is discussed.