Rapid detection and quantification of Alternaria solani in tomato

Early detection of infection is very crucial for efficient management of early blight of tomato caused by Alternaria solani. To monitor and quantify the occurrence of this fungus, a diagnostic tool based on real-time PCR was developed. Specific-primers were designed from β-tubulin gene and specificity was checked with A. solani isolates and related species obtained from different geographical origins. The primers were highly specific for A. solani, as no amplification signal was observed from thirteen other closely related taxa. Primer set, AS1 (5′-GCTCCCACTCCTTCCGCGC-3′) and AS2 (5′-GGAGGTGGAGTTACCGACAA-3′) amplified a specific amplicon of 289 bp from all A. solani isolates. The lowest detection limit of the real-time PCR assay with designed primer set (AS1 and AS2) was 0.5 pg. The assay was also successfully validated on artificially infested tomato seedlings and able to detect A. solani up to 20 days post inoculation. To the best of our knowledge, this is the first report of a quantitative real time PCR detection method for rapid and specific detection of A. solani with a primer set designed from β-tubulin region.

► QPCR system was developed for the detection and quantification of A. solani in tomato.
► The system is based on primers targeting β-tubulin gene of A. solani and amplified specific band of 289 bp.
► We obtain good correlation (R2 = 0.987) between the Ct-value and A. solani DNA concentration.
► Lowest detection limit of the assay is 0.5 pg.
► The developed system was used to analyze the occurrence of A. solani on artificially inoculated tomato plants.