Duplex PCR for detection of early and late blight coinfecting potato

Abstract

The disease identification at initial level is utmost factor for management of any disease. The detection of early and late blight at initial level is more important for its management due to fast spreading behaviour of these diseases as late blight devastates entire crop within a week. Although, morphological and serological methods are available, but due to their time consuming nature, sometimes there is failure in diagnosing latent infection with very low level inoculums. Moreover, diagnosis of both the diseases one by one is laborious. Therefore in the present investigation, a duplex PCR method was developed for simultaneous detection of Alternaria solani and Phytophthora infestans. PCR conditions and annealing temperature of both the primers [PINFTH2 (F)-GGGGGTCTTACTTGGCGGCG, PINFTH2(R)-CAAACCGGTCGCCAACTCGC for P. infestans and BIAMASbt (F)-GCTCCCACTCCTTCCGCGC, NBAIMASbt(R)-GGAGGTGGAGTTACCGACAA for A. solani] were standardized separately. Then the optimization of the PCR reactions and condition of duplex PCR was also standardized. The results revealed detection of single amplicon of 524bp with P. infestans while with A. solani 289bp amplicon was detected in genomic DNA from mycelium as well as in plants infected with both the diseases. This duplex PCR enabled the simultaneous detection of both the pathogens. In early stages of disease development, suspected samples (plants & tubers) were tested with this duplex PCR, which could diagnose both the pathogens correctly. The protocol will help in diagnosis of latent infection in potato tissues; and it will also be used as a diagnostic tool for researchers engaged in potato seed production and multiplication reducing cost and time thereby.