Development and validation of loop mediated isothermal amplification based detection assay for Puccinia striiformis f. sp. tritici causing stripe rust of wheat

Abstract

Wheat stripe rust caused by Puccinia striiformis f. sp. tritici is one of the most important diseases of wheat worldwide. Detection of latent infection in leaves is critical for estimating the initial inoculum potential for causing epidemics. A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the detection of P. striiformis f. sp. tritici DNA in urediniospores and wheat seedlings with latent infections. Reaction temperature, optimal Mg²⁺ concentration and ratio of outer/inner primers for reaction efficiency were optimized. The sensitivity of detection was 1 picogram of genomic DNA and marker could detect latent infection in wheat leaves as early as 48 h post inoculation. This colorimetric detection assay was indicated by color change from purple to violet color in positive reaction by adding 200 μM hydroxyl napthol blue solution (HNB) to the reaction mixture. LAMP products were further analyzed by visualizing EtBr stained reaction mixture on transilluminator that showed full brightness in positive reaction which was absent in negative reactions. Further confirmation of LAMP products through agarose gel electrophoresis showed ladder like banding pattern only in positive reactions. The developed method is efficient, rapid and sensitive for detection of Puccinia striiformis tritici causing stripe rust of wheat. No cross reaction with other related pathogens was observed. The developed technique is very useful in quantifying inoculum load during offseason which would help in development of prediction model and also be useful in development of diagnostic kit.