Duplex recombinase polymerase amplification assay for simultaneous detection of Pythium spp. and Ralstonia pseudosolanacearum from ginger rhizomes

Abstract

Soft rot and bacterial wilt are the major production constraints in ginger worldwide. As these pathogens are transmitted primarily through ginger rhizomes that are widely used as planting materials, effective and sensitive techniques are required for reliable and accurate diagnosis. In the present study, uniplex and duplex recombinase polymerase amplification (RPA) assays were developed for specific and sensitive detection of Pythium spp. and Ralstonia pseudosolanacearum from ginger rhizomes. The duplex RPA assay was 10 and 100 times more sensitive than duplex PCR assay in case of R. pseudosolanacearum and Pythium spp., respectively and were highly specific as they did not show any cross amplification with other rhizome-borne pathogens of ginger such as Fusarium spp., Macrophomina phaseolina and Sclerotium rolfsii. In addition, the assays could be performed under isothermal conditions at a temperature ranging from 37 to 40 °C in a heating block. In validation tests, these pathogens could be successfully detected using crude DNA extracted from ginger rhizome samples collected from the field, storage and market. In addition, the RPA assay developed could detect the pathogens in samples which were found negative in PCR assay. The present study could be the first report of simultaneous detection of fungal and bacterial pathogens using duplex RPA assay in ginger.