Development of real-time loop-mediated isothermal amplification for detection of Ralstonia pseudosolanacearum race 4 in rhizomes and soil

Abstract

Bacterial wilt in ginger caused by Ralstonia pseudosolanacearum race 4 causes significant economic loss in several Asian and Polynesian countries. Since the pathogen being soil and rhizome borne, an early detection would prevent the occurrence and spread of the disease. The present study aimed at developing a strain specific detection methodology for race 4 strain infecting ginger using Real Time Loop Mediated Isothermal Amplification (real-time LAMP). LAMP primers were designed from the gyrB gene, which can specifically detect race 4 R. pseudosolanacearum infecting Zingiberaceae plants. In real-time LAMP, a sigmoid amplification curve with a Ta value of 92 ± 1 °C was obtained only with race 4 with a detection limit of 103 CFU/g of soil or rhizomes. For on-farmdiagnosis, the protocol was customized with soil supernatant as template instead of genomic DNA, the extraction of which is cumbersome under field conditions. The real-time LAMP thus developed can be used as an indexing tool for seed rhizomes and soil for latent infection