Establishment of a loop-mediated isothermal amplification (LAMP) assay for the detection of phytoplasma-associated cassava witches' broom disease

Cassava (Manihot esculenta Crantz) is one of the most important food crops in the tropics; however, bacterial phytopathogens pose a serious threat to its farming. Cassava Witches' Broom Disease (CWB) is caused by the infection of phytoplasma and is manifested as reduction in tuber yield and starch content at harvest of 10 and 30 %, respectively. Although polymerase-chain reaction provides the gold standard in diagnostics, this method requires significant investments in infrastructure and training. Here, we developed a loop-mediated isothermal amplification (LAMP) assay that allows specific detection of phytoplasma from field-collected samples. Three primer sets were designed, of which two detected phytoplasma DNA sequence encoding 16S rRNA (16S rDNA), the other detected cassava actin. Following a 1 h LAMP reaction at 63 degrees C, a positive reaction can be visualized by agarose gel electrophoresis, hydroxynaphthol blue color change, or the presence of a precipitate. In a pilot field study, the assay was able to rapidly distinguish between healthy and CWBinfected cassava. With further development, a LAMP for routine on-site screening of cassava crops can be envisioned.