Loop-mediated isothermal amplification (LAMP) assay for detection of sesame phyllody phytoplasmas in Vietnam

Phloem-limiting phytoplasmas are known to be causal agents of phyllody, which is recognized by the abnormal development of floral structures resulting in serious yield losses in sesame plants. Currently, identification of the various groups of phytoplasmas that cause sesame phyllody (SP) is conducted by nested PCR, RFLP, and multiplex real-time qPCR assays. However, these methods require intensive labor and are costly and time-consuming so can only be undertaken in well-equipped labs. Here, diagnostic loop-mediated isothermal amplification (LAMP)-based assays allowing rapid detection of specific groups of phytoplasmas within 30 min were developed based on detection of the 16S rRNA sequence of phytoplasmas. Universal 16S rRNA phytoplasma primers and seven primer sets of different 16Sr group phytoplasmas (16SrI, 16SrII, 16SrIII, 16SrIV, 16SrV, 16SrX, 16SrXI) and universal plant cytochrome oxidase (cox) gene primers were used to detect 16S rRNA group phytoplasma sequences and the cox gene in sesame plants. The LAMP assays were carried out using a real-time fluorometer with amplification plots and annealing curves visualized directly. Results demonstrated that the 16SrI and 16SrII group phytoplasmas were causal agents of sesame phyllody in Vietnam. LAMP-based assays for in-field detection of sesame phyllody-causing phytoplasmas revealed advantages and potential applicability in comparison with conventional approaches. To the best of our knowledge, this is the first assessment of multiple phytoplasma infection associated with sesame phyllody disease in Vietnam using LAMP-based assays.