Development of Diagnostic Method for Phytoplasma in Sugarcane Leaves Using Nested-PCR Technique Combine with Fluorescent Dyes

Sugarcane (Saccharum officinarum L.) is an important field crop of Thailand. Thailand is the second largest sugar exporter in the world. However, the main problem in sugarcane production is sugarcane white leaf disease (SCWL), which is caused by Phytoplasma transmitted through the seed cane that is used for propagation. This disease causes stunted sugarcane, white leaves, and low yields. This research aimed to develop a rapid diagnostic method for phytoplasmas using the nested-polymerase chain reaction (PCR) technique and using fluorescent dye to detect the PCR products. The results showed that the 1st PCR with MLO-X and MLO-Y primers produced a single DNA band was 714 bp in SCWL but not in disease-free sugarcane. The 2nd PCR with P1 and P2 primers produced four DNA bands 714, 616, 302 and 204 bp in SCWL but not in disease-free sugarcane. The 2nd PCR products, which was added with a fluorescent dye, was found that fluorescence glow under UV light in SCWL tubes but not in disease-free sugarcane. The results indicate that the developed technique is accurate and can reduce the time for verifying nested-PCR products.