A multiplex PCR assay for rapid differentiation of sugarcane phytoplasmas reveals co-infection in samples from Thailand

Sugarcane white leaf (SCWL), sugarcane grassy shoot (SCGS) and sugarcane green grassy shoot (SCGGS) are the most devastating sugarcane diseases in Thailand that cause significant losses to the crop production and the sugar industries. SCWL and SCGS phytoplasmas were found to be closely related based on 16S rDNA gene analysis while SCGGS was distantly related to them. So far, differentiation of these phytoplasmas have been based on Sanger sequencing which is time consuming and costly. This study aimed to develop a rapid and simple method for simultaneous identification of these phytoplasmas by multiplex-PCR targeting the 16S rDNA gene and to investigate the incidence of co-infections. Three sets of primers were designed to produce the target amplification products at 340 bp, 230 bp and 120 bp respectively to SCWL, SCGS and SCGGS. The investigation in 20 asymptomatic samples revealed two samples showing mixed infection of SCWL and SCGS. These results thus indicated that this multiplex-PCR method provides a simple, rapid and reliable differentiation method for the three phytoplasma diseases in sugarcane in Thailand. Notably, this is the first report to detect co-infection of the two phytoplasmas. The sensitivity of this method thus demonstrated substantial potential as an effective tool for screening of sugarcane plants prior to propagation as well as providing a reliable method to avoid misinterpretation of the analysis in ambiguous samples.