A novel approach for simultaneous detection of Citrus yellow mosaic virus and Citrus greening bacterium by multiplex polymerase chain reaction

A method of multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection of Citrus yellow mosaic virus (CYMV) and fastidious Citrus greening bacterium, Candidatus Liberibacter asiaticus (CLa) in sweet orange trees. Initially total DNA from individual CLa and CYMV infected citrus plants were mixed and both pathogens were detected simultaneously by multiplex PCR. Subsequently, both pathogens were detected from the total DNA obtained after mixing midribs of CLa-infected and CYMV-infected leaf lamina of the sweet oranges. The finally adopted multiplex PCR protocol simultaneously detected CLa and CYMV from the total DNA extracted from the midrib of leaf of citrus plants infected by both the pathogens. Thus, the present protocol demonstrated the presence of mixed infections of CLa and CYMV in citrus orchards trees. The technique would also prove highly useful in disease survey, nursery certification and quarantine applications.