Development of a real-time RT-PCR method for the detection of Citrus tristeza virus (CTV) and its implication in studying virus distribution in planta

Tristeza is an economically important disease of the citrus caused by Citrus tristeza virus (CTV) of genus Closterovirus and family Closteroviridae. The disease has caused tremendous losses to citrus industry worldwide by killing millions of trees, reducing the productivity and total production. Enormous efforts have been made in many countries to prevent the viral spread and the losses caused by the disease. To understand the reason behind this scenario, studies on virus distribution and tropism in the citrus plants are needed. Different diagnostic methods are available for early CTV detection but none of them is employed for in planta virus distribution study. In this study, a TaqMan RT-PCR-based method to detect and quantify CTV in different tissues of infected Mosambi plants (Citrus sinensis) has been standardized. The assay was very sensitive with the pathogen detection limit of > 0.0595 fg of in vitro-transcribed CTV-RNA. The assay was implemented for virus distribution study and absolute CTV titer quantification in samples taken from Tristeza-infected trees. The highest virus load was observed in the midribs of the symptomatic leaf (4.1 x 10(7)-1.4 x 10(8)/100 mg) and the lowest in partial dead twigs (1 x 10(3)-1.7 x 10(4)/100 mg), and shoot tip (2.3 x 10(3)-4.5 x 10(3)/100 mg). Interestingly, during the peak summer months, the highest CTV load was observed in the feeder roots (3 x 10(7)-1.1 x 10(8)/100 mg) than in the midribs of symptomatic leaf. The viral titer was highest in symptomatic leaf midrib followed by asymptomatic leaf midrib, feeder roots, twig bark, symptomatic leaf lamella, and asymptomatic leaf lamella. Overall, high CTV titer was primarily observed in the phloem containing tissues and low CTV titer in the other tissues. The information would help in selecting tissues with higher virus titer in disease surveillance that have implication in Tristeza management in citrus.