Real Time PCR Based Quantification of Banana Bunchy Top Virus (BBTV) Titre in Banana cv. Grand Naine (Musa acuminata)

Abstract

Banana Bunchy Top Disease (BBTD) is one of the most severe viral diseases affecting major banana growing belts in India. Banana Bunchy Top Virus (BBTV) is transmitted by a black banana aphid (Pentalonia nigronervosa L Coquerel) in a persistent manner. BBTV virions are limited to the phloem tissue of banana resulting in low titre in banana. A reliable method to quantify the BBTV in the banana will be useful for monitoring the insect vector mediated BBTV transmission in banana, an essential requirement for characterizing the transgenic banana transformed for BBTV resistance. A protocol for real time PCR based absolute quantification of BBTV is reported in the present study. The partial BBTV coat protein gene (459 bp) was isolated, cloned into a plasmid vector and used to construct a standard curve using an SYBR green-based assay with known copies of BBTV coat protein gene. Using the standard curve, BBTV viral load was estimated in BBTV infected symptomatic and asymptomatic leaf samples of banana cultivar Grand Naine through SYBR green-based quantitative Polymerase Chain Reaction (qPCR). The study demonstrated that a higher viral tire was associated with BBTV disease symptoms appearance, whereas the low titre resulted in asymptomatic plants.