Real Time PCR Based Quantification of Banana Bunchy Top Virus (BBTV) Titre in Banana cv. Grand Naine (Musa acuminata)
N. Tanuja
Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore 641003, India.
A. Ramanathan
Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore 641003, India.
S. Varanavasiappan
Department of Plant Biotechnology, Centre for Plant Molecular Biology and Bioinformatics, Tamil Nadu Agricultural University, Coimbatore 641003, India.
E. Kokila Devi
Department of Plant Biotechnology, Centre for Plant Molecular Biology and Bioinformatics, Tamil Nadu Agricultural University, Coimbatore 641003, India.
L. Arul
Department of Plant Biotechnology, Centre for Plant Molecular Biology and Bioinformatics, Tamil Nadu Agricultural University, Coimbatore 641003, India.
D. Sudhakar
Department of Plant Biotechnology, Centre for Plant Molecular Biology and Bioinformatics, Tamil Nadu Agricultural University, Coimbatore 641003, India.
S. Vanitha
Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore 641003, India.
K. Soorianathasundaram
Department of Fruit Science, Horticulture College and Research Institute, Tamil Nadu Agricultural University, Coimbatore 641003, India.
K. K. Kumar *
Department of Plant Biotechnology, Centre for Plant Molecular Biology and Bioinformatics, Tamil Nadu Agricultural University, Coimbatore 641003, India.
*Author to whom correspondence should be addressed.
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.
Keywords: Grand Naine, BBTV, coat protein, PCR, real time quantification.