Recent advances in geminivirus detection and future perspectives
Nirbhay Kushwaha, Achuit K Singh, Brotati Chattopadhyay and Supriya Chakraborty
The detection and identification of viruses has been a challenge since the advent of the discipline of plant virology over a century ago.Agreat variety of methods have been developed that permit differentiation of viral pathogens.These methods, initially based solely on identifying the distinct biological characteristics of different viruses, were soon supplemented with methods based on light or electron microscopy and serology and subsequently by Enzyme Linked Immunosorbant Assay (ELISA) and finally the use of molecular (nucleic acid-based) techniques have revolutionized the diagnosis of plant viruses. While the technologies available to virologists have obviously become more diverse and improved, the challenges have also changed greatly.Detection of plant viruses is becoming more critical as globalization of trade,particularly in horticultural commodities increase. The potential effects of climate change have further aggravated the movement of viruses and their vectors, transforming the diagnostic landscape. Geminiviruses are a group of plant viruses characterized by circular, single-stranded (ss) DNA genomes (~2.7 kb size) and twined icosahedral virions (18 x 30 nm). Based on genome organization, host range and insect-vector, geminiviruses are divided into four genera:Mastrevirus, Curtovirus, Topocuvirus and Begomovirus. Begomoviruses are transmitted by whiteflies (Bemisia tabaci Gennadius) and usually possess a bipartite genome of twoDNAcomponents approximately 2.7 kb in size, designated as DNA-A and DNA-B. Monopartite begomoviruses are also known to occur. Recently, a novel class of molecule, known as satellite DNA-? of ~1.35 kb size are also found to occur in India. Whitefly transmitted geminiviruses categorized as begomoviruses are reported since long. During the last two decades, whitefly-transmitted geminiviruses have emerged as serious pathogens of several agricultural and horticultural crops (like tomato, chillies, cotton, pulses, papaya, cucurbits, okra etc.) causing enormous losses in the tropics, which provide ideal conditions for the perpetuation of viruses and the insect-vector. Despite concerted efforts to contain begomoviruses and their vectors, menacing disease epidemics caused by newly emerging or reemerging begomoviruses are becoming frequent and appearing even in new regions, previously free from such diseases. Techniques for geminivirus detection include biological indexing, electron microscopy, antibody-based detection, including ELISA, nucleic acid hybridizations like dot blot, squash blot and Southern blot techniques, polymerase chain reaction and other DNA polymerase-mediated assays, and microarray detection. Of these, microarray detection provides the greatest capability for parallel yet specific testing and can be used to detect individual or combinations of viruses with sensitivity comparable to ELISA. Methods based on PCR provide the greatest sensitivity among the listed techniques but are limited in parallel detection capability even in “multiplexed” applications. Better, easier and cheaper than polymerase chain reaction or antibody detection, rolling circle amplification using the bacteriophage 29 DNApolymerase allows for a reliable diagnosis of geminiviruses and presumably all viruses with small single-stranded circular DNA genomes. The results show the efficiency of this technique in characterizing viral DNA components of several geminiviruses from experimental and natural host plant sources. Nucleotide sequence data offers identification of viral molecule to strain/species level for accurate diagnosis.