Plant pathogens and pests are one of the major factors that limit crop productivity. Infection could be detected easily by visual inspection if the symptoms are clearly characteristic for the disease. However many factors play a major role in identification of infection like viral strain, cultivar, time of infection and other environmental factors (1). Geminiviridae and Potyviridae are the two largest families that constitute important plant viruses, which are highly devastating phytopathogens worldwide (2, 3). The family Geminiviridae was established in 1978 (4) and consists of 9 genera: Nine genera - Becurtovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus, Turncurtovirus [Monopartite genome] and Begomovirus [Bipartite genome] (5–7). The genetic material of geminiviruses is ssDNA and have either a monopartite (DNA-A) or bipartite (DNA-A and DNA-B) genome. Of the many geminiviruses infecting economically important crops, begomoviruses are one of the most destructive viruses and are transmitted by whitefly vector Bemisia tabaci (8–10).
Capsicum annuum (Chilli) and Solanum lycopersicum (Tomato) plants belong to the family Solanaceae and are grown in tropical countries. It is a family of flowering plants, consisting of many important crops like potatoes, tomatoes, peppers, tobacco, petunia and other crops of regional significance (11, 12). Plants belonging to the Solanaceae family are heavily affected by viruses and begomoviruses are the major pathogen (8, 13–16). Begomovirus symptoms in plants range from symptomless to different degrees of stunting and curling, distortion, mosaic, mottling, vein yellowing of leaves, flower abortion, small and unmarketable fruits (17). India is one of the top producers of chilli and tomato in the world and are cultivated across the country throughout the year (18). Begomoviral infection has been reported in different states of India in chilli and tomato plants and were shown to cause severe losses (19). Begomovirus infecting pepper (chilli) and tomato plants have narrow host ranges and were shown to infect plants of other families, e.g., Fabaceae [common bean and soybean (Glycine max)] and Cucurbitaceae (20, 21).
One of the basic requirements for the elimination and spread of viruses is detection of the presence of viruses at the earliest possible stage in plants (22). Many methods are available for the detection of plant viruses which could be classified into protein-based methods (immunotechniques) or nucleic acid-based methods (molecular methods). The various immunotechniques include enzyme linked immunosorbent assay (ELISA) (23–25), immunoblotting (26), immunosorbent electron microscopy (27, 28) and precipitin tests (29). These methods rely on specific interaction of antibody (monoclonal or polyclonal) with viral antigens (coat protein) and hence are prone for false negativity at low viral titres. The limitations of these methods also include cross-reactivity with similar antigens resulting in false positive results. The production of antibodies is tedious, expensive and requires expertise.
Molecular methods like PCR (30–33), Nucleic Acid Spot Hybridization (NASH) assay (34–40) depend on viral nucleic acids and viral specific primers or probes. PCR is the most commonly used detection technique for begomoviruses and primers were primarily designed on coat protein and replicase genes (41). Among the viral proteins, coat protein has been shown to be important for the infection and propagation of the virus. This gene was reported to be conserved among begomoviruses and many primers designed in this region were utilized for PCR based detection of begomovirus (42). The major advantages of molecular techniques are specificity, sensitivity, faster detection and multiplexing (43, 44). Though most molecular techniques are robust, there are drawbacks like non-specific amplification, inhibition of polymerases activity due to the presence of phenolics, polysaccharides in the nucleic acids (45).
Simple and convenient technologies for the identification of chemical and biological species are of great significance in environmental monitoring, public health and disease diagnosis (46, 47). Here we report the development of functionalized gold nanoparticles (AuNPs) based visual detection method that relies on hybridization principle. Gold nanoparticles (AuNPs) have been routinely used for the development of visual detection methods for many pathogens due to their unique colour properties and tunable localized surface plasmon resonance (SPR) (48). Surface plasmon resonance has been shown to be dependent on NP (nanoparticle) composition, size and shape (49, 50).
Detection methods based on functionalized nanoparticles were found to be highly sensitive with faster turnover than the other methods like PCR, ELISA. Early detection of plant pathogens is critical for the management of infections in plants as it would result in better segregation of healthy plants and infected plants at the earliest, thus preventing the spread of disease in a farm or field (51). Recently, a visual DNA method using integrated Recombinase Polymerase amplification (RPA) and a AuNP probe has been reported for detection of tomato yellow leaf curl virus and was found to be highly sensitive and stable (52). In our lab, we had developed a method for detection of Banana Bunchy Top Virus (BBTV) using functionalized AuNPs (53). In this work, we demonstrated the applicability of functionalized AuNPs in the detection of begomoviruses in plants and an Indian patent has been filed (Application No: 201941007582).