Detection and prevalence of viruses infecting field-grown tomato in North-western region of India were studied through serological and molecular approaches. Although visual assessment of symptoms is not accurate way of determining incidence of a viral disease, yet this method is widely used by many plant pathologists in monitoring the prevalence, incidence and intensity of a disease. Symptoms can also vary depending on virus strain, mixed infections, cultivar, light intensity, time of infection during the year, stage of plant growth, temperature and/or the presence of unknown pathogens .
Analysis of leaf samples collected from symptomatic tomato plants during surveys conducted for the present study (2015 to 2017) revealed occurrence of 8 RNA viruses viz. CMV, GBNV, PVM, PVS, PVX, PVY, ToCV, ToMV and a DNA virus viz. ToLCNDV. The surveyed fields were found to have same cropping cycles which mostly included solanaceous crops one after the other. In our earlier study, we reported natural infection of CMV, PVM and PVY on a weed species, Solanum nigrum, commonly found in tomato fields of the study area . However, none of the plants of another weed species (Chenopodium sp.) analysed in the present study showed occurrence of any of the above viruses
It was noticed that potato crop was harvested prior to tomato crop. Keeping this in mind, symptomatic plant samples were tested against eight potato viruses also, out of which four potato viruses (PVM, PVS, PVX and PVY) were detected. Out of these isolates, PVY positive isolate (B7-Asr) shared 100% sequence identity with another isolate of PVY from Egypt (MT242381.1) whereas PVM isolate (B9-Asr) shared 97.33% similarity with other isolates from Japan (LC511893.1) and New Delhi (KJ462137.1). K9-Una isolate of PVX showed 99.64% nucleotide similarity with an isolate from Bangladesh (MK587458.1). PVY isolates are divided into five strains viz. PVYO, PVYC, PVYN, PVYZ, and PVYE . PVYO (causes systemic mottling) and PVYN (causes veinal necrosis) are commonly found in potato fields, while PVYC is relatively less prevalent . PVY evolution has been aided by genomic recombination, which has resulted in the formation of novel genotypes/strains such as the recombinant PVYNTN, PVYN−Wi, and PVYNTN−NW . Lorenzen et al.  described multiplex assay utilising eight PVY primers capable of discriminating various strains of PVY (PVYO, PVYN, PVYN:O (Wilga) and PVYNTN). The primer combinations include PVY n2258 + o2439c for NTN, N:O strain, PVYo2172 + o2439c for O strain and PVYn2258 + n2650c for N strain. In our study, three different PCR assays were run to check the presence of target strains for Potato virus Y. Isolates of Una and Patiala gave amplification of 266 bp for strain O (GenBank Accession numbers MZ322852 and MZ322853) (Fig. 2H) and isolate of Amritsar confirmed band of 181 bp for strain NTN, N:O (Fig. 2G) whereas no bands were seen corresponding to strain N.
CMV has been found on several crops including cucumber, tomato, chilli, potato, brinjal, banana, pepper, vanilla, gherkin, gerbera, snake gourd, bottle gourd, etc. from India . Rybicki  listed CMV among the top ten economically significant plant viruses. During visual symptom based surveys conducted in various fields, typical shoestring symptoms on tomato leaves were observed. CMV isolate K3 shared 97.94% nucleotide sequence identity with other isolates from Ecuador (MW291545) followed by 97.20% sequence similarity with isolates from Uganda (MG021462.2). Bud necrosis disease in tomatoes is caused by Groundnut bud necrosis virus (GBNV), the most common tospovirus in India. Groundnut bud necrosis virus was named in 1992 after it was discovered through serology that the bud necrosis disease of groundnut was caused by a tospovirus other than TSWV . In the present study, only one sample from dist. Una was confirmed for GBNV by RT-PCR (Fig. 2B); nucleotide sequence of which was submitted to NCBI.
ToCV causes a deadly disease that primarily affects tomatoes, but it has also been seen infecting a variety of other commercially significant vegetables and wild plants . ToCV was first discovered in Florida (USA) in the mid-1990s and since then, it has spread to thirty five nations and territories, making it a typical example of being an emergent plant pathogen . Chlorotic regions are usually polygonal in shape in the early stages of infection, and are confined by main veins. Interveinal yellow regions can produce reddish-brown necrotic specks in advanced stages. Lower leaves are curled, thicker, and brittle, with a crisp texture when touched . In our study, whiteflies were observed in open-tomato fields. Only one sample responded positively in RT-PCR assay. Sequencing the cloned RT-PCR product finally confirmed the presence of ToCV in India (774 bp) (Fig. 2I). To the best of our knowledge, this is the first time that ToCV and CMV/PVY/ ToCV/ToLCNDV mixed infections are being reported from India. ToMV isolate (B10-Asr) shared 99.68% sequence identity at nucleotide level with an isolate of ToMV from Vietnam (MH393621.1).
Besides tomatoes, many other economically significant crops are being attacked by plant viruses, particularly begomoviruses, in Asia, which is one of the reasons causing frustration among small-scale farmers. Despite the fact that farmers are confused by plant viral diseases, only few research articles have addressed this issue, particularly in underdeveloped nations. ToLCNDV-infected tomato plants show typical symptoms such as upward or downward curling and crinkling of leaves, yellow mottling, vein clearing, leaf blistering, and leaf puckering. Plants become stunted with shortened internodes and poor fruit set as the infection progresses, resulting in full crop loss . Leaf curling was noticed to be the most common symptom in infected plants during field surveys, and it was later proven that it was caused by a Begomovirus. Although, in fields, mixed infections involving two or more plant viruses were prevalent, and they could have interacted in a variety of ways, complicating things further. Unrelated viruses that infect the same host cells are known to produce synergistic interactions. The mechanism underlying the synergistic relationships is unknown, but it is possible that a variety of viral and/or host products are involved.
A combined phylogenetic tree was constructed using nucleotide sequences (RNA & DNA) of different isolates of all the nine viral species analysed in the present study along with sequences obtained from NCBI (Fig. 3). Phylogenetic analysis of the viruses studied in the present investigation revealed that different isolates belonging to each specific virus clustered together irrespective of the geographical locations from where they have been detected. The tree consists of two large clades (L1 and L2) each of which is further comprised of two subclades (A and B). In clade L1, the coat protein sequence of Tomato chlorosis virus (TCV) is clustering with replicase gene sequence of Tomato mosaic virus (TMV), which hints toward their common ancestral origin. In clade L2, the coat protein sequences of Potato virus M (PVM) and Potato virus S (PVS) revealed some striking similarity, as these two RNA viruses belong to the same family. Phylogenetic analysis confirmed that CMV isolates (K3 Khanpur and B7 Jodhey) from this study were clustering together and were closely related to an isolate of Vietnam (VN-Bavi) identified from Nicotiana tabacum; Groundnut bud necrosis virus isolate (GNV-In1*; K3 Khanpur) was closely related to an Indian isolate (Tomato/UAS/Dharwad/UHSASB193); two PVY isolates, K8 Khanpur and KP7 Patiala, were clustering together whereas the third PVY isolate (PVY-In3*; B7 Jodhey) was closely related to a Macedonian isolate (KUA7-2013) and another isolate from Pakistan (PK); Tomato chlorosis virus isolate (TCV-In*; K4 Khanpur) was closely related to a South African isolate (ToCR-186); Tomato leaf curl New Delhi virus isolate (TLC-In1*; K12 Khanpur) was closely related to an Indian isolate (India:UP:Bahraich:Tomato1:2011) reported from Bahraich and identified from tomato; and TMV (B10 Jodhey) was closely related to an isolate from Uganda (ToMV-Ug). TMV isolates from India, Uganda, Zimbabwe, Slovakia, South Korea and Kazakhstan were forming one cluster which was further divided into a subcluster comprising TMV sequences from China, Japan, Australia and United States. The results of this study may be useful for further molecular evolutionary research on tomato or potato viruses in India.