Co-existence of chlorosis inducing strain of Cucumber mosaic virus with tospoviruses on hot pepper (Capsicum annuum) in India

Cucumo- and tospoviruses are the most destructive viruses infecting hot pepper (chilli). A diagnostic survey was conducted to assess the prevalence of cucumo and tospoviruses in chilli growing tracts of Tamil Nadu. Infected plants showing mosaic with chlorotic and necrotic rings, veinal necrosis, mosaic mottling, leaf filiformity and malformation were collected. Molecular indexing carried out through reverse transcription polymerase chain reaction (RT-PCR) with coat protein gene specific primer of Cucumber mosaic virus (CMV) and tospovirus degenerate primer corresponding to the L segment (RdRp). Ostensibly, amplifications were observed for both CMV and tospoviruses as sole as well for mixed infections. The sequence analysis indicated that the Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) to be involved with CMV in causing combined infections. The co-infection of CMV with CaCV was detected in 10.41% of the symptomatic plant samples and combined infection of CMV with GBNV was recorded in around 6.25% of the symptomatic plants surveyed. The amino acid substitution of Ser129 over conserved Pro129 in coat protein of CMV implies that CMV strain involved in mixed infection as chlorosis inducing strain. Further, the electron microscopy of symptomatic plant samples explicated the presence of isometric particles of CMV and quasi spherical particles of tospoviruses. This is the first molecular evidence for the natural co-existence of chlorosis inducing CMV strain with CaCV and GBNV on hot pepper in India.

www.nature.com/scientificreports/ hews the evolutionary dynamic of viral populations 26 . Unraveling the viruses concerned with mixed infections is vital for better understanding of the ecology and evolution of viral diseases which are essential for viral disease prevention and formulating management strategies 25 . The prime objective of the present study is to explore the mixed infections of CMV with tospoviruses in chilli under natural field conditions of Tamil Nadu in India.

Identification of viruses in the infected plants.
A total of 26 chilli samples of severe mosaic, mosaic mottling, leaf malformation and filiformity; 13 samples with concentric chlorotic ring spots and 9 samples with veinal necrosis and necrotic lesions were collected (Fig. 1). Of these, CMV was detected in 23 samples (47.91%); CaCV was recorded in 11 samples (22.91%) and GBNV in 5 samples (10.41%). The co-infection of CMV with CaCV was detected in 5 samples (10.41%) and CMV with GBNV was recorded in 3 samples (6.25%) (Fig. 2). The symptomatic leaf samples of mosaic mottling along with concentric chlorotic ring spots and mosaic with veinal necrosis were suspected for mixed infection based on symptomology. The symptomatic leaf samples were subjected to RT-PCR analysis with CMV coat protein gene specific primer and tospovirus degenerate primer pairs. The coat protein gene fragment of CMV and replicase protein (RdRP) fragment of tospovirus were amplified with an amplicon size of around 657 bp and 850 bp, respectively ( Supplementary Fig. 1 . 3a) which turned to necrotic lesions at 3-4 dpi (Fig. 3b). After 5 dpi, it exhibited veinal necrosis (Fig. 3c) followed by stem necrosis symptom (7 dpi) on inoculated plants (Fig. 3d). In contemplate, the symptomatic leaves started to wither after 9 dpi and the newly emerging leaves were observed to be having the systemic symptom of mosaic mottling followed by leaf filiformity after 10-12 dpi (Fig. 3e). The sap inoculated Nicotiana plants produced localized chlorotic lesions on 2-3 dpi (Fig. 3f) which turned into necrosis at 5 dpi (Fig. 3g). After 7 dpi, the systemic mosaic and necrosis were observed on newly emerged leaves (Fig. 3h,i). Furthermore, the co-existence of CMV with tospovirus in the inoculated plant samples was confirmed by RT-PCR using nucleocapsid gene specific primers of CaCV ( Supplementary Fig. 2a) and GBNV ( Supplementary Fig. 2b). Indeed, inoculated plant samples after 12 dpi were positively amplified for both CMV and tospovirus.

Discussions
Several epidemiological studies depicts that single host plant affected by more than one virus implies the occurrence of mixed virus infection. In general, mixed viral infections are more common 21 . Tospoviruses (CaCV and GBNV) infected chilli plants showed concentric rings on mature leaves and chlorotic spots on younger leaves 12 . Indeed, CMV infected chilli plants showed mosaic mottling, puckering, shoe string or filiformity and stunted growth 26,27 . Based on symptomology, symptomatic leaf samples of mosaic mottling along with concentric chlorotic ring spots and mosaic with veinal necrosis were suspected for mixed infection. Since, symptomology based detection would not be reliable and it has to be further confirmed and validated either by serological or by molecular method of detection 12 . RT-PCR based method will be the reliable to underpin the mixed infection and to index the associated viruses. It is more accurate to detect the viruses even at very low titer in the infected leaf samples than serology based assays 28 . From the analysis, it is clearly demonstrated that the tospoviruses associated with CMV were identified as Groundnut bud necrosis virus and Capsicum chlorosis virus. The symptomatological study depicts that GBNV and CaCV produces similar symptoms on chilli plant and it is very difficult to distinguish them based on symptomology. The sap inoculation of tospovirus (GBNV and CaCV) chilli isolate on Nicotiana and cowpea host plants produced localized chlorotic lesions followed by necrotic lesions at 4-5 dpi 12,29 . The CaCV certainly induces chlorotic spots initially and later developed necrosis around the central spot 8 . Intrinsically, mechanically inoculated plant samples after 12 dpi were positively amplified for both CMV and tospovirus in RT-PCR. Electron microscopic visualization and evaluation helps to study the particle morphology for detection of mixed viral infections. Substantially, diagnosis of viral diseases by TEM utters rapid and accurate identification www.nature.com/scientificreports/ towards mixed infection 30 . Hence, electron microscopic examination of infected plant samples corroborated the co-existence of isometric cored particle of CMV with quasi spherical particle of tospovirus. The results have been substantiated with previous findings 13, [31][32][33] . The nucleotide sequences of coat protein of CMV indicates that CMV isolate belong to subgroup IB. Based on serology and nucleic acid hybridization, CMV isolates are classified into subgroup I and II and further CMV subgroup I divided into IA and IB based on coat protein gene sequence and phylogenetic analysis 34 . Coat protein of CMV comprised of 657 nucleotides putatively translated into 219 amino acids. CMV can be categorized as chlorosis inducing strain (Ser 129 or Leu 129 ) and mosaic inducing strain (Pro 129 ) based on the position of 129 th amino acid of coat protein gene 35 . Certainly, amino acid 129 of coat protein is a genetic determinant responsible for symptom induction in host plant and in certain cases it act as virulence determinant 36 . Amino acid substitution of Ser 129 over conserved Pro 129 induces chlorosis in the host rather necrosis by disrupting the functions of chloroplast 37 . More likely, cholorosis induction in the infected plant determined by structural alteration in coat protein of CMV incited by conserved (pro 129 ) amino acid substitution 38 . Comparative amino acid sequence alignment vindicated CMV isolates associated with mixed infection is a chlorosis inducing strain (substituted with Ser 129 ). Whereas, CMV causing single infection are prevalently mosaic inducing strains (possess Pro 129 ). Increased flexibility of βE-αEF loop (129-136 amino acids) of coat protein has direct correlation with pathogenesis of virus. Moreover, flexibility of βE-αEF loop is regulated by 129 th amino acid properties 39 . In general, frequent reassortment and recombination occurs in segmented viruses over monopartite viruses 40 . Amicably, CMV undergoes prompt of genetic changes through reasssortment and recombination 27,41,42 . Thus, evolutionary mechanism is a characteristic attribution of multipartite viruses which plays significant role in emergence and interspecies transmission of viruses 43 . Any single variation in amino acid or nucleotides may effect on stability and infectivity of virus. Hence, thus apparent variation observed on multipartite TN CMV isolate suspected to be associated with co-adoption or co-existence of CMV with other viruses. Over all the accumulating evidences  www.nature.com/scientificreports/ suggests that amino acid 129 (chlorosis induction) besides symptom determinant may also have correlation with co-existence of CMV with tospoviruses.
Mixed virus infections will intensify the disease dynamic in host plants comparatively than single virus infection. In some cases, symptom induced by one virus can be masked by other virus in combined infections. Mixed infection in host plants will results in maintaining the genetic diversity of viruses. Thus may inflict to the emergence of novel genetic phenotypes or strains of virus. It is very crucial to study the significant role of mixed infection in altering genetic diversity of viral population. In the present study, we explicated the co-existence of Cucumber mosaic virus with Capsicum chlorosis virus and Groundnut bud necrosis virus in chilli under natural field conditions of Tamil Nadu in India. Indeed, CMV, GBNV and CaCV are the most notorious and economically important viruses reported to be causing mosaic and necrosis on most of vegetable crops. Hence, combined infection of these destructive viruses may menace to chilli cultivation. Moreover, frequency of occurrence of mixed infection of mosaic and necrosis disease in chlli becoming increasingly diverse and alarming currently in chilli growing areas of Tamil Nadu. To our knowledge, this is the first report for combined infection of cucumo-and tospoviruses in chilli. This preliminary study sought to help in further future studies on combined infections of CMV with other plant viruses. Although, the mechanism behind the interaction of viruses between different groups on a single host during mixed infections is yet to be studied in detail.   www.nature.com/scientificreports/ Subsequently, 2 µl of purified virus sample coated into grid kept for 3-5 min and then, washed thrice with sterile water. Consequently, virus particles have been stained with 2% phospho tungstic acid. The excess stain has been wiped off from edges of grid with filter paper and allowed for air dry. Eventually, grid was observed under TEM with EDAX (FEI, Technai) at 70,000-90,000 magnification and documented.

RNA extraction and RT-PCR analysis.
Total RNA was extracted from the collected symptomatic leaf samples using TRIzol reagent 45 . The first stand complementary DNA (cDNA) was synthesized from the extracted RNA to detect the viruses associated with infections. For the cDNA synthesis of CMV, 20 µl reaction mixture (9 µl-sterile water; 4 µl-5× reaction buffer; 2 µl-dNTPs; 1 µl-random primer; 1 µl-reverse transcriptase; 1 µl-RNase inhibitor; 1 µg-total RNA) was used (RevertAid, Fermentas, India) and incubated at 42 °C for 60 min followed by 70 °C for 5 min 46 . For tospovirus, the reaction mixture contains total RNA, random primer and nuclease free water up to 12.5 µl were incubated at 65 °C for 5 min followed by 5× reaction buffer, 10 mM dNTP mix, RNase inhibitor and reverse transcriptase were added and incubated for 1 h at 42 °C 47 . The presence of CMV was confirmed through RT-PCR with CMV coat protein gene specific primer (RsCMV-F and RsCMV-R) with a cyclic condition of 94 °C for 2 min followed by 94 °C for 30 s of denaturation, 59 °C for 30 s of annealing, 72 °C for 60 s with final extension of 72 °C for 10 min 48 . The tospovirus was confirmed through RT-PCR using tospovirus universal primer (gL3637 and gL4435C) corresponding to L segment (RdRp) with the cycling condition of 94 °C for 5 min followed by 35 cycles of 94 °C for 30 s, 56 °C for 1 min and 72 °C for 1 min with 72 °C for 10 min of final extension 44,49 . Cloning and sequence analysis. The positive amplicons of CMV (coat protein) and tospoviruses (replicase protein) were purified using GenJET PCR purification kit (Themo scientific Inc.) and cloned into pGEM-T easy vector (Promega). Two independent clones were sequenced at both orientations with M/s Barcode Biosciences, Bangalore. Data base searches performed with NCBI BLAST (http:// blast. ncbi. nlm. nih. gov) and comparative amino acid and nucleotide sequence analysis carried out using Clustal W (www. ebi. ac. uk). Sequences were aligned using Bio-Edit Sequence Editor program 7.2. The phylogenetic relationships among the isolates were analyzed using MEGA 7.0 software (www. megas oftwa re. net) by Neighbor joining tree method with 1000 bootstrap replication 12,50 .