Introduction

Capsicum (Capsicum annuum L. var. grossum Sendt) one of the most popular and highly remunerative crops is cultivated in most parts of the world, especially in temperate regions of Central and South America, European countries, tropical and subtropical regions of Asian continent mainly in India and China. India has an average annual production of 0.9 million tons from an area of 0.885 million hectare with a productivity of 1266 kg per hectare which accounts for one fourth of world production of capsicum (Sreedhara et al. 2013).

Capsicum is vulnerable to the attack of various fungal, bacterial and viral pathogens around the world but viral diseases take heavy toll of the crop under open as well as protected cultivation causing both yield and quality losses. About 13 viruses viz., Cucumber mosaic virus (CMV), Potato virus Y (PVY), Pepper mottle virus (PepMoV), Pepper severe mosaic virus (PeSMV), Pepper mild mottle virus (PMMoV), Tomato yellow leaf curl virus (TYLCV), Pepper veinal mottle virus (PVMV), Tomato spotted wilt virus (TSWV), Tobacco mosaic virus (TMV), Pepper yellow mosaic virus (PepYMV), Groundnut ringspot virus (GRSV), Tomato chlorotic spot virus (TCSV), and Pepper ring spot virus (PepRSV) are known to infect capsicum in different parts of the world (Gracia et al. 1968; Kang et al. 1973; Lockhart & Fischer 1974; Villalon 1975; Ong et al. 1980; Wetter et al. 1984; Sharma et al. 1993). Among these, PMMoV, a Tobamovirus is posing threat to capsicum cultivation due to its highly contagious nature and long survival in soil.

PMMoV was first recognized as the latent strain in USA (McKinney 1952), and then in Europe as a distinct strain (Wetter et al. 1984), is now distributed worldwide (Lamb et al. 2001) which includes Australia (Pares 1985), Taiwan (Green & Wu 1991), Egypt (Othmann 1991), Oregan (Hamm et al. 1995), North America (Beczner et al. 1997; Adkins et al. 2001), Japan (Honda & Kameya-Iwaki 1991; Ikegashira et al. 2004), Georgia (Martinez-Ochoa et al. 2003), China (Xiang et al. 1994; Wang et al. 2006), North Africa (Mnari-Hattab & Ezzaier 2006), Panama (Herrera-Vasquez et al. 2009), Brazil (Lima et al. 2011), and Turkey (Caglar et al. 2013). Moreover, Colson et al. (2010) detected PMMoV RNA in the food products, human stools and its association was also correlated with immune responses, fever, abdominal pains and pruritus in humans.

PMMoV, a member of genus Tobamovirus, is a rigid rod shaped virus belonging to family Virgaviridae (King et al. 2011). The virus is highly contagious and like other tobamoviruses also transmitted through seed and soil (Komuro & Iwaki 1969; Lanter et al. 1982; Brunt et al. 1996; Tan et al. 1997; Ikegashira et al. 2004; Toyoda et al. 2004). This virus can survive in crop debris including roots in soil, and contaminated equipments etc. Soil transmission during transplanting or seed transmission is considered to be the primary source of infection (Greenleaf 1986). The decontamination of contaminated field is extremely difficult as long as green pepper is continuously cultivated (Ikegashira et al. 2004). Seed borne infection in commercially available green pepper seeds has been reported by many workers (Tosic et al. 1980; Toyoda et al. 2004). This virus frequently results in significant crop losses or reduction both under field and greenhouse plantings (Jarret et al. 2008). Initially the pathogen causes mild foliar symptoms and many times it remains unnoticed until more evident symptoms appear on fruits resulting high yield losses. So far five pathotypes viz., P0, P1, P12 , P123, and P1234 of PMMoV have been recognized on the basis of their ability to break L gene-mediated resistance (conferred by L 1, L 1a, L 2, L 3, and L 4), allelic forms of the same locus on chromosome 11 (Boukema IW 1984; Sawada et al. 2004). A new resistance gene designated as Hk imparting resistance to Tobamovirus, has been discovered in capsicum by Sawada et al. (2005).

In Himachal Pradesh (HP), Bell pepper commonly known as “Shimla mirch” brings remunerative returns to the farmers and grown on an area of 2,447 ha with production and productivity of 31,810 t and 13.00 t/ha, respectively (Anonymous 2013). In recent years, this crop is becoming very popular as an off-season crop under polyhouse conditions. In HP, India, PMMoV was first intercepted by Sharma & Patiyal (2011) in polyhouse grown capsicum hybrids and the popularization of the polyhouse venture in the state has led to the escalated incidence of the virus. Recent surveys of polyhouses constructed in different districts of HP have shown that PMMoV is the most prevalent and destructive pathogen of polyhouse. In the present study, genome of Indian isolate of PMMoV has been sequenced and its evolutionary relationship with other members of family Virgaviridae has been discussed.

Materials and Methods

Infected capsicum plants exhibiting symptoms like mosaic, mottle, leaf and plant deformations were collected from various regions of HP under polyhouse conditions. The plants were indexed for the presence of different viruses viz., CMV, PepMoV, PMMoV, TMV and Tomato mosaic virus (ToMV) through DAS-ELISA (Bioreba, Switzerland) using polyclonal antibodies (Clark & Adams 1977) and the samples found positive for PMMoV antiserum were maintained on susceptible bell pepper variety “California wonder” through sap inoculation using standard leaf rub method. The virus was mechanically inoculated on California wonder, Datura stramonium and Nicotiana glutinosa plants at 3-4 leaf stage. The disease symptoms were studied on the artificially inoculated plants. Virus preparations were further subjected to transmission electron microscopy using leaf dip method described by Brandes (1964). The identity of the virus as PMMoV was confirmed by RT-PCR using coat protein gene (CP gene) specific primers (F5’CCAATGGCTGACAGATTACG-3’ and R5’CAACGACAACCCTTCGATTT-3’) and finally one isolate HP-1 was used for full genome sequence determination.

Total RNA was isolated from 100 mg of infected plant leaves using Trizol as per manufacturer instructions (Life Tech). The integrity of total RNA was analyzed on 1 per cent denaturing gel. Leaves from uninoculated healthy plants exhibiting negative reaction with PMMoV antiserum in DAS-ELISA were used as negative control. For RT-PCR, cDNA was synthesized using 1 μg of total RNA and oligodT(10-18) primer in 10 μl reaction using MMuLV reverse transcriptase (USB) as per manufacturer instructions. RT-PCR amplification based on CP region was carried out in 25 μl reaction volume using 2.5 μl of 10x Taq buffer, 1 μl of 25 MgCl2, 2.5 μl of 2mM dNTPs mix each, 1μl of 10 μM each CP gene specific primer, 0.2 μl of 5U/μl Taq polymerase (Banglore Genei). The final volume was adjusted using nuclease free water. The amplification was performed in GeneAmp PCR system 9700 (Applied Biosystems) with initial denaturation of 94°C for 4 min followed by 35 cycles of 94°C for 15 sec, 48°C for 40 sec and 72°C for 1 min with a final extension of 7 min at 72°C. PCR products were electrophoresed on 1.2% agarose gel stained with ethidium bromide and visualized in gel documentation system (Alpha Innotech, USA).

For complete sequencing of the genome, ten primer pairs were designed based on full genome sequence available in NCBI database (www.ncbi.nlm.nih.gov) using primer3 software (Table S1). For RT-PCR, the reaction was performed in 50 μl volume using master mix (Phusion High-Fidelity PCR NEB), containing 1 μl of 10 μM each gene specific primer, 2.5 μl of cDNA. The final volume was adjusted with nuclease free water. Amplification was performed in GeneAmp PCR system 9700 (Applied Biosystems) with initial denaturation of 94°C for 4 min followed by 35 cycles of 94°C for 15 sec, annealing at 45°C for 40 sec and extension at 72°C for 1 min with a final extension of 7 min at 72°C.

For the determination of complete sequencing of 5’ and 3’ ends, RACE-PCR was carried out using SMART-RACE based method as per the manufacturer instructions (Clontech). The amplicons were cloned into pGEM-T Easy vector system (Promega) and both the strands of recombinant plasmids were custom sequenced. The complete nucleotide sequence of each amplicon was deduced and BLAST analyzed (www.ncbi.nlm.nih.gov/blast). The sequences were also checked manually using bioEdit software (Altschul et al. 1997). The different fragments were aligned together and complete genome sequence was submitted to NCBI GenBank vide accession number KJ631123. The multiple alignment of the present isolate was done using ClustalW (www.ebi.ac.uk/Tools/msa/clustalw2/) and phylogenetic trees were constructed using MEGA5 program (Tamura et al. 2011) using the Neighbor-Joining method.

Results

The appearance of symptoms after sap inoculation indicated the viral nature of the pathogen. The inoculated plants showed mild mottling after about 8-10 days of inoculation followed by leaf deformation, yellow/ green mosaic and chlorosis of the leaves (Fig. 1). However, the symptoms were more prominent on young leaves. The infected plants remained stunted and developed small, distorted and lumpy fruits with colour variations. The virus produced chlorotic local lesions on Datura stramonium and Nicotiana glutinosa.

Fig. 1
figure 1

Symptoms produced by PMMoV-HP1 isolate on healthy susceptible plants of “California Wonder” upon artificially inoculation. 1: mild mosaic, 2: leaf puckering, mild mottling and mosaic, 3: leaf deformation, 4: lumpy and deformed fruit

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The identity of the virus as PMMoV was confirmed by DAS-ELISA using polyclonal antibodies ELISA kits, where a clear reaction was obtained with the PMMoV antiserum against controls. Electron microscopy of the symptomatic leaf tissue using crude sap extracts from infected leaves of “California wonder” showed the presence of short rigid rod shaped virus particles measuring about 300 nm in length similar to Tobamovirus particles. In RT-PCR, amplicon of ~730 bp corresponding to CP (Fig. 2) were obtained in the infected samples while no amplification was observed in healthy plants.

Fig. 2
figure 2

RT-PCR based confirmation of PMMoV using primer pair specific to coat protein region. M: 100bp DNA ladder, Lane 1-5: represents ampilcons of ~730 bp in infected plants, Lane 6 -7: represents healthy plants of capsicum

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PCR amplification of various genome regions of PMMoV-HP1 isolate (genbank accession number KJ631123) using virus specific primers (Table S1) revealed that the viral genome consist of 6356 nucleotides (nt) comprising of 5’ UTR, coding sequences and 3’ UTR (Fig 3). The 5’ UTR of the virus positioned upstream of the first open reading frame (ORF) is comprised of 69 nt and 3’ UTR consists of 198 nt (6159-6356). The coding region contains four long ORFs typical of tobamoviruses. The first ORF (126K protein) is between nt 70-3423, terminates with an amber read through codon, so that ORF2 (183K protein) finishes at nt 4908. The ORF3 encodes cell to cell movement protein gene (28K protein), consists of 774 nt starting at nt position 4909 without any nucleotide insertion after the stop codon of ORF2 and finishes at nt 5682. The CP gene of 17.2K protein size (ORF4) starts at nt 5685, two nucleotides after the stop codon of ORF3 and finishes at nt 6158. The CP gene codes for 157 amino acids.

Fig. 3
figure 3

A consensus PMMoV- HP1 genome map with primers positions used for full genome sequencing (A1) and A2 represents PCR based amplification of PMMoV genome using RT-PCR (A) and RACE PCR (B&C) respectively. M1: 100 bp DNA ladder, Lane 1-10: Represents RT-PCR based amplification of different regions corresponding to the genome of PMMoV, B & C: Represents the 5’ UTR and 3’ UTR amplification using RACE PCR, respectively

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The phylogenetic tree constructed by using MEGA5 software categorised the present isolate along with other members of the family Virgaviridae (Fig 4). Two major groups formed in this analysis, first comprised of all members of the family Virgaviridae except Passion fruit mosaic virus (PafMV) which constituted the second group. The group A was further divided into two clusters A1 and A2 accommodating Brugmansia mild mottle virus (BruMMV), Cactus mild mottle virus (CMMoV), Cucumber fruit mottle mosaic virus (CFMMV), Cucumber green mottle mosaic virus (CGMMV), Kyuri green mottle mosaic virus (KGMMV), Obuda pepper virus (ObPV), Odontoglossum ringspot virus (ORSV), Paprika mild mottle virus (PaMMV), ToMV, TMV, Tobacco mild green mosaic virus (TMGMV), Turnip vein-clearing virus (TVCV) and Zucchini green mottle mosaic virus (ZGMMV), respectively. The present isolate was grouped in the first sub cluster in the neighbourhood of ToMV, TMV, PaMMV, ObPV, BruMMV, TMGMV, ORSV, TVCV whereas the viruses viz., CGMMV, CFMMV, KGMMV and ZGMMV were placed in the second sub cluster.

Fig. 4
figure 4

Phylogenetic tree showing relationship of PMMoV-HP1 with other viruses of family Virgaviridae at nucleotide level using MEGA5 software

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The complete nucleotide sequence similarity between PMMoV-HP1 isolate and other PMMoV isolates ranged between 94-99 per cent (Table 1). Pair wise comparison of PMMoV-HP1 isolate with all 11 complete PMMoV genomes available in public databases revealed that the per cent identity of the present isolate was maximum with PMMoV-J (99.28%) with 46 nt differences distributed throughout the genome and minimum with PMMoV-Ia (94.15%) indicating its possible evolution with the Japanese isolate. There was a difference of only 11 amino acids (aa) in the coding regions again showing maximum variation in regions except CP which is most important for differentiation of PMMoV pathotypes due to its ability to overcome the L gene mediated resistance in capsicum.

Table 1 Pairwise similarity index of PMMoV-HP1 complete nucleotide sequence with other PMMoV isolates
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The phylogenetic tree constructed, grouped all PMMoV isolates into two main clusters placing PMMoV-HP1 isolate along with ten isolates identified into different pathotypes whereas only one isolate PMMoV-Ia was placed in the second group (Fig 5). The present isolate was placed in the first sub cluster in the neighbourhood of PMMoV-J, PMMoV-C1421, PMMoV-Pa18, PMMoV-TPO-2-19, PMMoV-S, PMMoV-CN, and PMMoV- BRDF01 whereas three isolates viz., PMMoV-Iw, PMMoV-L4BV, PMMoV-Kr were placed together in the second sub cluster. In this analysis we found that isolates representing different countries (PMMoV-S, PMMoV-CN, PMMoV- BRDF01) were placed in separate clades except Japanese isolates. The mutations in the CP gene of the tobamoviruses are advocated for the breakdown of the L gene mediated resistance and the PMMoV pathotypes have been differentiated on the basis of variations in aa sequence. In an attempt to place the present isolate in a given pathotype group, CP aa sequence of the test isolate was compared with known pathotypes. Pair wise comparison of the CP aa sequences (Table 2) revealed 100% similarity of the present isolate with pathotype PMMoV P12.

Fig. 5
figure 5

Phylogenetic analysis based on full genome sequence of PMMoV- HP1 (KJ631123) and other PMMoV isolates using Mega5 software

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Table 2 Pairwise similarity index of PMMoV-HP1 coat protein with other PMMoV isolates representing pathotypes P1234, P123 and P12
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Discussion

The symptomatology and transmission of the present isolate confirmed the viral etiology of the disease and its serological relationship with PMMoV antiserum established its identity as PMMoV. The electron microscopy of the virus preparations revealed the association of short rigid rod shaped particles, typical of Tobamovirus. The identity of the virus was further confirmed by RT-PCR using CP gene specific primers. The symptoms of the present isolate resembled with PMMoV symptoms described by different workers across the world (Wetter et al. 1984; Beczner et al. 1997; Antignus et al. 2008; Sevik 2011).The results pertaining to identification of test virus on the basis of DAS-ELISA and RT-PCR using the CP specific primers were in conformity with Wetter et al. (1984); Beczner et al. (1997); Kalman et al. (2001); Ikegashira et al. (2004); Mnari-Hattab & Ezzaier (2006); Herrera-Vasquez et al. (2009); Lima et al. (2011); Caglar et al. (2013); Zheng et al. (2013); Rong et al. (2013).

The genome of PMMoV-HP1 isolate comprised of 6356 nt having four ORFs typical of tobamoviruses and other PMMoV reported from other parts of the world (Alonso et al. 1991; Kirita et al. 1997; Velasco et al. 2002; Hagiwara et al. 2002; Yoon et al. 2005; Ichiki et al. 2005; Wang et al. 2006; Genda et al. 2007; Hamada et al. 2007; Oliveira et al. 2010). The present isolate shared maximum identity with PMMoV-J (Japenese) isolate and its comparison with the present isolate revealed 46 single nucleotide mutations where 37 were the transitions ( 21 out of 37 between pyrimidines C-T and 16 between purines A-G) followed by 10 transversion mutations ( 3 G-T, 6 A-T and 1 A-C). These mutations were non-silent and resulted in 11 amino acid differences: seven in the 126/183 KDa protein, three in the 28 KDa movement protein. There was no difference in 17.2 KDa coat protein. The maximum identity i.e. more than 99 per cent between the Japanese and the present isolate suggests that the two are derived from same origin and might have entered India through import of infected seed. Capsicum annuum L was introduced to Spain during fifteenth century, from where its cultivation spread rapidly to Europe. California wonder is the representative cultivar of Capsicum annuum L. cultivated across the world. This cultivar was introduced in Japan in mid twentieth century from America (Kirita et al. 1997) whereas, in India, it was introduced from Brazil by Portuguese. The path of Capsicum annuum L. might explain the long stable journey of PMMoV from Japan to India. The capsicum seed in India is imported from different countries like China, Japan, Netherlands, USA and Euorpe and the seed import might have facilitated the introduction of present isolate in India. This could be the reason for the placement of the present isolate in one cluster along with the representative isolates of Brazil, China, Japan and Spain. However, this aspect still needs further confirmation by involving more number of isolates

The results of phylogenetic analysis were similar to that obtained by Oliveira et al. (2010) where like the present isolate, PMMoV-BRDF01 found to be clustered with one Spanish (PMMoV-S), four Japanese (PMMoV-J, PMMoV-C1421, PMMoV-Pa18, PMMoV-TPO-2-19) and one Chinese isolate (PMMoV-CN). It has been reported that the ability of the tobamoviruses to overcome L gene-mediated resistance (L 1, L 1a, L 2, L 3 and L 4) in capsicum determines their pathogenic behaviour (P0, P1, P12, P123 and P1234) and the most important component of tobamoviruses responsible for this is the CP gene. The Tobamovirus CP is the elicitor of L gene-mediated resistance and act as a determinate in assigning a viral pathotype (Berzal-Herranz et al. 1995; Tsuda et al. 1998; Dardick et al. 1999; Hamada et al. 2002; Genda et al. 2007). It has been demonstrated that mutations in the CP gene are associated with breakdown of resistance inferred by the L gene (Tsuda et al. 1998; Gilardi et al. 1998, 2004; Hamada et al. 2002, 2007; Genda et al. 2007). Thus, based on CP gene homology, the present isolate has been tentatively identified as pathotype PMMoV P12 as aa sequence of the CP region showed 100 per cent similarity with that of pathotypes PMMoV-P12 reported by Alonso et al. (1991), Kirita et al. (1997), Hagiwara et al. (2002), Ichiki et al. (2005), Yoon et al. (2005), Wang et al. (2006), Oliveira et al. (2010). However, authentic validation as pathotype P12 requires phenotyping on the differential hosts (L alleles). In conclusion, this study constitutes the first report on characterization of any PMMoV isolate from India and its relationship with particular pathotype. The information generated in this study will facilitate the work on identification of effective sources of resistance in the capsicum germplasm and its use in breeding for resistance as no information is available on any aspect of this virus from India at least.