Introduction

Tospoviruses (order Bunyavirales, family Tospoviridae, genus Orthotospovirus) (Adams et al. 2017), cause serious diseases in a variety of economically important crops around the world (Ullman et al. 1993; Lovato et al. 2004; Pappu et al. 2009; Riley et al. 2011). These viruses are transmitted in a propagative manner by a limited number of thrips species (family Thripidae) (Ullman et al. 1993; Riley et al. 2011; Rotenberg et al. 2015). Virus particles are quasi-spherical, 80–110 nm in diameter (Whitfield et al. 2005; Oliver and Whitfield 2016). The genome is composed of three RNA segment of different lengths denoted as S, M and L. The L RNA has a negative sense while S and M RNAs are ambisense (Plyusnin et al. 2012). L RNA encodes an RNA-dependent RNA polymerase (RdRp), for virus replication (de Haan et al. 1991). The viral sense of M RNA encodes a nonstructural protein (NSM) which is involved in cell-to-cell movement (Kormelink et al. 1994) while the complementary strand encodes a precursor of the structural glycoprotein involved in transmission by thrips (Kikkert et al. 2001).

The viral sense of S RNA encodes a nonstructural protein (NSS) which is associated with viral suppression of gene silencing (Takeda et al. 2002) and transmission by its vector (Margaria et al. 2014), while the complementary sense codes for the nucleocapsid protein (N) (de Haan et al. 1990). The classification of tospoviruses is based on N protein serology, thrips specificity and more importantly, amino acid identity of the N protein (Plyusnin et al. 2012). Based on the latter, tospovirus species are divided into six distinct phylogenetic clades i.e. American 1, American 2, Eurasian, Asian 1, Asian 2 and lisianthus necrotic ringspot virus (LNRV). To date, at least 29 species in the genus Orthotospovirus have been reported, of which 11 are formally recognized as species, and 18 as tentative species (Hassani-Mehraban et al. 2016).

So far, five tospovirus species including Tomato spotted wilt virus (TSWV), Impatient necrotic spot virus (INSV), Groundnut bud necrosis virus (GBNV), Tomato yellow ring virus (TYRV) and Iris yellow spot virus (IYSV) have been reported in Iran from important crops such as tomato, potato, soybean, onion, alstroemeria and pepper (Bananej et al. 1998; Moini and Izadpanah 2001; Golnaraghi et al. 2002; Shahraeen et al. 2002; Ghotbi et al. 2005; Hassani-Mehraban et al. 2005).

Capsicum chlorosis virus (CaCV), a virus transmitted by three thrips species including Thrips palmi, Frankliniella schultzei and Ceratothripoide sclaratris (Premachandra et al. 2005; Persley et al. 2006), was first reported in tomato and pepper from Australia (McMichael et al. 2002), then in tomato in Thailand (Premachandra et al. 2005). The gloxinia isolate (HT-1) was characterized and reported from the USA (Hsu et al. 2000) and later recognized as an isolate of CaCV. In recent years, CaCV has been reported from new countries including Taiwan (Chen et al. 2007a), China (Chen et al. 2007b) and India (Kunkalikar et al. 2007) infecting tomato, pepper, peanut, amaryllis, blood lily, Amaranthus sp., Phalaneopsis orchid and calla lily (Chen et al. 2007a; Chen et al. 2009; Kunkalikar et al. 2010; Zheng et al. 2011; Sharma and Kulshrestha 2014). CaCV isolates have shown differences in host range (Chiemsombat et al. 2008) and genome sequence (Gamage et al. 2015).

During 2014, a survey was conducted for tospovirus infection in the central regions of Iran. Rudbeckia sp. showing chlorotic ring spot symptoms resembling tospovirus infection along with the presence of high population of Thrips tabaci were observed in Mahallat, (Markazi province, Iran) and samples were collected. In the present study, we have identified and analyzed the first CaCV isolate from Iran and the Middle East based on sequence data and host range studies.

Materials and methods

Serological detection

DAS-ELISA (Clark and Adams 1977) was used to test for the possible presence of the five tospoviruses previously reported in Iran from symptomatic rudbeckia plants. Polyclonal antisera against TSWV, INSV, GBNV, IYSV and TYRV (1:1000 dilutions) were kindly provided as gift by Dr. Dick Peters, Wageningen University, The Netherlands. Absorbance values were read at 405 nm using an ELx800 ELISA Reader (Bio-Tek Instruments, USA).

Virus source and host range

Leaves of Black-eyed Susan plants (Rudbeckia sp.; family Asteraceae) showing chlorotic ring spot and concentric oval patterns were collected in 2014 at the ornamental plant research center in Mahallat, Iran. Sap extracted from symptomatic plants was mechanically inoculated onto Vigna unguiculata using 0.01 M potassium phosphate buffer with 0.1% sodium sulphite. The host range of the virus was determined by inoculating 14 plant species using five plants of each species. The plants were kept in the greenhouse and monitored for symptom expression daily during a period of four weeks.

RNA extraction and RT-PCR

Total RNA was extracted from infected N. benthamiana leaf tissue by RNX Plus kit (CinnaGen, Iran) according to manufacturer’s instruction. Reverse transcription (RT) was carried out using Hyperscript RT (Gene All, Seoul, Korea) and two primers specific for the Asian, Eurasian (AS-EA-F) and American 1 (AM1-F) tospoviruses (Table 1), complementary to the eight conserved terminal nucleotides of the N gene 5′-untranslated terminal region (5’-UTR) of all tospoviruses (Hassani-Mehraban et al. 2016). EA-R, AS1-R and AM1-R primer sets (Table 1) designed on the conserved regions of the nucleocapsid gene of Eurasian, Asian 1 and American 1 tospoviruses, respectively (Hassani-Mehraban et al. 2016), were used with AS-EA-F and AM1-F primers to synthesize first cDNA strand by RT-PCR to obtain the corresponding 5’-UTR and partial N gene of Eurasian, Asian 1 and American tospoviruses, respectively. A degenerate primer set i.e. gL2740 and gL3920c (Chen et al. 2012) was used to amplify by RT-PCR a part of the L segment. The entire S RNA sequence consisting of 5′- and 3’-UTRs, N gene, intergenic region (IGR) and NSs gene was obtained with RT-PCR in combination with primer UHP and newly designed primers either from initial amplicon and/or available sequences from GenBank. Additionally, a primer set specific to the NSM ORF of CaCV was designed (Table 1). All PCR amplicons were cloned into pTZ57R/T (Fermentas, Lithuania) and two independent clones were sequenced (Macrogen, Republic of Korea). The assembled full-length sequences of S RNA, NSM gene and partial L gene were deposited in GenBank with accession numbers KX757228, KX757229 and KY319058, respectively.

Table 1 List of primers used for RT-PCR in this study
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Sequence analysis

Sequences were analyzed using BLAST with sequences available in GenBank and DNASTAR software 5 (DNAStar, USA). Data from CLUSTAL W sequence alignment were used as input for the construction of phylogenetic trees using the neighbor-joining method and bootstrap analysis with 1000 replications with MEGA 6 software (Tamura et al. 2013). The percentages of sequence identity between tospovirus isolates were calculated with BioEdit editor 7.2.5 (Tom Hall, Ibis Biosciences, USA).

Results and discussion

Symptomatic Black-eyed Susan (Rudbeckia sp.) leaf samples reacted positively only with the GBNV antiserum in DAS-ELISA. These ELISA results showed presence of either GBNV or a related serogroup member in the infected samples. Polyclonal antisera against nucleocapsid protein of serogroup IV members (WSMoV) may cross-react with one another, resulting in possible misidentification of the virus species. This situation has been observed within AM-1 (TSWV) and Eurasian (IYSV) serogroups (Hassani-Mehraban et al. 2016; Bald-Blume et al. 2017). Hence, serological diagnosis of tospovirus species may have drawbacks.

Sap from the symptomatic plants was mechanically inoculated onto Vigna unguiculata inducing local lesions, one of which was used for subinoculation of Nicotiana benthamiana and Capsicum annuum plants. The virus propagated and kept in these plants was designated RM.

RT-PCR was used to identify the RM isolate. No amplicon was produced using either Eurasian or American 1 primer sets (Table 1) from ELISA-positive samples. However, a fragment about 360 bp in size was amplified from mechanically infected N. benthamiana plants with RM isolate using the Asian 1 primer set. As expected, this product contained the 5’-UTR and partial N gene sequence of Asian 1 members. BLAST analysis of this sequence revealed 88–97% and 91–99% identity with comparable sequences of CaCV isolates at the nucleotide (nt) and amino acid (aa) levels, respectively. Therefore, the virus isolate was designated CaCV-RM.

The complete S RNA sequence of CaCV-RM consisted of 3648 nt containing 5′- and 3′- UTRs 66 and 67 nt long, respectively, and shared 72.4–100% nt sequence identity with other CaCV isolates. Comparison of the entire S RNA sequence of CaCV-RM with those of some other CaCV isolates showed 69.8 to 88.2% nt sequence identity (Table 2). The NSS ORF contained a 1320 nt sequence, starting at the AUG codon at position 67 and terminating with an UAA codon at position 1384–1386 nt that coded for a protein with a predicted molecular mass of 49.75 kDa. The viral complementary ORF coding for the N protein started with an AUG at nucleotide position 3581 and terminated with an UGA stop codon at nucleotide position 2754–2756. The N protein was 275 aa in size with a predicted molecular mass of 30.72 kDa. The non-coding IGR run from nucleotide positions 1387 to 2753 with an AU-rich content of 78.13% formed a hairpin structure.

Table 2 Comparison of S RNA and NSM gene and deduced protein of CaCV-RM with those of tospovirus species of the Asian clade 1 (As-C1)
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The NSS ORF of CaCV-RM shared 88.6–95.4% aa sequence identity with that of different CaCV isolates. The phylogenic analysis based on this protein showed that the Iranian CaCV isolate had the highest identity with the HT-1 isolate from the USA. However the aa sequence identity of the AIT (Thailand) and CP (China) isolates with CaCV-RM was less than 89.9 and 88.6% respectively. These isolates grouped in two separate branches. All three conserved motifs within NSS protein including Walker A (GXXXXGKT), Walker B (DEXX), and YL (Lokesh et al. 2010; Zhai et al. 2014) were present in CaCV-RM (data not shown).

The N ORF showed 92.3–97.8% aa sequence identity with other CaCV isolates. N protein sequence analysis showed that CaCV-RM belongs to the WSMoV (Asian 1) clade that comprises isolates from Australia (isolate Qld-3432) (Gamage et al. 2015), Taiwan (isolate Ph) (Zheng et al. 2008), India (isolate Ch-Pan) (Kunkalikar et al. 2010), and USA (Gloxinia HT) (Hsu et al. 2000) (Table 2; Fig.1a).

Fig. 1
figure 1

Phylogenetic relationship of CaCV-RM NSS, N and NSM predicted proteins with those of Asian Clade 1 (AS-C1) tospovirus species. Multiple sequence alignments were used as input to construct phylogenetic tree based on neighbor-joining algorithm with 1000 bootstrap replicates using MEGA 6 software. Bootstrap values less than 50% are not shown in the final phylogenetic trees. Corresponding TSWV sequences were used as outgroups. Sequence sources are mentioned in Table 2

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The most variable region in the S RNA sequence was the intergenic region (IGR), which showed the closest similarity (80.5%) to that of CaCV-Taiwan (Table 2). High variations in length and sequence of IGRs have been observed within different isolates of same species (Rao et al. 2013; Meng et al. 2015; Zhang et al. 2016) and variation in IGR and host range have been reported among TYRV isolates (Hassani-Mehraban et al. 2007).

The NSM of CaCV-RM shared 89.9–94.8% aa sequence identity with that of other CaCV isolates (Table 2). The conserved motifs of the 30 K superfamily, such as the D-motif and G-residue, occur in the NSM protein CaCV-RM and other CaCV isolates and also in those of members of the watermelon silver mottle virus (WSMoV) serogroup, while the phospholipase A2 (PLA2) catalytic site and P/D-L-X motif present in NSM of some TSWV serogroup members (Silva et al. 2001; Li et al. 2009), were absent in all WSMoV serogroups, including the CaCV isolates (data not shown).

The phylogenetic analysis constructed using N (Fig. 1a) and NSS (Fig. 1b) gene sequences revealed that CaCV-RM together with other CaCV isolates belong to the WSMoV-clade. Similarly, the phylogenetic analysis based on the NSM indicated that CaCV-RM is closely related to other CaCV isolates (Fig. 1c).

Recently, a natural reassortant (LGMTSG; G from GRSV and T from TCSV) in infected tomato plants has been identified in Florida, which may mislead proper identification (Webster et al. 2011). To investigate a possible reassortment event in CaCV-RM, in addition to S RNA and a part of M RNA (NSM), a part of L RNA was amplified and sequenced. This sequence shared 94.3–97.9% aa sequence identity and 83.8–95.4 nt sequence identity with that of other CaCV isolates. Also, this region showed 84.9–88% nt and 79.2–88.2% aa identity with selected serogroup IV species (Table 3). Therefore, these sequence data indicated that most likely there was no reassortment in the CaCV-RM isolate.

Table 3 Nucleotide and amino acid comparison of the partial RdRp sequence of CaCV-RM with available sequences of serogroup IV tospoviruses
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Eight out of 14 plant species tested showed local or systemic symptoms. Systemic infection of N. benthamiana and N. occidentalis induced chlorotic and necrotic lesions, leaf necrosis and plant death whereas C. annuum reacted with systemic chlorotic spots in the top leaves and fruit deformation. Gomphrena globosa, Chenopodium amaranticolor, C. quinoa, V. unguiculata and Solanum lycopersicum showed only local chlorotic or necrotic lesions. Six plant species did not show any symptom four weeks post inoculation (Table 4). C. annuum reaction to CaCV-Ph consisted of necrotic ringspots and deformations of both inoculated and non-inoculated upper leaves, and plant wilting (Zheng et al. 2008), while CaCV-RM caused systemic infection. V. unguiculata inoculated with CaCV-RM showed necrotic spots in inoculated leaves, which differed from the reaction induced by the Thai CaCV isolate, that consisted of chlorotic or necrotic concentric spots (Chiemsombat et al. 2008). S. lycopersicum showed local lesions in the leaves inoculated with CaCV-RM which resembled CaCV-Ph symptoms and differed from the systemic necrotic spots elicited by CaCV-958 (Zheng et al. 2008).

Table 4 Local and systemic reactions of plant species mechanically inoculated with RM isolate
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Unlike other CaCV isolates that have been reported to induce local or systemic symptoms in N. rustica (Zheng et al. 2008; Kunkalikar et al. 2010) CaCV-RM did not induce any symptoms in this plant. The variations in the symptoms might be due to the cultivar, virus isolate and environmental conditions. Back inoculation of CaCV-RM failed, as also reported earlier for other species e.g. ANSV (Hassani-Mehraban et al. 2010) and soybean vein necrosis virus (SVNV) (Khatabi et al. 2012).

This study confirmed the presence of CaCV as a member of the WSMoV clade in Iran and West Asia. The Iranian CaCV isolate was closely related to American, Australian, Indian and Taiwanese isolates in phylogenetic analysis based on N, NSS and NSM amino acid sequences.

In 2005, rudbeckia plants found to be infested with T. tabaci and Haplothrips sp. were examined for viral infection and TSWV was found in mixed samples of both species (Ghotbi et al. 2005) in serological tests. We identified T. tabaci as the only thrips present in rudbeckia plants and subsequently detected CaCV in the thrips body using RT-PCR (data not shown). However, transmission tests need to be performed to identify potential vector species. It is believed that tospoviruses are not transmitted by seeds, however it was shown that SVNV is transmitted by soybean seeds (Groves et al. 2016). To determine the possible transmission via seeds, a test using highly sensitive method is needed.

Currently CaCV is spreading from Australia and Thailand to other countries. In 2010 the virus was detected in a Kalanchoe sample from Tanzania (J.T.J. Verhoeven, personal communication) and TYRV was found in Poland (Zarzyńska-Nowak et al. 2015) and Kenya (Birithia et al. 2012). So far, CaCV has been found only in rudbeckia in Mahallat, which indicates the virus may have important hosts especially in ornamental crops, including gloxinia growing in this region. Therefore, intensive survey to monitor the virus spread and its vector in natural hosts is needed.