Introduction

The genus Capsicum (Solanaceae) is native to tropical Central and South America and comprises 27 species, although only five of them have been domesticated [1]. Among them, Capsicum annuum L., including pungent (chili or hot pepper) and non-pungent (sweet pepper) types, is the most widely commercially cultivated, being an increasingly important spice and vegetable crop worldwide [2]. Although there are more than 70 distinct viruses known to infect pepper, only about 20 of them have been reported to cause serious damage to this crop [3]. Most of the viruses infecting pepper are transmitted in nature by insects, including aphids (Hemiptera: Aphididae), whiteflies (Hemiptera: Aleyrodidae) and thrips (Thysanoptera: Thripidae) (reviewed in reference [4]).

In 1981, symptoms of vein yellowing and leaf roll were observed in pepper plants grown in greenhouses in Kitanakagusuku, Okinawa prefecture, Japan, although the disease was not reported until more than ten years later [5]. A similar disease was observed both in greenhouses and open fields in the Arava Valley, Israel, during 1998 [6, 7]. In addition to the symptoms observed in Japan, the Israeli group reported symptoms in fruit, which were smaller than normal and discolored, resulting in reduced commercial value. Both in Japan and Israel, the disease was shown to be transmitted by the aphids Aphis gossypii Glover and Myzus persicae (Sulzer) in a persistent manner [5, 7]. A luteovirus was shown to be the causal agent of the disease in both countries, and it was named pepper vein yellows virus (PeVYV) in Japan [5] and pepper yellow leaf curl virus (PYLCV) in Israel [7].

The family Luteoviridae is composed of three genera: Luteovirus, Polerovirus and Enamovirus, plus several species of unassigned viruses [8, 9]. Virions of members of the family Luteoviridae are 25 to 30 nm in diameter, icosahedral, and have no envelope. Poleroviruses are exclusively transmitted by aphids in a persistent circulative manner [10, 11]. Polerovirus genomes consist of a single molecule of linear, positive-sense ssRNA ranging from 5.6 kb to 6.2 kb. A small protein (VPg) is covalently linked to the 5´ end of the genomic RNA, which does not have a 3′-terminal poly(A) tract.

The typical genome organization of poleroviruses consists of seven open reading frames (ORF0 to ORF5 and ORF3a), which encode proteins P0 to P5 and P3a [8, 12]. P0 functions as a suppressor of RNA silencing. ORF1 and ORF2 overlap and encode the replication-related proteins (P1 and P2). P1 contains protease motifs, and the VPg is processed from its C-terminal region. P2 is an RNA-dependent RNA polymerase (RdRp). P3 and P5 are the major and minor coat protein, respectively, P5 being expressed as a C-terminal fusion to the core major coat protein. P5 acts as a factor for aphid transmission and virus particle stability. P4 is required for long-distance movement of some poleroviruses. P3a, translated from a non-AUG codon, seems to have an essential role in viral movement.

Nucleotide sequencing of the complete genome of two virus isolates causing the yellowing disease of pepper in Japan and Israel confirmed that they belong to the genus Polerovirus in the family Luteoviridae of plant viruses (PeVYV, [13]; PYLCV, [14]). Recently, two additional isolates assigned to PeVYV, from China and Australia, respectively, have been completely sequenced [15, 16].

In recent years, symptoms similar to those observed in Japan and Israel have been associated with the presence of poleroviruses in many countries worldwide: Turkey and Tunisia [17]; Spain [18]; India, Indonesia, Mali, Philippines, Taiwan, and Thailand [19]; Sudan [20]; the United States [21]; Ivory Coast [22]; China [23, 24]; Italy [25] and Australia [16]. The detection of a polerovirus associated with a yellowing disease of pepper in Spain [18] was the first in Europe. This virus was associated with a severe outbreak of the disease in Almería province, a region in southeastern continental Spain, where almost 10,000 ha of sweet pepper are cultivated in greenhouses for fresh consumption. Disease prevalence in many greenhouses in the area reached levels of 100%, resulting in discolored pepper fruits and reduced commercial value.

In this work, the complete nucleotide sequence of a polerovirus associated with the yellowing disease of pepper in Spain was determined. The genome (6125 nt in length) has the typical organization of poleroviruses and was most closely related (90.7% nt sequence identity) to a pepper polerovirus from Israel. A comparison of the sequence from Spain with the four complete sequences available for PeVYV/PYLCV isolates supports, following the officially accepted sequence demarcation criterion for species of the genus Polerovirus, i.e., differences in amino acid sequence identity of any gene product of greater than 10%, the existence of a complex of at least five polerovirus species whose members threaten pepper crops worldwide. Also, it was shown that recombination plays a key role in the speciation of this group of emergent poleroviruses.

Materials and methods

Virus isolate and aphid transmission

Adult aphids (Aphis gossypii Glover) naturally infesting symptomatic sweet pepper plants showing symptoms of interveinal yellowing and curling in leaves (Fig. 1A) were collected from a greenhouse in Almería province (southern Spain) in February 2013. The isolate was named Spain-Almería 2-2013 (ES-Alm2-13). Aphids were transferred to three sweet pepper (cv. California Wonder) plants at the four-leaf stage (Fig. 1B), where they were allowed to feed for 48 hours. At the end of this inoculation period, plants were sprayed with insecticide and maintained in an insect-proof greenhouse with temperature control (22–27 °C day/17–20 °C night). Also, grafting was used to transmit the virus from the three sweet pepper plants experimentally infected by aphids to four additional sweet pepper plants (Fig. 1C).

Fig. 1
figure 1

Aphid and grafting transmission assays. (A) Polerovirus-infected pepper plant from a greenhouse located in the Almería region (southern Spain). (B) Pepper (Capsicum annuum) plants (cv. California Wonder) inoculated with viruliferous aphids (Aphis gossypii) collected from the infected plants. (C) Pepper plants (cv. California Wonder) inoculated by grafting. (D) Tissue printing hybridization of petiole and stem from grafted pepper plants and of a healthy plant (H) included as a negative control

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Virus detection

The presence of polerovirus RNA in leaf samples was determined either by reverse transcription polymerase chain reaction (RT-PCR) or by tissue blot molecular hybridization. For RT-PCR, the pair of primers MA1754 (5′-CTG GAG CGT TGC GGA ATG-3′)/MA1755 (5′-GTG TCA GGC TGT TGG TAA TCA GAC-3′) was designed based on the available partial polerovirus sequence from Spain (isolate Almería-1) [18]. These primers amplify a region of 658 nt that includes part of the RdRp gene and the intergenic region. For tissue blot hybridization, freshly cross-sectioned leaf petioles and stems were squash-blotted on positively charged nylon membranes (Roche Diagnostics) and hybridized with a digoxigenin-labeled RNA probe specific for the RdRp/coat protein region of the Almería-1 isolate [18] cloned in pGEM-T Easy Vector (Promega). After linearization with EcoO109I, the plasmid was used for in vitro transcription using SP6 RNA polymerase (Roche Diagnostics) and DIG RNA Labeling Mix (Roche Diagnostics) to generate a RNA probe of negative polarity. Membranes were prehybridized for two hours with pre-hybridization solution (5x SSC [750 mM NaCl and 75 mM sodium citrate, pH 7.0], 1% N-lauroylsarcosine, 0.02% SDS and 2% blocking agent [Roche Diagnostics]) and 50% formamide. After pre-hybridization, the specific probe was added and incubated at 65 °C overnight. Hybridization was carried out under high-stringency conditions (washing steps at 65 °C in 0.1x SSC and 0.1% SDS) following standard procedures. Hybridization signals were detected on X-ray film (Mamoray HT, AGFA) after treatment with CDP-Star (Roche Diagnostics).

RNA extraction and RT-PCR amplification

Total RNA was extracted from symptomatic pepper leaf samples using TRIsure Reagent (Bioline) according to the manufacturer’s instructions. RT-PCR was carried out with AMV (Promega) and the Expand High Fidelity PCR System (Roche Diagnostic) with several pairs of primers (Supplementary Table S1) designed based on the three complete sequences of PeVYV/PYLCV deposited in GenBank when this study was initiated (Japan [AB594828], Israel [HM439608] and China [KP326573]) or on the sequence generated in this work. The primers were designed to obtain overlapping genome fragments. Both ends of the genome were polyadenylated and amplified using a 5′/3′ RACE Kit, 2nd Generation (Roche Diagnostic) (Supplementary Table S1). Amplicons of the expected sizes were cloned using the pGEM-T Easy Vector System (Promega), and at least five clones for each genome segment were sequenced at Macrogen Inc. (Seoul, South Korea).

Sequence and phylogenetic analysis

SeqMan and EditSeq (available in MEGALIGN [DNASTAR Inc., Madison, WI, USA]) were used for assembly and editing, respectively, of DNA sequences. BLAST (https://www.ncbi.nlm.nih.gov/blast/) was used for initial sequence similarity searches. Sequences were aligned with MUSCLE [26], and pairwise identity scores were calculated with SDT (Sequence Demarcation Tool) [27]. Phylogenetic analysis of the pepper polerovirus from Spain was performed by the neighbour-joining method (1000 replicates) available in the MEGA6 package [28].

Recombination analysis

To detect putative recombinant fragments in the polerovirus nucleotide sequence reported here, a search for potential parental viruses in the GenBank database was conducted using SWeBLAST [29]. Recombination analysis was conducted using RDP4 [30] from the alignment generated with MUSCLE implemented in MEGA6 [28]. To illustrate the mosaic nature of the polerovirus genome due to recombination, similarity plots were generated using SimPlot with the Kimura 2-parameter distance model [31]. Phylogenetic evidence of recombination was also analyzed using the neighbour-net method in the program SplitsTree4 [32] and statistically verified using the pairwise homoplasy index (PHI) test [33].

Results and discussion

The pepper polerovirus present in Spain is transmissible by Aphis gossypii

The three sweet pepper plants (cv. California Wonder) inoculated with A. gossypii adults collected from plants displaying virus-like symptoms showed the typical interveinal yellowing, upward leaf curling and internode shortening associated with PeVYV/PYLCV, identical to the symptoms observed in the plants from which the aphids were collected and to those previously reported in Spain [18]. The presence of a polerovirus was confirmed by RT-PCR analysis of the three inoculated plants using primers MA1754/MA1755, which rendered the expected DNA fragment of 658 bp, and by tissue blot hybridization (not shown). The isolate was named Spain-Almería 2-2013 (ES-Alm2-13). The plants initially inoculated by aphids were propagated by grafting onto healthy sweet pepper plants, the latter of which were confirmed infected by tissue blot hybridization (Fig. 1D).

PeVYV/PYLCV isolates from Japan and Israel were also shown to be transmitted by A. gossypii [5, 7]. These isolates were also transmissible by M. persicae, but this aphid species was not assayed in our work.

The pepper polerovirus present in Spain has a recombinant origin

Assembling of overlapping genomic PCR fragments from the polerovirus isolate from Spain yielded a consensus contig of 6125 nt (Fig. 2A). The highest nucleotide sequence identities were with PeVYV/PYLCV from Israel (90.7%), Japan (89.5%), China (89.1%) and Australia (88.4%). The genome of the polerovirus isolate from Spain has the typical structure of members of the genus, containing seven ORFs (ORF0 to ORF5 and ORF3a) (Fig. 2B) putatively encoding proteins P0 to P5 and P3a. Percent identities of the deduced amino acid sequence of each protein with the equivalent proteins of other pepper polerovirus isolates are shown in Supplementary Figure S1.

Fig. 2
figure 2

Genome organization and recombination analysis of the pepper polerovirus from Spain (pepper vein yellows virus 5, PeVYV-5). (A) cDNA fragments cloned and sequenced to obtain the complete genome sequence of PeVYV-5. See Supplementary Table S1 for details on the primers used for amplification and the number of clones sequenced. (B) Schematic representation of the genome organization of PeVYV-5. The boxes represent the predicted ORFs, and their coordinates are indicated. (C) Similarity plot diagram made using the program SimPlot comparing the nucleotide sequence of PeVYV-5 (Spain) with those of pepper polerovirus isolates from Israel, Australia, Japan and China. (D) SimPlot diagram comparing the nucleotide sequence of PeVYV-5 (Spain) with those of tobacco vein distorting virus (TVDV, EF529624) and cucurbit aphid-borne yellows virus (CABYV, X76931). The main putative recombinant fragment (major parent: TVDV, minor parent: CABYV, p = 1.193 × 10−68) is indicated by a double-headed arrow. (E) Schematic diagram showing putative recombinant fragments for pepper polerovirus isolates. Yellow and blue rectangles indicate sequences related to TVDV and CABYV, respectively. Similar black and white shading indicates sequences shared by different sets of pepper poleroviruses

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Phylogenetic analysis of the complete genome sequences of pepper poleroviruses, supported by high bootstrap values, showed that the polerovirus isolate from Spain clusters with the Israeli isolate (Fig. 3). Isolates from China, Japan and Australia grouped together in a different clade. When the phylogenetic analysis was carried out separately for the amino acid sequences of the six proteins putatively encoded by the pepper polerovirus genomes, some inconsistencies were observed (not shown). Thus, the isolates from Spain and Israel clustered together only in the case of P0, P1 and P2 proteins. In the case of P3, P4, P5 and P3a, the isolate from Israel branched separately from the cluster formed by the rest of the isolates.

Fig. 3
figure 3

Phylogenetic tree illustrating the relationship of the complete genome sequence of the pepper polerovirus from Spain (pepper vein yellows virus 5, PeVYV-5) to isolates of all accepted polerovirus species. The tree was constructed using the neighbour-joining method available in the MEGA7 package, and bootstrap values (1000 replicates) are shown at the nodes. BChV, beet chlorosis virus; BMYV, beet mild yellowing virus; BWYV, beet western yellows virus; CtLRV, carrot red leaf virus; CYDV-RPS, cereal yellow dwarf virus-RPS; CYDV-RPV, cereal yellow dwarf virus-RPV; CpCSV, chickpea chlorotic stunt virus; CLRDV, cotton leafroll dwarf virus; CABYV, cucurbit aphid-borne yellows virus; MYDV-RMV, maize yellow dwarf virus-RMV; MABYV, melon aphid-borne yellows virus; PeVYV-1 to PeVYV-4, pepper vein yellows virus 1 to pepper vein yellows virus 4; PLRV, potato leafroll virus; SABYV, suakwa aphid-borne yellows virus; ScYLV, sugarcane yellow leaf virus; TVDV, tobacco vein distorting virus; TuYV, turnip yellows virus. The enamovirus pea enation mosaic virus 1 (PEMV-1) was used as an outgroup. The bar below the tree indicates 0.1 nucleotide substitutions per site

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To detect putative recombinant fragments in the novel genome, a search for potential parental viruses in the GenBank database was conducted using SWeBLAST. The sequences with the highest SWeBLAST scores were aligned and analyzed using the RDP4 package. This analysis showed several putative recombination events between PeVYV/PYLCV genomes known to date as well as the genomes of the poleroviruses tobacco vein distorting virus (TVDV, EF529624) and cucurbit aphid-borne yellows virus (CABYV, X76931), as illustrated in Fig. 2C, D, and E. Several small putative recombinant fragments exchanged between pepper poleroviruses were detected with p-values higher than 10−10 (not shown). Only one putative recombination event with a very low p-value (1.193 × 10−68 using the RDP method) was detected in the polerovirus isolate from Spain (see Fig. 2D). This recombination event would involve TVDV as major parent and CABYV as minor parent. Evidence of phylogenetic inconsistencies, as shown by reticulate networks after neighbour-net analysis (Fig. 4), additionally supported the involvement of recombination in the origin of the pepper poleroviruses (p = 0.0, PHI test).

Fig. 4
figure 4

Phylogenetic network (neighbour-net method) of the complete genome sequences of the five pepper vein yellows poleroviruses (PeVYV-1 to PeVYV-5), tomato vein distorting virus (TVDV, EF529624) and cucurbit aphid-borne yellows virus (CABYV, X76931) generated using the SplitsTree4 program. Networked relationships among viruses with a box-like structure instead of a bifurcating tree are indicative of recombination. The bar below the network indicates 0.1 nucleotide substitutions per site. See Table 1 for names and GenBank accession numbers of the pepper poleroviruses

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The putative major parental virus of the pepper poleroviruses TVDV was originally described in China, and isolates of CABYV, the putative minor parent, are also widely distributed in Asia. This suggests that poleroviruses infecting pepper could have an Asian origin, which is in agreement with the first detection of symptomatic plants in Japan [5]. A recombinant origin for the PYLCV isolate from Israel involving TVDV and CABYV has been suggested previously [14].

The yellowing disease of pepper emerging worldwide is caused by a complex of different poleroviruses

The current species demarcation criteria for members of the genus Polerovirus include differences in amino acid sequence identity of any viral gene product higher than 10% [8]. The genome of the polerovirus isolate from Spain described here codes for two proteins which are less than 90% identical to the equivalent proteins of all previously characterized pepper poleroviruses, P0 (77.5-86.7%) and P5 (74.1-84.1) (Supplementary Figure S1). Thus, according to the above-mentioned criterion, the virus isolate characterized in this work belongs to a new species in the genus Polerovirus. Also, comparison of the deduced amino acid sequence of all gene products of the five available complete sequences of the poleroviruses causing the yellowing disease in peppers worldwide (PeVYV/PYLCV from Japan, China, Israel, Australia and Spain) revealed that they belong to five distinct polerovirus species (Supplementary Figure S1). The sequence of P0 would differentiate the pepper poleroviruses in four species-level units (Australia, Japan, China/Israel and Spain). Additionally, isolates from China and Israel would be separated at the species level according to differences in the sequence of P5.

To date, there is only one recognized species containing pepper-infecting poleroviruses, Pepper vein yellows virus, which is represented by the isolate described in Japan [9]. Given that the symptoms caused by all pepper poleroviruses are similar, if not identical, we propose to name them “pepper vein yellows virus 1” to “pepper vein yellows virus 5” (PeVYV-1 to PeVYV-5) according to date of description, with PeVYV-5 corresponding to the polerovirus from Spain described in this work (Table 1).

Table 1 Proposed nomenclature for the poleroviruses infecting pepper
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Taken together, our results and those published over the last few years [13,14,15,16] have shown the emergence of different poleroviruses affecting pepper crops around the world, with a high degree of complexity due to recombination events. The biological and pathological properties of isolates belonging to these species should be investigated in the future. The availability of infectious clones will facilitate investigation of whether differences in symptoms, host range or transmission specificity exist between the different species revealed in this work.