Background
Geminiviruses (family
Geminiviridae) are phytopathogenic viruses that cause huge crop losses every year throughout the world by infecting diverse plant species, including food and fiber crops, weeds. and ornamental plants [
1]. In the past few decades, the severity and incidence of diseases caused by geminiviruses has increased tremendously. Geminiviruses having circular, small single stranded DNA (ssDNA) genome encapsidated in twinned-icosahedral particles [
2,
3]. Based on differences in genome organization, mode of transmission through insect vectors, and host range, the family
Geminiviridae is divided into nine genera [
4,
5]. Among these, the genus
Begomovirus is the largest, most wide-spread, and economically most important [
6]. Begomoviruses in the Old World are usually associated with pathogenicity- and symptom-determining betasatellites, classified in the newly-established family
Tolecusatellitidae (genus
Betasatellite) [
7]. The begomovirus-betasatellite complex not only infects several economically important crops around the world, but also weeds and ornamental plant species.
Pedilanthus leaf curl virus (PeLCV), a Begomovirus that was first observed in 2009 on an ornamental shrub
Pedilanthus tithymaloides, in association with
Tobacco leaf curl betasatellite (TbLCuB) [
8], has swiftly increased its host range in Pakistan and India. The PeLCV-TbLCuB complex has also been found infecting soybean (
Glycine max) [
9],
Sesbania bispinosa (a leguminous weed)
, and
Raphanus sativus (radish), showing continuous expansion in its host range [
10,
11]. PeLCV has monopartite genome (single ssDNA molecule of ~ 2.8 kb), with four complementary strand genes specific for virus replication (AC1, AC3), transcription (AC2) and pathogenicity (AC4), and two virion strand genes (AV1 and AV2) for movement of virus molecules within and between plant cells.
Since begomoviruses pose a serious threat to agroecosystems worldwide [
12], they have become a topic of considerable attention among molecular biologists in the agriculture sector, particularly because of their increasing host range and severity of losses to economically important crops each year [
13,
14]. Since, the discovery of betasatellites in 1999, they have been found in Pakistan, India, China, Japan, Vietnam and several other South Asian and African countries. [
15]. The betasatellites encode a single gene of nearly 256 nucleotides, which is responsible for symptoms induction [
16,
17]. Although a lot of information is available on viruses infecting agricultural crops, comparatively less attention has been paid to the viruses that infect weeds, wild, and ornamental plant species, which are potential reservoirs of these viruses. Therefore, this study was aimed at evaluating
Petunia atkinsiana, as an alternative host and potential reservoir for begomoviruses.
Discussion
In the present study, phylogenetic, recombination, and infectivity analysis of an important begomovirus, Pedilanthus leaf curl virus was performed. The ornamental plant P. atkinsiana was found to be infected with this virus. Petunia atkinsiana is grown all over the world due to its attractive flower and leaf shape. Based on high nucleotide identity (96–98%) with the previously reported soybean strain of PeLCV, we considered the isolated virus to be a variant of PeLCV. Previously, PeLCV was found in combination with TbLCuB, but in the current study it was associated with DiYVB. Therefore, it can be considered as a new natural combination of a begomovirus with a betasatellite.
The literature analysis of PeLCV identified it as a very interesting virus. The data suggested that PeLCV infects at least 15 different hosts (Additional file
2), the majority of which are from Pakistan. Therefore, it can be predicted that PeLCV originated in Pakistan. Although, several begomoviruses have been reported to infect a single host, only few begomoviruses that infect multiple hosts have been identified yet [
26,
27]. In case of RNA viruses, a single virus like
Cucumber mosaic virus (CMV) has been shown to infect multiple hosts [
28]. However, for begomoviruses, such a broad host range has rarely been observed. To the best of our knowledge, PeLCV is among the begomoviruses with the broadest host ranges.
RaLCuV is only reported from India, and in the phylogenetic trees of
CP and
Rep genes, it appeared to be closely related to CLCuKoV. CLCuKoV is a highly recombination-prone virus and is considered to be major factor in the evolution of cotton leaf curl disease [
29]. From the current data, it can be considered that RaLCuV in India might have evolved as a recombinant virus with high nucleotide similarity to PeLCV or CLCuKoV. Although experimental evidence will be needed to confirm the origin of RaLCuV, it can be hypothesized that RaLCuV evolved due to positive selection for point mutations in CLCuKoV. In recent studies, there is an agreement that the majority of the
Rep genes of begomoviruses are more variable than
CP [
30]. However, in the case of PeLCV, the genetic variation is equally distributed in both the genes.
The recombination analysis of closely related begomoviruses revealed that PaLCuV in Pakistan possibly originated from PeLCV. Molecular phylogeny of DNA-A indicated that viral isolates of PeLCV originating from Pakistan are different from Indian isolates and constitute separate clades (with the exception of PeLCV-[PK:Fai:Chenopodium:05] and PeLCV-[PK:Mul:Pedilanthus:04]). Therefore, it can be concluded that PeLCV originated from Pakistan and then later spread to India. However, it is surprising that PeLCV is associated with different betasatellites in India and Pakistan. In Pakistan, PeLCV is associated with Tobacco leaf curl betasatellite (TbLCuB), Cotton leaf curl Multan betasatellite (CLCuMuB), or Digera yellow vein betasatellite (DiYVB). In contrast, in India it is associated with DiYVB, ToLCPaB and TbLChB. The complexity of betasatellites can be observed in the phylogenetic tree. Unlike, DNA-A component, the betasatellites (TbLCuB) from both the countries constitute separate groups. The Indian isolates of TbLCuB have been named as Radish leaf curl betasatellite in GenBank, but according to the species criteria of betasatellites from ICTV, they are more than 91% similar to Pakistani isolates of TbLCuB. Therefore, we have renamed them as TbLCuB. The separate group of TbLCuB from India suggests that the TbLCuB present in Pakistan has not yet spread to India. The majority of the DiYVB isolates were found in Pakistan. However, in a single incidence, DiYVB is reported from India, suggesting the possible recent introduction of this particular betasatellite from Pakistan to India. These observations indicate that begomoviruses and their satellites are spreading into different territories.
Although, PeLCV has been reported from several different hosts, we conducted the first infectivity experiments with N. benthamiana and P. atkinsiana. Successful infection in N. benthamiana and the original host, P. atkinsiana plants inoculated with partial dimeric constructs of PeLCV and its associated DiYVB proved them to be the causative agents for leaf curl disease. Moreover, plants inoculated with PeLCV alone showed upward curling and swelling of leaves, whereas, more severe symptoms were observed in the host plants with the inoculation of PeLCV along with its cognate betasatellite, DiYVB. These results suggest that PeLCV alone is sufficient to cause infection but addition of betasatellite increases the virus pathogenicity.
Conclusions
We have characterized Pedilanthus leaf curl virus (PeLCV) and Digera yellow vein betasatellite (DiYVB) as a new natural combination associated with leaf curl disease on petunia plants. Through the phylogeny of PeLCV, we found complex evolutionary pattern of the virus, with great diversity in genes encoding coat protein and replication associated protein. Phylogenetic analysis predicted that PeLCV is closely related to RaLCuV and PaLCuV and recombination analysis demonstrated that PeLCV was involved in a natural recombination event and is the donor parent for the evolution of two recombinant virus species, RaLCuV and PaLCuV. Infectivity analysis with infectious dimeric constructs of PeLCV and DiYVB reproduced leaf curl disease symptoms in N. benthamiana and P. atkinsiana plants, proving them as natural disease-causing agents. Recent reports of PeLCV infection on different plant species suggests that it is an emerging Begomovirus that has spread to other weed and vegetable plants within last few years, as well as crossing the continental barriers from Pakistan to India. Moreover, PeLCV showed relaxed trans-replication with five different betasatellite species, make it a particularly interesting begomovirus. High genetic variability in the virus suggests that it is evolving at an alarming rate. The natural existence of broad host ranges and the recombination propensity of different betasatellites may result in a future epidemic of this virus. Therefore, this study highlights the importance and flexibility of PeLCV in terms of infection and spread to other plant species.