Molecular and biological characterization of Chilli leaf curl virus and associated betasatellite infecting Cucurbita maxima in Oman

Abstract

During a survey in February 2016, leaf curl disease symptoms were witnessed in Cucurbita maxima plants in Al-Batina commercial farm in Oman. Symptoms exhibited were characteristic of begomovirus infection as leaf curling, yellowing, and color breaking followed by mosaic pattern. The transmission electron microscopy confirmed the presence of typical twinned geminate typical of Geminate virus particles. Rolling circle amplification (RCA) was employed to characterize the unknown causal agent of C. maxima disease. In molecular identification RCA produced nearly 2.8 and 1.4 kb DNA molecules corresponding to begomovirus and satellite molecules, cloned and sequenced them. In Blast, species demarcation tool and phylogenetic analysis revealed the begomovirus and satellite isolates were determined as Chilli leaf curl virus (ChLCV) and tomato leaf curl betasatellite (ToLCB). In biological analysis by agrobacterium mediated inoculation, ChLCV displayed upward leaf curling and vein swelling symptoms in Nicotiana benthamiana plants; however, in presence of ToLCB enhanced downward leaf curling and crumpling symptoms were revealed. This study provides the first evidence that ChLCV and ToLCB caused leaf curl disease of C. maxima in Oman.

Plant-infecting single stranded DNA viruses (ssDNA) belongs to genus begomoviruses (family: Geminiviridae) are important pathogens infecting mostly dicotyledonous plants. They are transmitted by whiteflies (Bemisia tabaci) vector and have either a DNA-A or DNA-A and DNA-B genomic components. The DNA-A contain six open reading frames (ORFs), which encode protein for replication, encapsidation and movement. Four (AC1–AC4) ORFs are located in the complementary sense. The AC1 encodes for a Rep (replication associated protein) and AC2 for a TrAP (transcriptional activator protein) while the protein encoded by AC3 is the Ren (replication enhancer protein) whereas the protein encoded by AC4 functions as a suppressor of RNA silencing. The other two ORFs (AV1 and AV2) are located in the virion sense, where AV1 codes for coat protein and the AV2 for a protein of unclear function. Similarly, DNA-B component also contains two ORFs (BC1 in complimentary and BV1 in virion sense). BC1 and BV1 detached by an intergenic region (IR) approximately 300 nucleotides (nt) that contains fundamental elements required for the replication and transcription of the viral genome [2]. Mostly monopartite single stranded DNA (ssDNA) viruses reported from Asia, Europe, Middle East and Australia have been shown to be associated with satellites molecules known as betasatellites (previously recognized as DNA β); [4]. This study provides evidence that Chilli leaf curl virus (ChLCV) and associated tomato leaf curl betasatellite (ToLCB) are causing the leaf curl disease of C. maxima in Oman.

During a survey in February 2016, leaf curl disease symptoms exhibiting yellowing, color breaking and mosaic typical of begomovirus infection were noticed on C. maxima plants in Al-Batina commercial farm in Oman (Fig. 2, panel a). Medium to high density of B. tabaci followed by 55–65% disease incidence was also recorded. The transmission electron microscopy confirmed the presence of typical twinned geminate typical of Geminate virus particles. To isolate the pathogen infected (three) and non-infected (two) leaf samples were collected and genomic DNA was extracted using CTAB buffer according to Doyle and Doyle [8]. The DNAs were used in rolling circle amplification (RCA) to amplify all circular genomic components associated with the disease samples. The concatamers of RCA were subjected to endonuclease restriction analysis with different restriction enzymes, with BamHI restriction enzyme ~ 2.8 and 1.4 kb DNA monomer molecules were produced. Both the monomer fragments were cloned into pGEMT-3Zf vector at BamHI restriction site (Promega, USA), and were subsequently confirmed in a single or double endonuclease restriction analysis. The selected full-length clones for individual sample were sequenced bidirectionally and obtained nucleotide sequences are 2760–2761 nt long and were submitted in the DNA GenBank under the following accession numbers (MN119490–MN119492). The predicted open reading frames (ORFs) and features of these clones were recognized using ORF Finder program run on-line (Table 1). Further molecular analysis of the complete DNA genome of the begomovirus showed 99.4% nucleotide sequence identity to previously characterized isolates of Chilli leaf curl virus (ChLCV:HG969264) associated with tomato leaf curl disease (ToLCD), while less than 87% nucleotide identity with other begomoviruses. Species demarcation tool (SDT) confirmed the isolates are of ChLCV which were further shown by the phylogenetic analysis to group into ChLCV cluster (Fig. 1, panel a). In recombination analysis no recombination event was found from these isolates, according to the applicable species segregation criteria [6], the virus identified here is an isolate of ChLCV. Potentially three full-length betasatellite clones (P1–P3) were sequenced and final sequences were submitted into the GenBank under the unique accession numbers (MN119487-9). The sequences are at 1378–1379 nt in length. Analysis of these sequences confirmed to typical of earlier betasatellites having SCR; a satellite conserved region, A-rich; a sequence rich in adenine nt and a single complementary-sense ORF known as βC1 gene [3]. Pairwise sequence evaluations displayed that these sequences have greater than 94% nt sequence identity to tomato leaf curl betasatellite (ToLCB) associated with ToLCD, mungbean yellow mosaic disease [10, 14, 16]. Further in phylogenetic analysis our isolates clustered with other ToLCB from Arabian Peninsula (Fig. 1, panel b).

Fig. 2.

Field infected cucumber (a) and healthy (d) plants. Symptoms exhibited by N. benthamiana plants infected with ChLCV (b), ChLCV and ToLCB (c), mock inoculated (e) and non-inoculated (f) N. benthamiana plants are shown for comparison

Table 1.

Features of Chilli leaf curl virus and tomato leaf curl betasatellite isolated from Cucurbita maxima plants

Isolate/virus type	Acc. No.	Size (nt)	Begomovirus Position of genes (coordinates)/no. of amino acids (predicted coding capacity in kDa)						Isolate/satellite type	Betasatellite
			CP	V2	Rep	TrAP	REn	C4		Acc. No.	Size (nt)	Position of BC1 gene(coordinates)/no. of amino acids (predicted coding capacity in kDa)
P6/ChLCV	MN119490	2760	308–1081 257 (29.71)	148–513 121 (11.69)	1530–2615 361 (40.23)	1241–1627 128 (14.55)	1078–1482 134 (16)	2165–2458 97 (10)	P1/ToLCB	MN119487	1378	201–557 118 (13)
P7/ChLCV	MN119491	2761	309–1082 257 (29.71)	149–514 121 (13.5)	1531–2616 361 (40.23)	1242–1628 128 (14.55)	1079– 1483 134 (16)	2166–2459 97 (10)	P2/ToLCB	MN119488	1378	201–557 118 (13)
P8/ChLCV	MN119492	2761	309–1085 258 (29.71)	149–514 121 (13.5)	1531–2616 361 (40.23)	1224–1628 134 (16)	1061–1483 140 (17)	2166–2459 97 (10)	P3/ToLCB	MN119489	1379	202–558 118 (13)

Fig. 1.

Phylogenetic dendrograms based upon alignments of the complete nucleotide sequences of the ChLCV (a) or ToLCB (b) clones obtained from Cucurbita maxima in Oman with selected begomovirus and betasatellite sequences obtained from the databases. Vertical branches are arbitrary, horizontal branches are proportional to calculated mutation distance. Values at nodes indicate percentage boot strap values (1000 replicates). The trees were arbitrarily rooted on the sequence Tomato pseudo-curly top virus (X84735), for the virus tree, and Ageratum yellow vein Singapore alphasatellite (FJ956707), for the betasatellite tree, as outgroup

For biological characterization, ChLCV (clone# P6) was restricted with BamHI and PstI to discharge a fragment of 1450 bp holding hair pin sequences. This fragment was ligated into pG0029 at the compatible restriction sites. Then full-length monomer of ChLCV was released from P6 digested with BamHI and ligated into pG0029 at BamHI site to produce 1.4 mer recombinant molecules (pGP2-1.4). To produce the infectious construct for betasatellite, similar strategy was adapted, restricts the clone# P2 with BamHI and XbaI to obtain a 578 bp partial fragment and ligated into pG0029 restricted vector. Then full-length molecule of ToLCB was released from P2 digested with BamHI and ligated into pG0029 to produce 1.3 mer recombinant molecule (pGP2-1.3). The selected recombinant plasmids for ChLCV (pGP6-1.4) and ToLCB (pGP2-1.3) were transformed into the agrobacterium (GV3101) supplemented with antibiotics and inoculated the Nicotiana benthamiana seedlings and kept under controlled conditions [9]. Inoculation of just the ChLCV construct to N. benthamiana seedlings (n = 10) resulted in severe leaf curling (upward) and vein swelling on the undersides of young, newly developing leaves initiating at 16 days post-inoculation (Fig. 2, panel b). Co-inoculation of N. benthamiana seedlings (n = 12) with the constructs for both ChLCV and ToLCB resulted in severe leaf curling (downward) followed by crumpling young, newly emerging leaves at 12 dpi (Fig. 2, panel c). In PCR using specific primers, 100% genomic components of ChLCV and/or ToLCB were detected in agroinoculated N. benthamiana plants. The severity of symptoms of ChLCV is due to pathogenicity determinant gene of ToLCB. Earlier studies were shown that the betasatellite produce proficient infection in cotton [5], specifically βC1 is responsible to produce characteristic symptoms of leaf curl disease of cotton [14].

Chilli leaf curl symptoms were first observed in early nineteen fifties in India, and later the virus was identified in the 1963s [13]. However, due to recent phenomenon of agriculture in Oman ChLCV has recently been identified infecting tomato and pepper in Oman, [12]. Although, ChLCV is identified recently, in the Sultanate Oman, yet there is possibility that this virus could be distributed several years back but discover recently. Cucurbita maxima is a vegetable crop grown commonly at commercial farms in Al-Batina region of Oman. We identified a ChLCV and it’s associated ToLCB molecules infecting this economically important vegetable crop. This study provides the first evidence of ChLCV and ToLCB infecting C. maxima in Al-Batina, Oman. The host range of ChLCV has been spreading into chilli (the original host) and pepper in India [7, 13, 15], pepper in Pakistan [19], tomato, watermelon and mint in Oman [11, 17, 18]. Thus, it shows that ChLCV has been spreading into different geographic areas to infect different host plants. Since, distribution and diversity of begomoviruses mainly due to geographical and human migration into different regions, this is the case with Oman. For instance, approximately 2.5 million expats workers, majority of them from south Asian countries (India, Bangladesh and Pakistan) are working in the Sultanate of Oman, having affinity for hot spices (chillies, pepper in daily diet) which was not produce in Oman, brought for their own kitchen gardening. This could be one reason of distributing this very important begomovirus to Oman. However, in recent years different begomoviruses are reported from different cucurbitaceous crops, viz. bitter gourd, pointed gourd, pumpkin and sponge gourd from South Asian countries [1, 20].

The pathogenicity test proves ChLCV has the ability to induce disease, in other host plants, either individually or with betasatellite. The demonstration of ChLCV and associated ToLCB in C. maxima host demonstrates the potential threat to this crop and there are possibilities that this virus complex may expedite their spread into different crops. Since, Oman has extensive trade with different countries, there is possibility that this virus can spread to other countries through air or sea and perhaps through borders, especially to the border sharing countries (United Araba Emirates, Saudi Arabia and Yemen). It is extremely important to avoid any risk in spreading this virus through exporting material to new hosts in other countries, an efficient quarantine laws should be implemented.