Tomato leaf curl New Delhi virus (ToLCNDV) is a member of the family Geminiviridae belongs to the Genus Begomovirus. ToLCNDV is a bipartite begomovirus constituting DNA A and DNA B as their genomic components and are efficiently transmitted by whitefly, Bemisia tabaci to a wide range of host plants. ToLCNDV is causing serious threats to various horticultural crops in the countries of the Asian continent viz., India, Pakistan, Bangladesh, Philippines and Thailand [5, 6, 17]. ToLCNDV was originally reported to infect solanaceous vegetables since 1980s, however in recent days it has extended its host range towards the cucurbitaceous vegetables viz., pumpkin, bottle gourd, bitter gourd, ash gourd, cucumber, ridge gourd, watermelon, long melon and ivy gourd throughout the globe [4, 9, 12, 13, 15]. Predominantly, squash leaf curl China virus (SLCCNV) and ToLCNDV are the two major begomoviruses reported on the cucurbits from India.

Chayote (Sechium edule) is an cucurbitaceous vegetable cultivated on a large scale in the high altitude zone (Kodaikanal region) of Tamil Nadu. During 2012–2014, a survey was conducted on mosaic diseases of cucurbits cultivated in Tamil Nadu, severe mosaic disease associated with the chayote crop, which is imposing serious threat to the crop cultivation was observed. Despite being a perennial crop, farmers are cultivating chayote as annual crop in this region because of the occurrence of this disease for the past 5 years consecutively. During a survey in and around the Kodaikanal region which include Thanndikudi, Thandiankudisai, Pannaikadu and Pallangi, a severe mosaic disease was recorded with a maximum disease incidence of 100% under natural field conditions. The disease was characterized by severe mosaic, cupping and enation on the leaves with reduced fruit size (Fig. 1). Farmers are facing yield loss of more than 90% because of this mosaic disease [14]. Also diseased fields were observed with enormous whitefly population. Earlier Mandal et al. [7] had recorded the associated of ToLCNDV with chayote yellow mosaic disease from Northern India. The objective of this study was to study the causal virus associated with the mosaic diseases of the chayote crop and to complete the characterization of the virus in Southern India.

Fig. 1
figure 1

Symptoms of severe mosaic on chayote (i mosaic, ii whiteflies on leaves, iii enation and iv reduction of fruit size)

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Nearly thirty leaf samples showing severe mosaic symptoms were collected from the Thadiankudisai—TN TDK CHOU1 (Kodaikanal region) were subjected to the DAS-ELISA using the squash leaf curl virus (SLCV) antiserum obtained from DSMZ, Germany along with apparently healthy leaf for preliminary screening to test the association of begomoviruses. Infected leaf samples alone showed positive reaction with mean absorbance range between 0.352 and 0.486 whereas healthy samples recorded 0.103. Based on symptoms, presence of whiteflies in infected field and positive result in ELISA, association of begomovirus was suspected with the disease. Hence total DNA was extracted using CTAB method and PCR amplification has been carried out with the specific primer pair (GK ToLCV F/R) to amplify the complete coat protein of associated begomovirus [10]. The PCR resulted in the successful amplification of ~ 950 bp in the symptomatic leaves of chayote plants only but not in the non-symptomatic samples (Fig. 2). The ~ 950 bp fragment was cloned using TOPO T/A cloning kit (Invitrogen Inc., USA) and sequenced. Sequence results of 950 bp spanning complete coat protein showed that 93% identity towards the ToLCNDV (KF551576).

Fig. 2
figure 2

PCR amplification of yellow mosaic diseased samples of chayote using specific primer pair (GK ToLCV F/R) from different locations of Kodaikanal region. M: 100 bp ladder marker, lanes 1-4: Thanndikudi, lanes 5-6: Thandiankudisai, lanes 7-8: Pannaikadu, lane 9: Pallangi, lane 10: Healthy control

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Rolling circle amplification (RCA) was performed to amplify the complete genome of DNA A using Ø29 DNA polymerase (Thermo Scientific, USA) following the standard protocol [16]. RCA product was digested with different restriction endonucleases (BamHI, EcoRI, HindIII, and XbaI). The ~ 2.7 kb fragment was produced by the XbaI corresponding to the genome size of the begomovirus and no other enzymes (HindIII, BamHI and EcoRI) produced. The XbaI digested linearized 2.7 kb fragment separated on agarose gel (1%) and were purified using Quick gel extraction kit (Qiagen, Germany) and ligated to XbaI linearized pUC19 vector. The ligated product was transformed in Escherichia coli strain DH5α, and total of 26 colonies were obtained and all the colonies were subjected for plasmid miniprep extraction. Plasmid DNA was restricted with cloning site enzyme and BglI with an aim to release the 2.7 kb insert. From the double digestion of recombinant plasmid DNA with BglI and XbaI, two positive clones were obtained; both the clones were sequenced by primer walking.

The sequence of 2739 nucleotides was obtained from both the clones sharing 99.5% identity among themselves. The nucleotide sequence was initially subjected to BLAST and the sequence shared maximum identity of 91% towards ToLCNDV reported from India, Pakistan and Spain. The genome structure was determined using Vector NTI (Invitrogen Inc., USA) and the sequence was submitted in the in the NCBI database (KP191047). The analysis of the sequences shows the genome organization of the present begomovirus isolate resembling old world begomoviruses with two open reading frames (ORFs) on viral sense strand and five ORFs on the complementary sense strand.

Phylogenetic analysis of the complete DNA A nucleotide sequence of ToLCNDV isolate infecting chayote with the other ToLCNDV isolates reported from different parts of the world available in the database was determined using MEGA6.0 software (Fig. 3). Striking feature of the dendrogram is the formation of separate cluster by our isolate (TN TDK CHOU1) with the other isolates reported from Southern India and Spain infecting cucurbits and tomato. Also phylogeograph shows chayote isolate as distinct from other ToLCNDV strains. Based on sequence analysis and considering the ICTV demarcation criteria for begomovirus [3], it is evident that the present isolate of begomovirus, associated with severe mosaic disease of chayote, is an isolate of ToLCNDV.

Fig. 3
figure 3

Phylogenetic analysis of ToLCNDV (TN TDK CHOU1) isolate infecting chayote in Tamil Nadu based on complete DNA A nucleotide sequences with other ToLCNDV isolates. The trees were constructed using the NJ algorithm implemented by MEGA 6.0 with SLCCNV as an outgroup. The bootstrap consensus tree values from 1000 replicates are given at the branch nodes. Branches corresponding to partitions reproduced in less than 70% of bootstrap replicates are collapsed

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Sequence identity matrix was generated using Bioedit Sequence Alignment Editor (version 5.0.9). Upon comparison of complete DNA A sequence with other ToLCNDV isolates, it was found to exhibit more than 92% identity with ToLCNDV (KT426903 and KT426906) reported from Karnataka (Supplementary Table 1). Similarly, different ORFs on DNA A such as AV1, AV2, AC2, AC3 and AC5 were showing maximum identity with ToLCNDV infecting ridge gourd from Karnataka. Whereas ORF AC1 was having maximum identity (92.5%) with ToLCNDV (KP235541) from Tamil Nadu infecting chilli and ORF AC4 had 96% identity with other Indian isolates reported on poppy, cucumber, chilli and tomato (KC513822, KC545812, HM007113 and U15016, respectively). The inter-generic region between ORF AV1 and AC1 showed a maximum identity of 89.8% with ToLCNDV (KT426906) (Supplementary Table 1).

The recombination detection programme (RDP4) tool was for detection of probable recombination events, identification of likely parents and localization of possible recombination breakpoints. The analysis was performed with default settings using a 0.05 P value cutoff [8]. Two algorithms, GENCONV (P 1.243 × 10−01) and SiScan (P 7.510 × 10−37) showed the intraspecific recombination and identified as a ToLCNDV variant. One recombinant fragment (coordinates 40–72 in the IR region) was detected for ToLCNDV-infecting chayote in Tamil Nadu (TN TDK CHOU1). ToLCNDV-PkT5/6 (AF448058) from Pakistan infecting Tomato was indicated as the major parent, and ToLCNDV-RG5 (KT426907) infecting ridge gourd in Karnataka was minor parent. The recombination analysis results suggest that the ToLCNDV (TN TDK CHOU1) isolate evolved from the Pakistan strain and Indian strain by recombination.

In the present study, we report the association of ToLCNDV on the natural occurrence of severe mosaic disease on chayote based on the complete DNA-A sequence for the first time from Southern India. There are reports that showed ToLCNDV (DNA A and B) cause disease in a severe form upon association with DNA β molecule [18]. Since XbaI alone produced ~ 2.7 kb fragment in the restriction the digestion, attempt was made for the detection of DNA B and satellites (DNA α and DNA β) through PCR analysis. DNA B was detected in the samples with the two primer pairs (ToLCBD971F 5′-GTGGCAGAACGCCACCATGAACG-3′; ToLCBD2142R 5′-GCTGCGCGGCCAATATGTCAATAG-3′ and ToLCBD2081F 5′-GCGTACTCWACGCGCTCAGATTG-3′/ToLCBD656R 5′-GTGTTTCACAGA TTTCCTTACGCG-3′) as described by Venkataravanappa et al. [19]. All the tested samples were found to be positive for the presence of DNA B by producing amplification of ~ 1.2 kb with both the primer pairs. Similarly, samples tested were negative in the PCR analysis for the association of α-satellite and β-satellite with the universal primer pairs UN101/UN102 and β01/β02, respectively [1, 2]. The natural occurrence of mosaic disease on chayote in severe form is observed in the successive years under the farmer’s field at lower Palani Hills region (Kodaikanal) of Tamil Nadu. Distribution of ToLCNDV on tomato was mainly observed in North India while in southern part it is present on cucurbits. Patil et al. [11] reported occurrence of ToLCNDV on ridge gourd from South India causing ridge gourd yellow mosaic disease. Similarly [9, 10] had also documented ToLCNDV on bitter gourd and pumpkin. Previously [7] reported the occurrence of ToLCNDV on chayote from North Western India only based on the partial coat protein gene and the present study further confirms the occurrence of ToLCNDV on chayote leading to major threat for the cultivation of chayote in Southern India.

Roy et al. [13] reported that the natural occurrence of ToLCNDV on ash gourd in North India. It found to have its perpetuation even in the absence of main host and its subsequent spread to tomato. In North India, cucurbits are grown mainly during the Kharif season as sowing time of tomato coinciding the harvesting of cucurbits. But in Tamil Nadu, cucurbits are cultivated throughout the year concurrently with the tomato. In the epidemiological point of view, the occurrence of ToLCNDV on cucurbits is easily transmitted by the whitefly from the tomato crops grown near to the cucurbits field. Hence ToLCNDV in Tamil Nadu might be simultaneously perpetuating on both the tomato and cucurbits crops without any interruption. This finding will be helpful in deriving the management strategy for the begomovirus infection on cucurbits.