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Association of Tomato leaf curl Sudan virus with leaf curl disease of Squash in Saudi Arabia

ABSTRACT

During the field survey of cucurbits field in 2014-15, severe leaf curl disease of Squash was observed in the Kingdom of Saudi Arabia. The naturally infected plants exhibited typical upward severe leaf curling with stunted plant growth. The causative agent was identified by PCR using coat protein gene specific primers. Whiteflies were also observed in the cucurbits field indicated the possible begomovirus infection. An amplicon of 750 bp from naturally infected leaf samples was obtained which confirmed the association of a begomovirus with squash leaf curl disease. The full length betasatellites were also amplified from infected samples by using betasatellites specific primers. The rolling circle amplification was used to amplify the full viral genome. The full-length as well as betasatellites genomes were cloned and sequenced bidirectionally.

The highest sequence identity (93.7%) of full genome was observed with Tomato leaf curl Sudan virus-Tomato- Hail isolates. The Phylogenetic tree analysis of full genome formed closed cluster with Tomato leaf curl Sudan virus -Tomato-Jeddah, Hadasham and Tabuk which provided the strong evidence about the association of Tomato leaf curl Sudan virus with squash leaf curl disease in Saudi Arabia.

Keywords: Tomato leaf curl Sudan virus, Squash leaf curl disease, Begomovirus, Saudi Arabia.

INTRODUCTION

Cucurbits are important vegetable crops grown worldwide and their production limits by various Sayed Sartaj Sohrab

1Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Post Box No. 80216, Jeddah 21589, Saudi Arabia.

*Corresponding author: [email protected], [email protected] Received : 14 May 2012 Accepted : 10 June 2017

diseases. Begomoviruses (family Geminiviridae, genus Begomovirus) are recognized as infectious agents and due to global spread of whiteflies vector, begomovirus causing economic losses in crops in tropical and sub- tropical zones have been major threat to worldwide . The family Geminiviridae comprises small circular single-stranded DNA viruses that cause severe diseases in major crop plants worldwide. Based on genome organization, host range, and type of insect vector, currently the family Geminiviridae is divided into nine genera Becurtovirus, Begomovirus, Capulavirus, Curovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocovirus and Turncurotovirus. Members of the largest genus, begomovirus have both bipartite and monopartite genome which is transmitted by whitefly (Bemisia tabaci). The bipartite begomovirus genome has two ssDNA molecules (2.7 kb), known as DNA-A and DNA-B while mono-partite begomovirus has only DNA-A with satellite molecule known as betasatellites and alphasatellites. Betasatellites are small (1.4 kb), highly diverse with circular ssDNA. The new world virus contains only bipartite begomoviruses while Old World comprises monopartite begomovirus and DNA- 1 satellites molecules known as alphasatellites, betasatellite and delta satellites. A satellite molecule, known as â DNA is circular ssDNA with (~1350 nt) with bC1 gene has been reported to be associated with some monopartite begomoviruses . The replication and encapsidation of betasatellite is fully dependent on their helper virus and they play an important role in symptom expression and disease development .

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farmer's field at multiple locations (Jeddah, Hadasham, Tabuk, Hail) in Kingdom of Saudi Arabia. The disease was observed as marginal rolling and curling of leaves and stunting of plants (Fig. 1A). Naturally infected symptomatic and non-symptomatic top emerging leaves were collected from field infected squash crops and immediately kept in ice with self-sealing plastic bags and brought to lab for further processing. The causative agent was transmitted by whiteflies to squash seedlings. The affected plants produced less flowers and fruits. The presence of whiteflies (B. tabaci) was also observed in and around the field and under the infected plants. The causal virus was successfully transmitted to healthy squash seedling. The adult whiteflies were allowed to feed on infected squash plants for a 24-h for virus acquisition and then transferred onto healthy squash seedlings (twenty B. tabaci per seedling) to transmit the causal virus by giving 24-h inoculation access period. As a result, the causal virus was successfully transmitted from naturally infected to healthy squash seedlings which further developed similar leaf curl symptoms after 18–23 days post inoculation (dpi), suggesting Koch's postulates.

Full genome amplification, cloning and sequencing The total genomic DNA was extracted from 100 mg freshly collected young leaf samples exhibiting severe leaf curl symptoms on squash plants by using a DNeasy Plant Mini Kit (QIAGEN, Valencia, CA, USA) and finally eluted in 50 µl nuclease free water. Extracted DNA (100 ng) was used for PCR amplification. The begomovirus infection was confirmed by coat protein (CP) gene amplification using specific CP gene primers ( T o L C S D V ) ( F o r w a r d : T A A G G G C C C G T G A T T A T G T T G ) ( R e v e r s e : T T T A T T A A T T C G ATATTGAATCAT-. The polymerase chain reaction (PCR) reaction mixture consisted of 2.5 units of Taq DNA polymerase (MBI Fermentas, USA) 5 µl of 10xPCR buffer and 1 µl of 10 mM dNTPs, 1 µl of forward and reverse primers. Total reaction volume was made up of 50 µl using sterile distilled water. PCR amplification conditions were standardized employing different temperature of denaturation, annealing, and extension based on Tm value of the primers. The successful In the early 2000s, only two begomoviruses

were introduced into the eastern Mediterranean basin:

Squash leaf curl virus (SLCV) and Watermelon chlorotic stunt virus (WmCSV) and their frequent migration have been observed in Middle Eastern countries like Egypt, Israel, Jordan, Lebanon and Palestine (Lapidot et al;

2014). The presence of Squash leaf curl virus (SLCV) in Saudi Arabia has been reported for the first time in 2002 and further reported in 2012 . Tomato leaf curl Sudan virus (ToLCSDV) a monopartite begomovirus containing DNA-A and associated betasatellites and is transmitted by whitefly vector (Bemisia tabaci). In 1950s–1970s, begomovirus disease in cucurbits was of minor importance in India. The association of begomoviruses causing disease in squash have been reported from various parts of the world and they are designated as Squash yellow mild mottle virus (SYMMoV) and Pepper golden mosaic virus (PepGMV) from Costa Rica, Squash leaf curl China virus (SLCCNV), Squash leaf curl China virus-[Wax gourd] from Thailand ((SLCCNV-TH], Squash leaf curl virus (SLCV-Eg) from Egypt (Chickpea chlorotic dwarf virus (CpCDV)- Egypt, and from India, many begomoviruses are reported to cause disease in squash known as Pumpkin yellow vein mosaic virus (PYVMV), Squash leaf curl china virus (SLCCNV), Tomato leaf curl New Delhi virus (ToLCNDV), Tomato leaf curl Palampur virus (ToLCPMV) and Pepper leaf curl betasatellite (PepLCB) and a variant of Squash leaf curl China virus (SLCCNV) has been reported recently.

Currently, the natural infections of ToLCSDV on tomato and weeds have been reported earlier and it is possible that causative agent has been transmitted to squash through whiteflies from tomato as well weeds growing near by the squash field. The objective of this study was to characterize the causal virus associated with the squash leaf curl disease.

MATERIALS AND METHODS

Field survey, sample collection and whiteflies transmission

To collect the virus infected samples, a survey was conducted in the month of July-August 2014-2015 in and around the vegetable field. During the cropping season, leaf curl disease of squash was observed in the

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amplification of CP gene was obtained using the following program. First, denaturation of DNA at 94 C o

for 2 min then 35 cycles of denaturation (94 C for 1 min), o

o o

annealing (55 C for 30 s) and extension (72 C for 1 min).

At the end of cycles, final extension step at 72 C for 5 min o

was given. The presence of betasatellites was also confirmed by betasatellites specific primers and an amplicon of full-length betasatellites (~1.4 kb) were obtained from infected samples and cloned into pGEMT-easy vector (Promega Corporation, Madison, USA). The PCR amplified products were visualized on Agarose gel (1%) by using an Ultraviolet transilluminator. The purified genomic DNA from

symptomatic squash plant was used for amplification of full-length begomoviral genome by rolling circle amplification using TempliPhi 100 Amplification Kit (GE Healthcare, Life Sciences, Piscataway, NJ, USA) following the manufacturer's instructions. The amplified product was further digested by using selected restriction enzymes like Eco RI and Hind III and analyzed on a 1% Agarose gel. The expected size (~2.7kb) restricted product was gel purified and cloned into a pUC18 vector and transformed into competent E.

coli strain DH5a. The complete genome sequences assembled and submitted into GenBank under various accession numbers (Table 1).

Table 1. Pair wise (%) identities of full genome of ToLCSDV-Squash-Hadasham- KT760555 with selected begomoviruses

Accession Nos Abbreviation Location Host % Identity (nt) KT033712

KT728751 KT728750 KT728749 KT728748 KT728742 KT728741 KT033711 KT033708 KT728747 KF444467 KT760556 KT033714 KT033710 HG530539 JN591386 HE819244 JN591385 JN591386 AY044139 JX483708 KC763630 AY044137 AY044138 GU180085 JF919733 JF919734 JF919731 EF110891 KC106648

ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV ToLCSDV TYLCV

Hadasham Tabuk Tabuk Hail Hail Jeddah Jeddah Jeddah Hadasham

Hail Hail Tabuk Hadasham Hadasham

Usfan Oman Oman Oman Oman Sudan Sudan Sudan Sudan Sudan Sudan Yemen Yemen Yemen Yemen Iran

Squash Squash Squash Squash Squash Squash Squash Tomato Tomato Tomato Tomato Tomato Corchorus Amaranthus

Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato

91.2 90.9 90.9 90.8 90.9 91.0 91.0 93.2 91.2 90.9 93.7 91.2 91.1 91.2 91.3 92.7 92.8 92.7 92.7 91.0 90.2 88.0 87.8 82.6 87.5 92.1 91.5 89.6 89.5 79.2

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Sequence and Phylogenetic analysis

The newly generated sequences were aligned and resulting complete nucleotide sequence was initially searched for similarity using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/).

The highly similar begomoviral sequences data were selected and retried from GenBank and compared for their sequence identity matrix and similarity by using BioEdit software (http://www.mbio.ncsu.edu/

BioEdit/BioEdit.html). A phylogenetic tree was constructed using MEGA6 program from the aligned nucleotide sequences with neighbor joining and maximum parsimony methods using maximum composite likelihood for DNA substitution test .

RESULTS

Field survey, whiteflies transmission and virus detection

The field survey was conducted in many agricultural fields in August 2014-15 and severe leaf curl disease as the natural infection was observed on many squash plants (Fig 1A) growing nearby agriculture field. Approximately, 80% disease incidence was observed in the area surveyed. The causal agent was detected by PCR. An amplicon of 750 bp was successfully obtained from symptomatic samples and no amplicon was obtained from healthy samples. There were no any symptoms observed on negative control plants even after 40 days (Fig 1B).

Sequencing and phylogenetic analysis

The complete genome sequences assembled and submitted into GenBank under accession numbers (Table 1). The complete DNA genome contained six potential open reading frames (ORFs): V1 (coat protein), V2 (pre-coat protein) in virion sense strand while C1 (replication associated protein), C2 (transcription activator protein), C3 (replication enhancer protein) and C4 (C4 protein) in complementary sense, which were typical of Figure 1 A: Begomovirus infection on Squash

Figure 1 B: Detection of begomovirus by PCR from Various locations. (M. 1Kb ladder, 1-3, Jeddah,4-5- Hadasham, 6-7, Tabuk, 8-9, Hail.

Figure 2: Phylogenetic tree based on full genome of ToLCSDV-Squash with selected begomoviruses.

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begomovirus genome. The betasatellites sequences were submitted in GenBank with various accession numbers (KT728729, KT728730, KT728731, KT728735, KT728736, KT728737, KT728738, KT728739, KT199104).

The betasatellites sequences showed highest (99%) identity with ToLCSDV-Tomato isolates from Saudi Arabia and Yemen. The full genome nucleotide (nt) sequence analysis was conducted with selected begomovirus sequences by using ClustalW program.

The aligned nucleotide (nt) sequences of identified virus along with selected begomovirus isolates were used to construct phylogenetic tree using maximum likelihood in MEGA6 program. The complete DNA-A sequence shared maximum nucleotide sequence identity (93.7%) with ToLCSDV-Tomato-Hail (KF444467) followed by (93.2 %) ToLCSDV-Tomato- Jeddah isolate (KT033711) and (92.7%) with isolates from Oman. Interestingly, the ToLCSDV identified from weeds like Corchorus showed 91.1%- (KT033714) while Amaranthus showed 91.2%- (KT033714) identity with ToLCSDV-Squash-Hadasham-(KT760555) isolate (Table 1). The Phylogenetic tree based on the complete nucleotide sequences of DNA-A showed that, the virus isolates from Squash clustered with ToLCSDV-Tomato- Jeddah, Hadasham and Tabuk. Interestingly, ToLCSDV-Squash-had (KT760555) formed closed cluster with ToLCSDV-Tomato-Hail (KF444467) (Fig.2).

DISCUSSION

Although, begomovirus is a major viral pathogen in vegetables and Solanaceous crops and it has reported from several locations in Asia. ToLCSDV is an emerging problem in multiple vegetables and fruits crops as well as weeds and widely distributed in the Kingdom of Saudi Arabia. The present study established the association of ToLCSDV with the Squash leaf curl disease based on complete DNA-A sequence and betasatellites. During field survey leaf curl symptoms on squash and tomato crops growing in neighboring were observed. Interestingly, some weeds were also found to be affected with leaf curl disease. The percent nucleotide identity among complete genome sequences of the ToLCSDV isolates detected in this study was between 90-91%, indicating a low variability between

them. The phylogenetic relationships indicated that the full sequences of the ToLCSDV isolates are closely related to ToLCSDV-tomato isolate infecting tomato.

On the other hand, natural infections of many begomoviruses have been reported from various locations but the occurrence of ToLCSDV on Squash has not been reported earlier. The results discussed herein may be an example of the adaptation of begomoviruses to new hosts and environments. Movement of these viruses over large distances is likely occurring through movement of infected whiteflies and not by infected plant material. Consequently, differences in migration rates may be due to biological factors affecting whitefly movements within each country, perhaps related to the different time of year when the cucurbits crops are planted. Since, both pathogens share the same vector and can coinfect the same cucurbit and tomato plants in the same locations. A detailed study is required to confirm this hypothesis.

Due to frequent mutation, adaption and recombination in the existing viruses, the new viral strains and species emerges globally. So, it is an urgent need to perform characterization of begomoviruses periodically circulating in this region to determine the relationships between preexisting begomoviruses species and their hosts, and to identify arising new isolates/species.

Additionally, factors playing a key role in the evolution of begomovirus strains/isolates are urgently required to avoid their dispersal to other region and crops and future economic losses. However, this is the first observation of a ToLCSDV infecting Squash in the Kingdom of Saudi Arabia. Based on results obtained in this study and previously published reports, it is concluded that the tomato and weeds are playing an important role in the disease spread on cucurbit and this virus can be further spread to other crops in coming years during cropping season with more economic loss.

The prevalence of this disease needs further investigation at more locations in Kingdom of Saudi Arabia.

ACKNOWLEDGMENT: Author would like to thank General Directorate of Research Grants (GDRG), King Abdulaziz City for Science and Technology (KACST-

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Unit, King Fahd Medical Research Center (KFMRC), King Abdulaziz University, Jeddah, Saudi Arabia.

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