Molecular Characterization of Begomovirus on Cucumber in Java

Cucumber (Cucumis sativus L.) is one of the most widely cultivated horticultural plants in Indonesia. Cucumber production in 2013–2016 tends to decrease every year from 491.636 tons, 477.976 tons, 447.677 tons, and 430.201 tons, respectively (BPS, 2017). In addition, cucumber in the fields always showed unhealthy due to infection by many types of pathogens, including viruses. Some of these viruses have been reported to infect horticultural plants in several countries, especially Begomovirus which caused a serious problem in cucumber (Brown et al., 2002). Begomovirus is a genus member of Geminiviridae family that causes important diseases in horticultural plants, i.e. Solanaceae, Cucurbitaceae, and Malvaceae (Phaneendra et al., 2012; Venkataravanappa et al., 2012) and whitefly (Bemisia tabaci) is vector of Begomoviruses (Jones, 2003). Begomovirus infection causes curling, yellowing, and mosaic leaves in Cucurbitaceae (Ali–Shtayeh et al., 2014). Begomoviruses that infect the Cucurbitaceae namely, Tomato leaf curl New Delhi Virus (ToLCNDV), Squash leaf curl virus (SLCV), Squash leaf curl China virus (SLCCNV), Ageratum yellow vein virus (AYVV), and Pepper yellow leaf curl Indonesia virus (PYLCIV) (Bandaranayake et al., 2014; Wilisiani et al., 2014; Ali–Shtayeh et al., 2014). Moreover, Begomoviruses infection showed diverse symptoms such as curly leaf on melon in Bantul, Yogyakarta caused by PYLCIV, mosaic with leaf distortion symptoms on Cucurbitaceae caused by ToLCNDV (Wilisiani et al., 2014; Khameneh, 2016) and leaves yellowing, curl, blister, mottle, and small fruit caused by SLCV (Ali-Shtayeh et al., 2014). In addition, ToLCNDV is an important virus in horticultural crops in Asia (Mizutani et al., 2011; Pratap et al., 2011). Recently, ToLCNDV have been reported to infect cucumber in West Java and Bali (Mizutani et al., 2011; Haerunisa et al., 2016). Further, ToLCNDV reported on cucumbers in Tegal, Kalasan, Ngemplak, ABSTRACT


INTRODUCTION
Cucumber (Cucumis sativus L.) is one of the most widely cultivated horticultural plants in Indonesia. Cucumber production in 2013-2016 tends to decrease every year from 491. 636 tons, 477.976 tons, 447.677 tons, and 430.201 tons, respectively (BPS, 2017). In addition, cucumber in the fields always showed unhealthy due to infection by many types of pathogens, including viruses. Some of these viruses have been reported to infect horticultural plants in several countries, especially Begomovirus which caused a serious problem in cucumber (Brown et al., 2002).
We conducted field surveys on 2016 at several cucumber cultivation areas in different locations from previously reports (Septariani et al., 2014;Haerunisa et al., 2016) to collected cucumber leaf samples. Based on field survey, we found Begomoviruslike symptoms such as yellow leaves, blister leaves, mosaics, and upward curling of the leaf edges symptoms. This research aimed to detect the virus frequency, its distribution and identification of the causal of those Begomovirus(es) symptoms.

Determination of Virus Frequency
Serological detection was conducted using the Dot Blot Immunobinding Assay (DIBA) using specific antiserum of ToLCNDV and SLCV (DSMZ, Germany) according to protocol provided by Asniwita (2013). 0.1 gram of leaf tissue was crushed in 1 ml of tris buffer saline (TBS) to produce sap. Then, 2 µl of sap per sample was dropped onto the nitrocellulose membrane. The positive and negative controls were used in DIBA detection. After the sap dried, the membrane was immersed in a blocking solution of non-fat milk in TBS containing Triton X-100 with a final concentration of 2% for 2 hours. The membrane was washed with dH 2 O, then immersed in TBS containing 1 µl first antiserum (1:5,000 v/v) for 3 hours. The membrane was washed with TBST (TBS + tween 0.05%) and then the membrane was soaked in TBS buffer containing 1 µl second antibody (1:5,000 v/v) for 2 hours. The membrane washed again with TBST, then it was immersed in alkaline phosphatase buffer (pH 9.5) containing BCIP/NBT chromogenic substrates for 30 minutes until the color of positive samples on membrane change to purple. Further, the staining reaction was stopped by immersing the membrane in dH 2 O. Detection result was used to determine the virus frequency (VF) according to the following formula:

Virus Detection using Polymerase Chain Reaction (PCR)
The samples with dark purple color by DIBA test from each location were selected for detection of nucleic acid by PCR. The total DNA was extracted from infected leaves using CTAB method (Doyle & Doyle, 1987) and the total DNA was diluted in 50 µL nuclease free water.

DNA Cloning and Sequencing
DNA cloning was conducted by TA-cloning of PCR product into pTZ57R/T vector plasmid (InsTAclone PCR Cloning Kit, Thermo Scientific). The cloning steps consisted of ligation, transformation, and confirmation of DNA plasmid. The ligation and transformation steps were performed according to protocol provided by Thermo Scientific.
The preparation of DH5α competent cells was carried out following the method by Sambrook & Russel (2001). The transformation was conducted by the chemical method (InsTAclone kit, Thermo Scientific). The E. coli DH5α bacterial pellet was dissolved in 300 µl T-solution (A-solution mixed with B-solution with a ratio of 150 µl/150 µl, v/v), then incubated on ice for 5 minutes. Bacterial culture was centrifuged at 3,000 rpm for 5 minutes and the pellet was collected and was diluted in 120 µl T solution, then it was incubated on ice for 5 minutes. The ligated DNA plasmid was taken 2.5 µl and added 50 µl of competent cells, then the mixture was homogenized and incubated on ice for 5 minutes. The DNA transformants were cultured in LB agar containing ampicillin (100 mg/L), 10 µl IPTG 100 mM, and 50 µl X-gal 2%. The bacterial culture was incubated at 37°C for 16 hours.
DNA plasmid carrying recombinant was confirmed by colony PCR of white colonies using universal primer pairs M13F (-20) and M13R (-26) pUC. The positive DNA plasmid containing target insert then cultured in LB-broth containing ampicillin and incubated at temperature 37°C on shaker at 150 rpm for overnight. Isolation DNA plasmid recombinant was carried out according protocol provided by Crosa et al. (1994). Bacterial cultures was centrifuged for 1 minute at 5,000 rpm and the supernatant was discarded. The bacterial pellet was added by 40 µl TE pH 8.0 and 600 µl lysis buffer (TE + 4% SDS pH 12.45), then incubated for 30 minutes at 37°C. Subsequently, the suspension was added by 30 µl Tris 2 M pH 7.0 and 240 µl NaCl 5 M, and incubated for 4 hours on ice. The suspension was centrifuged for 1 minute at 10,000 rpm. The supernatant was collected and transferred to a 1.5 ml micro tube, then added by equal volume of ethanol absolute and centrifuged for 15 minutes at 10,000 rpm. The supernatant was discarded, and the plasmid DNA pellet was washed by adding 500 µl 70% ethanol and centrifuged for 10 minutes at 10,000 rpm.
Further, the 70% ethanol was discarded, and the plasmid DNA was dried for 30 minutes and then diluted in 50 µl TE buffer pH 8.0.
The plasmid DNA was send to First Base Malaysia for DNA sequencing. The matrix identity of nucleotide and amino acid was analyzed by BioEdit software, while the phylogenetic tree was constructed by MEGA V6.0 software using 1,000 times bootstrap.

Symptoms and Disease Incidence in the Field
During field surveys in several cucumber cultivation areas in Java, we found varying viruslike symptoms on cucumbers such as yellow mosaic with leaf malformation, leaf chlorosis, upward curling of the leaf edges, yellowing with vein banding, leaf blister, and stunting ( Figure 1). Previously, those types of symptoms were reported on Cucurbitaceae in India, Indonesia and Sri Lanka (Tiwari et al., 2010;Haerunisa et al., 2016;Sohrab et al., 2013).
Serological detection using specific antiserum of ToLCNDV and SLCV showed the average frequency of cucumbers positively reacted with ToLCNDV antiserum in East Java, Central Java, Yogyakarta, and West Java were 97.77%, 100%, 100%, and 92.77%, while SLCV were 93, 33%, 85.83%, 78.33%, and 89.99%, respectively (Table 1). It is indicated that the frequency of ToLCNDV and SLCV is very high detected on cucumbers in Java. ToLCNDV was previously reported to infect cucumber in Bali, Central Java, Yogyakarta, and West Java with disease incidence up to 100%, (Septariani et al., 2014;Haerunisa et al., 2016), while until present the existence of Squash leaf curl China virus (SLCCNV) found only in Bali with a disease incidence up to 80% (Wiratama et al., 2015).
ToLCNDV infection on cucumbers in East Java is the first report in this study. The highly frequency of ToLCNDV infection in the fields might due to highly whitefly population as vector with favorable environment temperature for the vector (25-40°C), and resistant cucumber varieties against ToLCNDV not available yet. The highly incidence of the disease is affected by the use of hybrid seeds throughout the growing season and the monoculture cultivation system (Maruthi et al., 2007). These facts in accordance with the conditions in the fields which are most farmers cultivate cucumber in monoculture system. In addition, the rapid spread of ToLCNDV in Java was facilitated by highly population of its vector Bemisia tabaci on cucumber, especially on underside leaves. Previously, Wiratama et al. (2015) reported that the high incidence of ToLCNDV and SLCCNV on cucumber in Bali might due to the high population of B. tabaci. Massive spread of ToLCNDV in cucumber in Java might be also caused by several factors, such as efficient ToLCNDV transmission by B. tabaci with persistent manner (one vector can transmit the virus to healthy plants), either virus or its vector have wide host range, and the presence of weeds surround cultivation area as reservoir plants as previously report by Tiwari et al. (2010).

Begomovirus DNA Amplification by PCR
Amplification of total DNA from samples positively reacted with ToLCNDV antiserum by PCR successfully amplified a DNA amplicon with size ±600 bp using a specific primer of ToLCNDV (Figures 2). However, samples positively reacted with SLCV antiserum did not amplified any DNA using SLCV specific primer, but positively amplified by ToLCNDV specific primer (data not shown). This indicates that SLCV antiserum cross-reacting against samples infected by ToLCNDV. The similarity of SLCV CP gene is closely to ToLCNDV . The similar results occurred in Soybean mosaic virus (SMV) antiserum reacted positively against samples infected by Bean common mosaic virus (BCMV) on soybean (Rahim et al., 2015), and Tomato spotted wilt virus (TSWV) antiserum reacted positively with samples infected with Chrysanthemum stem necrosis virus (CSNV) (Boben et al., 2007). Cross-reaction serologically can occur between viruses species belongs to same genus.
The primer pair T-2F/TS-2R and S2F/TS-2R successfully amplified ToLCNDV and SLCV on cucumber samples in West Java and Bali, respectively (Haerunisa, 2016). However, in this study, SLCV did not amplify by specific primer of SLCV, indicating that SLCV might not present yet to infect cucumber in Java.

Molecular Characterization of Begomovirus
The colony PCR of white colonies containing plasmid DNA of ToLCNDV confirmed that ToLCNDV successfully cloned. Ten ToLCNDV DNA clones from Nganjuk, Kediri, Tulungagung, Brebes, Klaten, Kulon Progo, Bogor, Indramayu, and Subang amplified the DNA with size ± 750 bp (Figure 3), which is consist of pTZ57R/T plasmid sequence ±150 bp and ToLCNDV DNA insert ± 600 bp.   Homology of nucleotide and amino acid sequences among ToLCNDV Java isolates was ranging from 95.6-99.2% and 99.7-100%, respectively (Table 2). These results revealed that all ToLCNDV Java isolates are same species as previously stated by Brown et al., (2012) that the similarity of nucleotide sequences of Begomovirus isolate more than 89% belongs to the same virus species. The ToLCNDV Java isolates showed highest nucleotides (96.1-98.1%) and amino acids (99.7-100%) homology to Klaten isolates (AB613825) and lower homology to isolates from other countries ( Table 2). Based on the sequences demarcation, Begomovirus is grouped in the same strain if the nucleotide sequences homology is more than 94% (Brown et al., 2012).
Phylogenetic analysis revealed that ToLCNDV Java isolates from Nganjuk, Kediri, Kulon Progo, Bogor, and Brebes are in the same cluster with ToLCNDV isolates from Klaten, Indonesia, and separated from ToLCNDV isolates that infected Cucurbitaceae from other Asian countries (Taiwan, Thailand, Japan, Bangladesh, Pakistan, India, and Iran) (Figure 4). DNA-A ToLCNDV from Klaten that infects cucumbers is classified as one strain of ToLCNDV, called ToLCNDV-Indonesia (Mizutani et al., 2011). ToLCNDV isolates from Indonesia that infect cucumbers are in one same cluster and those isolates separated distinctively to corresponding isolates from other countries. Based on the host plant, ToLCNDV Cucurbitaceae isolates are in a separate cluster from ToLCNDV Solanaceae isolates (Figure 4). These findings showed the existence of different ToLCNDV strains based on the area of origin and host plants. In addition, since the first report of ToLCNDV infection in Central Java by Mizutani et al., (2011), Septariani et al., (2014, Haerunisa et al., (2016), and also the present report clearly showed the rapid spreading of ToLCNDV in Java and Bali.

CONCLUSION
ToLCNDV was successfully detected with highly frequency from cucumber cultivations in Java. The ToLCNDV distributes evenly, while SLCV is not detect yet in Java. Phylogenetic analysis of nucleotide sequences revealed that the ToLCNDV Java isolates from Nganjuk, Kediri, Kulon Progo, Bogor, and Brebes are in the same cluster with ToLCNDV isolates from Klaten, Indonesia and separated distinctively to corresponding isolates from other countries.