Introduction
Because tomato is a good source of vitamin C, potassium, and vitamin K which contribute to a healthy diet in many countries (Lapidot et al., 2010), it is one of the most widely grown vegetable crops globally and is a highly cultivated crop in South Korea. Tomato cultivation has increased rapidly to match increased demand for consumption. However, warming temperatures caused by climate change and tomato-infecting viruses transmitted by insect vectors negatively affect the production and profitability of tomato in Korea (Lee et al., 2010; Kil et al., 2014a; Kil et al., 2015). Among these viruses, Tomato yellow leaf curl virus (TYLCV) was the primary cause of reduced tomato yields, and damage caused by TYLCV has been reported in Korea every year since 2008 (Lee et al., 2010; Kil et al., 2014b). TYLCV is a whitefly-transmitted member of the genus Begomovirus in the family Geminiviridae, and the genome consists of a single-stranded circular DNA of about 2.8 kb encapsidated in a twinned icosahedral virion (Moriones and Navas-Castillo, 2000). TYLCV-infected tomato plants show severe symptoms such as stunting, leaf curling and yellowing, which reduce both tomato yield and fruit quality (Papayiannis et al., 2011). Recently not only has introduction of a new biotype of the whitefly vector Bemisia tabaci to Korea been identified (Lee et al., 2010), but seed transmission has been reported as an added cause of TYLCV spread in Korea (Kil et al., 2016). TYLCV has been reported in mainly tropical and subtropical areas where the whitefly Bemisia tabaci is present (Czosnek and Laterrot, 1997). Before 2008, there were no reports of TYLCV occurrence in Korea; however, TYLCV was initially detected in Tongyeong, Gyeongsang-Do in 2008 and viruliferous B. tabaci was identified in 2008, after non-viruliferous whiteflies were initially isolated in Korea in 2005 (Lee et al., 2010).
Studies of the genetic diversity of TYLCV related to resistance breaking and seed transmission have suggested that recombination, which occurs frequently among geminiviruses, contributed to the genetic diversity of their populations and has led to the emergence of new viruses and diseases (Padidam et al., 1999; Sanz et al 2000; Pita et al., 2001; Saunders et al., 2002), including among TYLCV and other tomato-infecting begomoviruses (Padidam et al., 1999; Kirthi et al., 2002; Fauquet et al., 2005; Idris and Brown, 2005; Sawangjit et. al, 2005). More recently, the first TYLCV isolates reported from Korea were found to be most closely related to either Chinese isolates, to Japanese isolates, or to both, and all were putative recombinants between isolates from Portugal and Myanmar (Kim et al., 2011). In this report, we recently surveyed tomato cultivation areas in Chungchungnam-do and Daejeon for TYLCV, and sequenced TYLCV coat protein (CP) coding sequences to investigate virus divergence using phylogenetic analysis.
Materials and Methods
Sampling
Tomato tissues showing TYLCV-like symptoms of stunting, leaf curling and yellowing (Fig. 1) were collected production greenhouses in Chungchungnam-do (two sites) and Daejeon city (four sites); multiple symptomatic leaves were collected from each site, and bulked by location for DNA and total RNA extraction (see below).
Total RNA extraction and Reverse transcription-polymerase chain reaction assay for tomato-infecting RNA viruses
Total RNAs were isolated by a manual method using Trizol reagent (Life Technologies, Carlsbad, USA) from bulked leaves of collected samples. Extracted total RNAs were kept at - 70℃ and used as template to synthesize cDNA using SuPrimeScript RT Premix (2X) (Genetbio, Daejeon, Korea) with random hexamer priming. PCR primers for the detection of viruses were designed based on the nucleotide sequence of the coat protein gene of each virus by referring to the nucleotide sequences available from the National Center for Biotechnology Information Genbank (Table 1). PCR (RT-PCR) was performed with cDNA from total RNA using Blend Taq DNA polymerase (TOYOBO, Japan). Total volume of PCR was 20 μL containing 10 X buffer 2 μL, 2 mM dNTP 1 μL, forward primer 1 μL (10 pmol), reverse primer 1 μL (10 pmol), Blend Taq polymerase 0.1 μL, Total DNA 3 μL, DW. 11.9 μL. Conditions for PCR were: 2 min at 94℃, 30 cycles of denaturation at 94℃ for 30 sec, 58℃ for 30 sec, and extension at 72℃ for 1 min. The final PCR products were visualized under UV following 1.2% agarose gel electrophoresis.
Total DNA extraction and polymerase chain reaction of TYLCV CP coding seqeunce
Total DNA was isolated by a manual method using CTAB Extraction Buffer (iNtRON Biotechnology, Seongnam, Korea). From each composite sample, 200 mg of tissue was placed in 2 ml microcentrifuge tubes with two 4 mm stainless steel beads, frozen with liquid nitrogen, and homogenized to powder using a Vortex Genie 2 (Scientific Industries, Inc., USA) on setting 2700 rpm, before setting the tubes on ice. CTAB Extraction buffer (500 μL) including 1% β-mercaptoethanol by volume was added and the sample vortexed again. The CTAB/plant extract mixture was then incubated for 30 min at 65℃ in a water bath. After incubation, 500 μL phenol:chloroform:isoamyl alcohol (25 : 24 : 1) was added and mixed by inversion. After mixing, tubes were centrifuged at 4000 rpm for 10 min at room temperature. Then 350 μL of the upper, aqueous phase was transferred to a clean 1.5 mL microcentrifuge tube. To each tube, one tenth volume of 3 M sodium acetate (35 μL) and then three volumes of 100% EtOH (1155 μL) were added and mixed by inversion. The mixture was incubated for 10 min at room temperature. After incubation, the tubes were centrifuged at 12,000 × g for 10 min and the supernatant was carefully discarded. Pellets were washed by the addition of 80% EtOH and centrifugation at 12,000 × g for 5min at room temperature. The supernatant was discarded, and the DNA pellet was dried for 30 min. After drying, the pellets were dissolved in sterile distilled water (DW) and stored at - 20℃ until used as PCR templates.
PCR primers for the detection of TYLCV are shown in Table 1. PCR was performed with total DNA using Blend Taq DNA polymerase (TOYOBO, Japan). Total volume of PCR was 20 μL containing 10X buffer 2 μL, 2 mM dNTPs 1 μL, forward primer 1 μL (10 pmol), reverse primer 1 μL (10 pmol), Blend Taq polymerase 0.1 μL, Total DNA 3 μL, and DW 11.9 μL. Conditions for PCR were: 2 min at 94℃, 30 cycles of denaturation at 94℃ for 30 sec, 58℃ for 30 sec, and extension at 72℃ for 1min. The final PCR products were visualized under UV following 1.2% agarose gel electrophoresis.
Cloning and Sequencing CP amplicons
The CP coding region of TYLCV was amplified from the sample extracts using as above, and the c.777 bp amplified PCR product was purified with the Wizard® SV Gel and PCR Clean-Up System (Promega Inc., Korea), and cloned into the All in One™ Vector (All in one PCR Cloning Kit, BIOFACT Inc., Korea). In order to confirm the size of the cloned products, purified plasmids were digested with restriction enzyme EcoRI (New England Biolabs Ipswich, Massachusetts, USA) and visualized under UV following 1.2% agarose gel electrophoresis. After size verification by electrophoresis, sequences of CP clones were identified by Macrogen Inc. (Seoul, Korea) using universal primer M13F (- 20).
Phylogenetic Analysis
Phylogenetic trees of TYLCV CP nucleotide (nt) and amino acid (aa) sequences were constructed using the neighbor joining method with 1,000 bootstrap replicates in MEGA version 6.0. Additional TYLCV CP sequences were obtained from NCBI GenBank (Table 2). Tomato yellow leaf curl Mali virus (TYLVMV; genus Begomovirus) CP nt and aa sequences were used as the outgroup to root trees.
Results and Discussion
Because previously reported symptoms of TYLCV infection in tomato included severe leaf rolling, yellowing and stunting (Moriones and Navas-Castillo, 2000), we collected tomato samples that showed similar symptoms in Daejeon and Buyeo-gun Chungchungnam-do, South Korea in 2018 (Fig. 1). Tomato leaf tissues that showed typical TYLCV symptoms were all positive for the anticipated 777 bp TYLCV PCR product, and no other tested viruses (Cucumber mosaic virus [CMV], Tomato infectious chlorosis virus [TICV], Tomato chlorosis virus [ToCV], Tomato mosaic virus [ToMV], and Tomato spotted wilt virus [TSWV]) were detected (data not shown). No tests were carried out for either Tomato bushy stunt virus or Southern tomato virus, both of which have been reported in Korea in recent years (Kim et al., 2007; Oh et al., 2018). To compare the sequence variability of the newly collected isolates to those of other isolates and previous reported TYLCV CP sequences that were collected in same region in 2017 (Choi et al., 2018), cloned TYLCV CP amplicons were sequenced. Five and ten TYLCV CP clones were obtained from tissues collected from Daejeon and Buyeo-gun Chungchungnam-do, respectively. Sequence analysis indicated one type among the five Daejeon clones and seven different sequences types among ten Buyeo-gun Chungchungnam-do clones. CP sequence analysis showed 97.17 - 98.84% nt and 98.45 - 99.22% aa identity, respectively (Table 2). Interestingly, only eight CP aa positions differed among 2018 and 2017 Buyeo-gun Chungchungnam-do isolates (Table 3). Comparison to the 2017 Buyeo-gun TYLCV-BK1 CP sequence, revealed highest aa identity up to 99.2% was revealed in 2018 Buyeo-gun Chungchungnam-do collected samples, while the CP clone from Daejeon (about 45 km away from Buyeo-gun Chungchungnam-do) showed 98.45% aa sequence identity; a 2017 isolate from Nonsan (TYLCV-N; MG787543) (Choi et al., 2018) shared 98.83% aa identity to TYLCV-BK1 (Table 3). Recently, Kil et al. (2015) reported that TYLCV can overwinter in greenhouses in a weed host (Stellaria aquatica), and weeds Lamium amplexicaule, Achyranthes bidentata, and Veronica persica, and sweet peppers (Capsicum annuum) were also identified as potential TYLCV reservoirs in Korea (Kil et al., 2014a, b, 2015). Multiple other naturally TYLCV-infected weed species have also been identified in other countries (e.g., Ioannou et al., 1987; Jordà et al., 2001; Salati et al., 2002; Papayiannis et al., 2011), so further examination of weeds near infected tomato crops may result in identification of additional potential weed reservoirs in Korea. It should also be noted that viruliferous B. tabaci can overwinter in greenhouses (Ohnesorge et al., 1980), but that a plant host for TYLCV would also need to be present as a virus source.
Additional phylogenetic analysis of nt sequences of the viral CP coding sequence deposited in National Center for Biotechnology Information (Table 2) showed that all of the new isolates of TYLCV were related to Korean isolates from Hwaseong, Goseong, Busan, Boseong, Nonsan, Jeonju, and Jeju (Fig. 2). Notably, despite all having ≥ 97.1% nt and ≥ 98.4% aa CP identities to 2017 isolate BK1 (Table 3), the phylogenetic tree in Fig. 2 shows that BK1 is in a ‘China’ clade with the two Jeju isolates, plus Nonsan (GU325632), Jeonju, and Chinese isolates Sh10 and ZJHZ12, each of which have genomes of 2781 nt (Lee et al., 2010; Kim et al., 2011); this suggests that isolate BK1 may also have a 2781 nt genome. The Hwaseong isolate (which has a 2775 nt genome) is separated from other Korean isolates and appears to belong to the ‘Japan + China’ group (Kim et al., 2011). All of the other 2018 Korean isolates (JP-B1 to JP-B7, plus JP-D5), and 2017 isolate TYLCV-N (MG787543) fall within a separate ‘Japan’ clade including the Boseong, Busan, and Goseong isolates, which all have genomes of 2774 nt (Kim et al., 2011; Kil et al., 2014a). The primary distinction between the 2774 nt and 2781 nt genomes is not in the protein coding regions, but the presence of an additional 7 nt in the Intergenic Region (IR) (Kim et al., 2011). That the isolates with full genome sequences are still differentiated in a phylogenetic tree based on the CP region implies that the new TYLCV isolates would also be divided into similar clades in trees based on their full genome sequences and IR lengths – and that they are therefore less uniform than the pairwise sequence comparisons to 2017 isolate BK1 in Table 2 suggest. In the report of Kim et al. (2011), the 2774 nt Boseong and Busan isolates fell into the ‘Japan cluster’, the 2781 nt Jeju, Nonsan (GU325632), and Chinese Sh10 and ZJHZ12 isolates into a ‘China cluster’, and the Hwasoeng isolate (2775 nt) with Japanese Han and Chinese AH1 isolates (each 2781 nt), into a separate ‘Japan + China cluster’ (Kim et al, 2011). In our tree (Fig. 2), the Portugese Mld-PT isolate (2793 nt) also grouped with the ‘Japan + China’ cluster, which is thus most diverse in both genome length and geographic origin. These results suggest that the Buyeo and Daejeon isolates of the current study and the 2017 isolates BK1 and N (Choi et al., 2018), despite being collected within a small region, may represent multiple introductions, as previously suggested for isolates collected over a much larger area within Korea (Kim et al., 2011).
TFig. 2. he phylogenetic tree was constructed by the neighbor joining method with 1000 bootstrap replicates, using the complete nucleotide sequences of the Tomato yellow leaf curl virus (TYLCV) coat protein (CP) of the 2018 Korean isolates (indicated with ●) and the 2017 Buyeo-gun and Nonsan isolates (▲). CP sequences of 14 additional TYLCV isolates from Korea, China, Japan or Portugal were obtained from National Center for Biotechnology Information GenBank. The CP gene of Tomato yellow leaf curl Mali virus (TYLCMV) was used as the outgroup to root the tree. Isolates are indicated in the tree by isolate name/accession number/country/host of origin. The ‘Japan’, ‘China’, and ‘Japan + China’ clusters are based on those of Kim et al. (2011).
Conclusion
We found that TYLCV infections were the main cause of tomato leaf-curling viral diseases in 2018, without any of the mixed infections detected in 2017 (Choi et al., 2018). The most important feature in the epidemiology of TYLCV is that this virus is transmitted by the whitefly B. tabaci (Cohen and Harpaz, 1964). The geographic range of this insect is closely related to temperature, and its density increases with increasing temperature (Zhou et al., 1997). In addition, viruliferous whiteflies and overwintering weed hosts are potential factors affecting transmission (Ohnesorge et al., 1980; Kil et al., 2015). The local farm environment factors, such as weeds and whitefly populations, could be affecting TYLCV incidence and resulting in yield losses in the tomato farms analyzed, and it will be important to determine the range of plants which act as reservoirs for both TYLCV and the whitefly vectors between tomato crops. The apparent presence of three different groups of isolates (including TYLCV-Hwaseong) within a small area suggests that TYLCV has become established within Korea, resulting from multiple introductions, and TYLCV is likely to persist as a significant problem in tomato production in the absence of rigorous control measures. Therefore, farming management practices that control whitefly pests and indigenous overwintering alternative host plants of both virus and vector are very important for minimizing the damage caused by TYLCV in tomato cultivation in Korea.
Acknowledgements
This study was financially supported by research fund of Chungnam National University in 2017.
Authors Information
Oh June-Pyo, Chungnma National University, Department of Applied biology, Master student
Choi Go-Woon, Chungnma National University, Department of Applied biology, Master
Kim JungKyu, Chungnma National University, Department of Applied biology, Researcher
Oh Min-Hee, Chungnma National University, Department of Applied biology, Master student
Kim Kang-Hee, Chungnma National University, Department of Applied biology, Undergraduate student
Park Jongseok, Chungnma National University, Department of Horticulture Science, Professor
Lesile L. Domier, University of Illinois at Urbana-Champaing, Department of Crop Sciences, Professor
John Hammond, United States Department of Agriculture-Agricultural Research Service, Researcher
Lim Hyoun-Sub, Chungnma National University, Department of Applied biology, Professor
