Field surveys for the Bemisia tabaci complex were conducted from 2009 to 2013 in Korea, and the results were compared with published data of the B. tabaci complex. Three species, MED, MEAM1, and JpL, were collected from several provinces. The MED was mainly collected in greenhouses, displacing the earlier invasive species, MEAM1, and the JpL species was collected in the field. JpL is newly confirmed as a unique species of B. tabaci species complex in Korea and Japan.
Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a globally distributed species complex, which includes several cryptic species (Brown et al. 1995; De Barro et al. 2000; De Barro et al. 2011). This species complex has been known to damage commercially important plants by direct feeding (Byrne & Bellows 1991) or by the transmission of begomoviruses (Geminiviridae) (Brown 2000). Currently, because of the lack of morphological characters, which can be used for distinguishing the 31 species of the B. tabaci complex (De Barro et al. 2011), several species of the complex have been mainly distinguished based on a threshold of genetic differentiation in one mitochondrial gene, cytochrome oxidase subunit I (COI) (Dinsdale et al. 2010), and a genetic differentiation of 3.5% has been used as the species genetic boundary. However, Lee et al. (2013) revealed that the species boundary is changeable with increasing reports of COI sequences and suggested a new genetic boundary of 4.0%.
Until now, 2 tentative species, Middle East-Asia Minor 1 (B biotype; ME AMI) and Mediterranean (Q biotype; MED), have been recorded in Korea: the MEAM1 species, which was identified based on 2 mitochondrial genes (large subunit ribosomal RNA (IrRNA) and small subunit ribosomal RNA (srRNA)), was reported in 2000 (Lee & Paul 2000), while the MED, identification based on IrRNA, was reported in 2005 (Lee et al. 2005). Until now, some research papers dealing with the B. tabaci complex have been published; however, these were mostly concentrated on responses to insecticides (Lee et al. 2012) and/or transmitted viruses (Lee et al. 2010; Park et al. 2012). As a result, information is not sufficient to understand the current status of the B. tabaci complex in Korea.
Recently, the number of invasive alien species has continuously increased in Korea because of increased global trade and developments in transportation (Hong et al. 2012). Thus, possibly, other species of the B. tabaci complex may have invaded Korea. Currently there are 6 species in Japan (Asia I, Aisa II, China, JpL, MED and ME AMI) (Ueda et al. 2008), and 14 species in China (Asia I, Asia II 1–4, Asia II 6–7, Asia II 9–10, China 1–3, MED, and MEAM1) (Hu et al. 2011). It is necessary to determine the distribution of other species (excluding MED and MEAM1) of the B. tabaci complex in Korea. Thus, in this study, we examined the distribution and diversity of the B. tabaci complex through a large-scale survey.
Sampling was conducted from Dec 2009 to Jul 2013 throughout 7 provinces of Korea: Gyeonggido (GG), Jeollanam-do (JN), Chungcheongbuk-do (CB), Chungcheongnam-do (CN), Gyeongsangnam-do (GN), Gyeongsangbuk-do (GB), and Jejudo (JJ). Adults, nymphs, and eggs were collected from vegetables, ornamental plants and weeds, and from urban as well as agricultural landscapes. Collection details, geographical locations, host plants and dates of collection are summarized in Table 1. A total of 276 whitefly adults, nymphs, and/or eggs were collected, and individual samples were preserved in 99% ethanol. Voucher specimens are deposited in the collection of the Institute of Insect Sciences at the National Academy of Agricultural Science, Korea.
Genomic DNA extraction was performed using DNeasy® Blood & Tissue Kit (QIAGEN Inc., Dusseldorf, Germany), according to the manufacturer's protocol. Each sample for extraction consisted of a single individual from the same colony. PCR amplification was conducted with one primer set, Cl-J-2195 (5′-TTGATTTTTTGGTCATCCAGAAGT-3′) and TL2-N-3014 (5′-TCC C AT G C ACTA AT CT G C C ATATTA-3′) (Simon et al. 1994), using AccuPower® PCR PreMix (Bioneer, Seoul, Korea) with the following thermal cycle parameters for 20 amplification reactions: initial denaturation for 5 min at 94 °C, followed by 34 cycles of 1 min each at 94 °C, 1 min at 52 °C, and 1 min at 72 °C, with a final extension for 5 min at 72 °C. PCR products were visualized on agarose gels after electrophoresis. Single bands were purified using a QIAquick PCR purification kit (QIAGEN, Dusseldorf, Germany). PCR products were sequenced in both directions by ABI 3730xl sequencer (Applied Biosystems). Resulting chromatograms were evaluated for miscalls and ambiguities and assembled into contigs in SeqManTMPro (version 7.1.0, 2006; DNAstarlnc., Madison, Wisconsin, USA). The sequences were visually checked individually for protein coding frame-shifts to avoid pseudogenes (Zhang & Hewitt 1996). Consensus files were aligned using Clustal X 1.83 (Thompson et al. 1997). All sequences are deposited in the GenBank (accession numbers given in Table 1). These sequences are not unique to previously reported COI sequences of B. tabaci.
For identifying samples, a neighbor-joining tree was constructed based on 47 new but not unique COI sequences together with 212 COI sequences of B. tabaci (including 31 species) from the GenBank ( http://www.ncbi.nlm.nih.gov/genbank/) and 4 COI sequences of B. atriplex, B. subdecipiens, and B. afer, as an outgroup. Alignments of nucleotide sequences were performed using CLUSTALX with default conditions. A neighbor-joining (NJ) analysis was conducted for the combined data set, in MEGA 5.0 (Tamura et al. 2011). Intra-specific genetic divergences were calculated by using a K2P distance model (Kimura 1980) of MEGA 5.0.
In the NJ tree, the 47 COI sequences were categorized into 3 species, MED, MEAM1, and JpL (Fig. 1). Among the 47 COI sequences, 29 COI sequences belonged to the MED species, with no genetic variations, while the 17 COI sequences belonged to the JpL species, in which divergences ranged from 0.0% to 0.2%. The one remaining COI sequence was referred to as MEAM1. Among the 33 reported haplotypes of MED (Fig. 1), the 29 COI sequences from Korea were identical to COI sequences reported from China, Croatia, Taiwan, (Dinsdale et al. 2010), France (Dalmon et al. 2008), Greece (Tsagkarakou et al. 2007), Japan (Ueda 2006; Boykin et al. 2007), North America (Mckenzie et al. 2012), Spain, U.S.A. (Shatters et al. 2009), and Uganda (Sseruwagi et al. 2005). Among the 5 haplotypes of JpL (Fig. 1), the 17 COI sequences from Korea were identical to either of 2 types, AB308114 and AB308116 of Japan (Ueda et al. 2008), and among the 28 haplotypes of MEAM1 (Fig. 1), the one COI sequence was identical to the COI sequence from USA, Spain, Australia, China, Colombia, Dominican Republic, France, Guadeloupe, India, Italy, Sicily, Saudi Arabia (Dinsdale et al. 2010), Israel (Hsieh et al. 2006), Reunion (Delatte et al. 2006), and Argentina (Viscarret et al. 2003).
From the large scale sampling, we observed that MED is widely distributed across Korea, being found in 7 of the country's 9 provinces, GG, GB, GN, JJ, JN, CB, CN. Also JpL was detected from GG, JJ, and JB (Fig. 2A). On the other hand, MEAM1 was only detected in GG. We compared our results with prior research papers (Lee et al. 2000; Lee et al. 2005; Park et al. 2010; Lee et al. 2012) and confirmed that there has been a considerable change in the relative abundance of MEAM1 and MED (Fig. 2) in that MED has been displacing the earlier invader, MEAM1. The displacement of an earlier invasive B. tabaci race by a new invasive race has been reported in several countries such as China (Liu et al. 2007) and Australia (De Barro et al. 2011). JpL had been reported only in Japan only until now (Ueda et al. 2008). In this survey, JpL was recorded in Korea for the first time. In Japan (Ueda et al. 2008) and Korea (in this study), most of the JpL samples were collected on the Japanese honeysuckle, Lonicera japonica Thunb. (Dipsacales: Caprifoliaceae). This is a native plant in temperate eastern Asia regions including Japan and Korea (Williams et al. 2001), suggesting that JpL may be mainly distributed in the East Asian region.
Collection of Bemisia tabaci samples in The Republic of Korea from 2009 to 2013.
Recently, Lee et al. (2010) reported that the Korean MED had the same IrRNA sequence as those from Iran (AF247525) and Nigeria (AF247526), suggesting that this species was introduced either from Africa or the Near East to Korea. However, because these are unpublished sequences, this finding is not conclusive. In this study, we observed that the 29 COI sequences of the MED species from Korea were identical to those from wide areas of the world (including China, Croatia, France, Greece, Japan, North America, Spain, Taiwan, U.S.A., and Uganda), indicating that the place of the origin of this putative species is unsettled.
This study was supported by a grant of the Research Program for Agricultural Science & Technology Development (Project No. PJ008946), National Academy of Agricultural Science, Suwon, Korea. A grant was also supported by the Ministry of Food, Agriculture, Forestry and Fisheries.