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MOLECULAR CHARACTERIZATION OF TomaTo yellow leaf curl virus (Tylcv) AND ITS INSECT VECTOR Bemisa tabaci

Shigenori UedaLowland Farming and Horticulture Research Division,

Kyushu Okinawa Agricultural Research Center, NARO, Japan 1823 Mii, Kurume, Fukuoka 839-8503, Japan

Email: sued@affrc.go.jp

ABSTRACT

Tomato yellow leaf curl disease (TYLCD) caused by Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) is one of the most devastating viral diseases of plants worldwide. TYLCV apparently arose in Israel in the 1930s and spread from the Middle East into the Mediterranean basin, North and South America, the Indian Ocean islands, Australia, and Asia. In Japan, both TYLCV-Israel and TYLCV-Mild strains are now widely distributed and have caused serious reductions in tomato fruit production. TYLCV is transmitted by Bemisia tabaci (Gennadius), an infamous insect pest distributed in the tropics and subtropics. It is a cryptic species for which genetic variation can be demonstrated in the absence of morphological variation. Some populations of B. tabaci biotypes, such as the B and Q biotypes, have developed high levels of resistance to certain insecticides. Currently, both populations have proliferated and become vector insects for TYLCV. We describe here the relationship between TYLCV and Bemisia tabaci based on molecular analyses.

Keywords: Tomato yellow leaf curl virus / Bemisia tabaci / molecular phylogenetic analysis

INTRODUCTION

Tomato fruit production is important worldwide, but viral diseases cause substantial economic losses. Tomato yellow leaf curl disease (TYLCD), caused by Tomato yellow leaf curl virus (TYLCV; genus Begomovirus, family Geminiviridae), is one of the most devastating viral diseases affecting tomato plants globally (Fig. 1. Salati et al., 2002, Mansoor et al., 2003). In Japan, TYLCV first caused significant yield losses in 1996 (Kato et al., 1998, Haga & Doi, 2002, Onuki et al., 2004), the first report of the virus' invasion into East Asia. Both the Israel (TYLCV-IL) and the Mild (TYLCV-Mld) strains of TYLCV have been detected in Japan (Kato et al., 1998, Ueda et al., 2004, 2005). Begomoviruses are transmitted be the Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) in a circulative manner. The vector insect of TYLCV, B. tabaci is also an important insect pest worldwide. A major research goal is to control TYLCV and B. tabaci without reducing the yields of affected vegetable and ornamental crops. This report summarizes my lab's previous research on the relationship between TYLCV and B. tabaci based on molecular phylogenetic analyses.

Molecular characterization of TYLCV and related begomoviruses

TYLCV is a monopartite begomovirus species with a single-stranded DNA genome of approximately 2.8 kb. Based on the recent taxonomy and nomenclature of geminiviruses, TYLCV consists of five strains — Israel (TYLCV-IL), Mild (TYLCV-Mld), Oman, Iran, Gezira — and is distinct from other TYLCV-like viruses, such as Tomato yellow leaf curl Sardinia virus (TYLCSV) (Fauquet et al., 2008, Abhary et al., 2007). In Japan, TYLCV was first recorded in 1996 in Shizuoka and Aichi Prefectures and in Nagasaki Prefecture (Kato et al., 1998). Analyses of complete nucleotide sequences from various TYLCV isolates in Japan revealed that the Shizuoka (TYLCV-Mld[JR:Shizuoka]) and Aichi (TYLCV-Mld[JR:Aichi]) isolates were closely related to the TYLCV-Mild strain (TYLCV-Mld), while those from Nagasaki (TYLCV-IL[JR:Nagasaki]) were closely related to TYLCV-IL (Kato et al., 1998, Onuki et al., 2004, Ueda et al., 2004). Since then, TYLCV quickly spread to Kyushu. In 2009, TYLCV had spread into 37 prefectures, including Okinawa (Ueda et al., 2008a), which has subtropical weather (Fig.

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2). TYLCV has also spread into the major tomato growing areas of Japan. In some districts, both TYLCV-IL and TYLCV-Mld have been detected. TYLCV has a high, RNA-like, rate of nucleotide substitution, which is independent of its frequent recombination. The mean genomic substitution rate of TYLCV was estimated to be 2.88 × 10-4 nucleotide substitutions per site per year (subs/site/year) (Duffty & Holmes, 2008). In 2003–2004, a genetically distinct isolate of TYLCV-IL, TYLCV-IL[JR:Tosa], was identified in Kochi Prefecture, on Shikoku. TYLCV-IL[JR:Tosa] was thought to be the third isolate introduced into Japan, because its chimeric genome suggested recombination between TYLCV-IL and TYLCV-Mild (Ueda et al., 2005). Sister isolates of TYLCV-IL[JR:Tosa] were recorded from Okinawa and Tochigi Prefectures in Japan and from some other countries. In China, TYLCV-IL was first reported in Shanghai in

2005 (Wu et al., 2006). The DNA-A nucleotide sequence of TYLCV-IL[CN:Shanghai 2:05] was almost identical with TYLCV-IL[JR:Tosa]. The TYLCVs isolated from eastern North America and the Caribbean were thought to have been introduced from Asia (Duffy & Holmes, 2007). In Spain, a natural recombinant between TYLCV and TYLCSV exhibiting a novel pathogenic phenotype has become prevalent (Monci et al., 2002). TYLCV-like viruses such as Tomato yellow leaf curl Axarquia virus (TYLCAxV) and Tomato yellow leaf curl Malaga virus (TYLCMalV) also represent novel recombinant begomoviruses (Abhary et al., 2007). It is unclear how this genetic group (TYLCV-IL[JR:Tosa] and sister isolates) is distributed worldwide, but their broad distribution suggests that they may have acquired advantageous traits for infecting host plants (Fig. 3).

Fig. 1. Tomato yellow leaf curl disease caused by Tomato yellow leaf curl virus.

Fig. 2. Map showing prefectures in which TYLCV has been detected in Japan

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Vector insect Bemisia tabaci

The insect vector of TYLCV, B. tabaci (Fig. 4), is the most economically important and widely distributed insect pest in the tropics and subtropics (Brown et al., 1995, De Barro et al., 2000). Taxonomic studies have shown that the B. tabaci complex is a cryptic species for which genetic variation can be demonstrated in the absence of morphological variation using molecular markers (Brown, 2000, Costa & Brown, 1991, Perring, 2001). In the southwestern United States, the B biotype displaced the indigenous A biotype in the field within a few years of its introduction (Brown & Bird, 1995), possibly because of differences in susceptibility to insecticides in use to control whitefly at the time. The B biotype transmits begomoviruses to a large number of crops, ornamentals, and weed species (Brown & Bird, 1995). In Japan, the B biotype of B. tabaci was first detected in 1989 and was associated with imported poinsettia plants (Ohto, 1990). Since then, it has dispersed rapidly throughout Japan (Matsui 1992, 1995). The Q biotype was detected in Japan in 2004 (Ueda & Brown, 2006, Matsuura, 2006). The use of neonicotinoid or pyriproxyfen insecticides selects

for the Q biotype, which exhibits greater resistance to these insecticides than does the B biotype (Nauen et al., 2002, Horowitz et al., 2005). By 2009, the Q biotype had become widely distributed throughout Honshu, Shikoku, Kyushu, and Okinawa islands (Kijima et al., 2011) in Japan (42 prefectures). In many regions, the displacement of the B biotype by the Q biotype has been particularly noticeable (Higuchi et al., 2007), perhaps due to selection by pesticides applied to cultivated crops. As a result, the Q biotype has become the major vector for TYLCV in Japan. Mitochondrial cytochrome oxidase I (mtCOI) sequences were previously used to reconstruct a phylogeography for representative populations or biotypes of B. tabaci (Flohlich et al., 1999). Therefore, we developed a simple mtCOI PCR-RFLP method to identify the Q biotype in field samples (Fig. 5; Ueda, 2006). The mtCOI PCR products (866 bp) from the Q biotype were digested with EcoT14I (StyI) into two fragments of 555 and 311 bp, while the PCR products from the B biotype were cleaved with StuI into two fragments of 560 and 306 bp. Japanese honeysuckle, Lonicera japonica (Caprifoliaceae), an evergreen perennial weedy vine native to eastern Asia, is the natural over-wintering host of Tobacco leaf curl Japan virus (TbLCJV), Honeysuckle yellow vein mosaic virus (HYVMV), and related Far East Asian begomovirus species (Kitamura et al., 2004, Ueda et al., 2008b). The indigenous Japanese honeysuckle population of B. tabaci (JpL biotype), which was earlier described as B. lonicerae Takahashi, feeds on Japanese honeysuckle growing in wooded areas (Miyatake, 1980). The JpL biotype of B. tabaci is thought to be the vector of indigenous begomoviruses in Japan (Osaki et al. 1976, 1979).

Fig. 3. Neighbor-joining phylogenetic trees showing relationships among complete DNA-A sequences of 17 TYLCF isolates. DNA-B sequence of a TGMV isolate (Acc. no. M73794) was used as the outgroup. Isolates reported from Japan are shaded. Fig. 4. Bemisia tabaci (Gennadius)

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Fig. 6. Phylogenetic tree of Bemisia tabaci mitochondrial cytochrome oxidase I gene sequences reconstructed using the Bayesian method and map showing the distribution of populations in Japan. The posterior probabilities support values >50% are shown at the nodes. Horizontal branch lengths are drawn to scale; the bar indicates 0.1 nt substitutions per site. The sequences of Trialeurodes vaporariorum and Bemisia afer were used as the outgroup. Populations from Japan are shaded (Ueda et al., 2009).

Fig. 5. Simple mtCOI PCR-RFLP method to identify the identification of Q biotype of Bemisia tabaci. The 866 bp mtCOI PCR products (A) from the Q biotype were digested with EcoT14I (StyI) into two fragments of 555 and 311 bp (B), while the PCR products from the B biotype were cleaved with StuI into two fragments of 560 and 306 bp (C).

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Phylogenetic analyses of B. tabaci globally separate it into 12 major groups, as follows: Mediterranean/Asia Minor/Africa (includes the B biotype), Mediterranean (includes the Q biotype), Indian Ocean, sub-Saharan Africa silverleafing, Asia I, Australia, China, Asia II, Italy, New World, sub-Saharan Africa non-silverleafing, and Uganda sweet potato (Boykin et al., 2007). In Japan, little was known about the relationship between the distribution of B. tabaci and its molecular genetic background since the invasion and dispersal of TYLCV. Using the Bayesian method, we analyzed sequences of mtCOI from B. tabaci populations collected from most regions of its Japanese distribution. The tree (Fig. 6) distinguished 12 major clades, together with an independent clade consisting only of JpL-biotype populations. In addition, we identified populations belonging to the Asia I, Asia II, and China genetic groups, together with invasive populations such as the B and Q biotypes (Ueda et al., 2009). Those populations were distributed in the subtropical region of Japan. Several distinct populations of B. tabaci have been reported from eastern Asia (De Barro et al., 2005, Hsieh et al., 2006, Li, 2006). The An, Nauru, and some other biotypes have been identified in Taiwan and on China’s mainland (Hsieh et al., 2006).

CONCLUSION

Several management strategies have improved the control of TYLCV and B. tabaci in Japan. To prevent whiteflies from invading greenhouses, improved equipment, such as fine-mesh screens (0.4–0.6 mm mesh size) and UV-absorbing plastic sheets, together with the application of insecticides have become popular. Recently, TYLCV-resistant cultivars have been planted widely in more areas of Japan. However, TYLCV-resistant cultivars are susceptible to other tomato-infecting viruses transmitted by B. tabaci, such as Tomato chlorosis virus (Crinivirus, Closteroviridae), Ageratum yellow vein virus, and Tobacco leaf curl Japan virus (Begomovirus, Geminiviridae). Recently, a novel begomovirus satellite DNA molecule, known as DNA-β, was shown to be associated with some monopartite begomoviruses. My lab demonstrated that TYLCV could trans-replicate with Ageratum yellow vein betasatellite and be transmitted by B. tabaci among tomato plants (Ueda et al., 2012). There is currently an urgent need to investigate and establish effective, sustainable management practices to control TYLCV and other whitefly-transmitted viruses, as well as B. tabaci itself, to

avoid further spread of these diseases in Japan and around the world.

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