Occurrence of Tomato infectious chlorosis virus (TICV) in Jordan

Blackwell Publishing Ltd

G. H. Anfoka and M. K. Abhary

Department of Biotechnology, Faculty of Agricultural Technology, Al-Balqa Applied University, 19117 Al-Salt (Jordan); e-mail: anfoka@wanadoo.jo

A new disease on tomato (Lycopersicon esculentum L.) caused by Tomato infectious chlorosis virus (TICV) has been detected for the rst time in Jordan. Disease symptoms consisted of interveinal yellowing areas in older leaves followed by generalized yellowing. Using specic primers, Tomato infectious chlorosis virus was detected in symptomatic plants by RT-PCR. The amplied fragment (416 bp) was cloned and sequenced. Results of sequence analysis showed that the Jordanian isolate of TICV shared high nucleotide similarity (> 98%) with two other isolates from Japan and France. The distribution of TICV has been investigated in four regions in the Jordan Valley by non-radioactive dot blot hybridization. Data from the study showed high incidence of the disease in all surveyed regions. In addition, the expected size of the coat protein gene of TICV could be amplied from two symptomatic weeds species, Chenopodium album and Chenopodium murale, indicating that these weeds are natural hosts for the virus.

Introduction
Tomato infectious chlorosis virus (TICV) belongs to a group of bipartite, whitey transmitted closteroviruses, which includes Lettuce infectious yellows virus (LIYV) ( Klaassen et al., 1995) and Sweet potato sunken vein virus (Hoyer et al., 1996). The virus is transmitted in a semi-persistent manner by the greenhouse whitey, Trialeurodes vaporariorum (Westwood) (Li et al., 1998) and has long, exuous, lamentous particles that measure about 850 900 12 nm ( Duffus et al., 1996). The virus induces cytoplasmic vesicles characteristic of closteroviruses in the phloem of infected plants ( Wisler et al., 1996). TICV has a bipartite, single-stranded RNA genome, and the length of its genomic RNA 1 and RNA 2 strands are 7.8 and 7.4 kb, respectively. The virus infects several important crops and ornamental plants, including tomato (Lycopersicon esculentum L.), tomatillo (Physalis ixocarpa Brot.), potato (Solanum tuberosum L.), artichoke (Cynara scolymus L.), lettuce (Lactuca sativa L.), and petunia (Petunia hybrida Vilm.) (Li et al., 1998). Tomato plants affected by TICV show interveinal chlorosis symptoms that begin on lower leaves and gradually progressed to the upper ones. As symptoms develop, interveinal areas of leaves eventually become bright yellow except for the veins, which remain green (Hartono et al., 2003). Symptoms on tomato plants include interveinal yellowing, necrosis, and brittleness of leaves accompanied by severe yield loss. When TICV was rst found in the Irvine area of Orange County, California and in 1993, symptoms of the disease affected virtually 100% of tomato plants in every eld. The disease was associated with a high incidence of T. vaporariorum (Duffus et al., 1996). In one season in Orange County, growers suffered 2 million USD in losses (Wisler et al., 1998). The

disease has recently been reported in many countries in the world (Dovas et al., 2002; Verhoeven et al., 2003; Hartono et al., 2003; Segcv et al., 2004; Tsai et al., 2004; Delatte et al., 2005). For example, 80100% infection incidence was reported in some tomato elds in Greece (Dovas et al., 2002). Another virus, Tomato chlorosis virus (ToCV), that causes disease symptoms similar to those caused by TICV is transmitted by the whitey Bemisia tabaci and has been reported in many countries in the region, including Israel and Lebanon (Segcv et al., 2004; Abou-Jawdah et al., 2006). In 2004, unusual yellowing symptoms resembling those caused by nutrient deciencies have been observed in tomato crops grown in Jordan Valley. Disease symptoms were associated with a high population of whiteies and infections of tomatoes caused by Tomato yellow leaf curl virus (TYLCV) (genus Begomovirus, family Geminiviridae) were widespread in the same year (Anfoka et al., 2005). However, the symptoms induced by TYLCV in tomato differed considerably from the yellowing disease reported in this article. Plants affected by the yellowing disease exhibited symptoms that resembled those described in the United States for infections caused by Tomato infectious chlorosis virus (TICV) and ToCV (Duffus et al., 1996; Wisler et al., 1996, 1998). Therefore, attempts have been made in this study to analyze the etiology of the yellowing disease.

Materials and methods

Sample collection

In 2004, symptoms resembling those caused by TICV were observed on tomato plants grown under greenhouses. The intermediate and lower leaves of diseased plants showed brittleness, rolling and marked interveinal yellowing (Fig. 1);

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using the following parameters: one cycle at 94C for 2 min followed by 40 cycles at 94 C for 1 min, 58C for 45 s, and 72C for 1 min; with a nal extension at 72C for 5 min. RT-PCR products were separated by electrophoresis on 1% agarose gel in TBE (45 mm Tris-borate and 1 mm EDTA), stained with ethidium bromide, and photographed. DNA molecular weight markers (Promega, Co., Madison, WI, USA) were used to determine the size of the amplied fragments.
Detection of TICV by dot blot hybridization

Fig. 1 Leaet of tomato plant showing interveinal yellowing caused by Tomato infectious chlorosis virus.

plants appeared less vigorous, and in some cases fruits showed delayed ripening. These symptoms were not observed in young plants or fruits and the disease was associated with high populations of whiteies. Weeds such as Chenopodium album and Chenopodium murale showing chlorotic spots and blotches were observed in many tomato elds. Based on the fact that fully developed young tomato leaves at the onset of yellowing have the highest TICV concentration (Li et al., 1998), fully developed symptomatic leaves of tomato, C. album and C. murale were collected and kept at 20C for further use.
RNA extraction and amplication by reverse transcriptionpolymerase chain reaction

In an attempt to develop a test more suitable than RT-PCR for mass screening, an alkaline phosphatase labelled probe was synthesized by PCR with the AlkPhos Direct Hybridization Kit (Amersham Pharmacia, Piscataway, NJ, USA) using the TICVspecic primers and a RT-PCR-amplied fragment as the template according to the manufacturers instructions. The probe was used to detect TICV in tomato samples collected from four locations in Jordan Valley. Total nucleic acids were extracted from healthy or symptomatic tomato leaves as described above and diluted with 20 SSC to a nal concentration of 0.2 g/L. A positively charged nylon membrane (Biodyne B, Pall-Corporation, Pensacola, FL, USA) was spotted with 5 L of extracted RNA and the nucleic acids were xed using a UV cross linker (FLUO-LINK FLX). The probe was hybridized to the blot at 65C overnight and rinsed under high stringency conditions. Blots were exposed to lm for 35 h.
Cloning, sequencing and alignment analysis

Total nucleic acids were extracted from symptomatic and healthy tomato plants, as well as from symptomatic C. album and C. murale leaves that show chlorotic spots according to the method described by Gauthier et al. (1997). Healthy tomato plants were used as negative controls. Approximately 5 g of total RNA were used as a template for RT-PCR. The Access RT-PCR System kit (Promega Corp., Madison, WI, USA) was used to amplify a fragment (416 bp) of the coat protein (CP) gene of TICV. The upstream primer TICV-F: (5-AATCGGTAGTGACACGAGTAGCATC-3) is identical to the nucleotide sequence 282306 nt of the CP gene of TICV (DQ355217) and the downstream primer TICV-R (5-CTTCAAACATCCTCCATCTGCC-3) is complementary to the nucleotide 676 697 nt (Hartono et al., 2003). 50 L of the following RT-PCR mixture was made up: 10 L of 5X AMV/T PCR buffer, 1 L of 20 mm dNTP RT-PCR mixture consisted of 2 L of 25 mm MgSO4, 200 nm of each primer, 33 L of sterile distilled water, 1 L of T DNA Polymerase (5 /L), 1 L of AMV reverse transcriptase (5 /L), and added to 2.5 /L of extracted RNA. To allow cDNA synthesis, the tubes were incubated at 50C for 45 min and PCR was performed in a programmable thermal controller (PTC-200, MJ Research Inc., Watertown, MA, USA)

To conrm the identity of the RT-PCR amplied fragments, the TICV-specic 416 bp fragments amplied from tomato samples were ligated to pGEM-T Easy Vector (Promega, Co., Madison, WI, USA) and cloned according to the manufacturers instructions. Recombinant plasmids were isolated from transformed Escherichia coli JM109 according to Sambrook et al. (1989) and screened by restriction digestion with EcoR1 followed by electrophoresis in 1.2% agarose gel. The nucleotide sequence of one clone was determined in both directions at the Biotechnology Center (Madison, WI, USA) using the Big Dye sequencing kit (Applied Biosystems; Foster City, CA, USA). Sequences were checked for homologous sequences available from the GenBank using the BLAST program available at the National Center Biotechnology Information BLAST programme.

Results
Detection of TICV by RT-PCR

Leaf samples taken from symptomatic tomato, C. album and C. murale, and from healthy tomato plants were tested for TICV infection by RT-PCR. The expected size (416 bp) of the CP gene could only be amplied when the total nucleic acids obtained from the symptomatic tomato plants C. album and C. murale were used as templates (Fig. 2). No bands were observed

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When aligning amino acid sequence of TICV from France (DQ355217.1) with the sequence of TICV from Jordan, one base change was found at nucleotide position 215. This change resulted in an amino acid change from G to D.

Discussion
Tomato is grown extensively in Jordan Valley, however, infection with plant viruses such as Tomato yellow leaf curl virus, Cucumber mosaic virus, Tomato spotted wilt virus, Tobacco mosaic virus and Potato virus Y are considered the most limiting factors affecting their production. Recently, a new disease was observed in Dir Alla region, consisting of irregular chlorotic areas that evolved to interveinal yellowing, beginning on lower leaves and gradually progressing to the upper parts of the plant. These symptoms were more severe at the end of the growing season when plants become old. Farmers thought that these symptoms were due to nutritional disorders, pesticide toxicity, or natural senescence. This might explain the reason why TICV has not previously been reported in Jordan. Diseased plants were less vigorous, causing severe yield losses due to reduced fruit growth and delayed ripening. Similar symptoms were previously reported for TICV and ToCV (Wisler et al., 1996, 1998). Recently, many studies reported on the distribution of TICV worldwide. However, this disease has never previously been reported in Jordan. RT-PCR is a sensitive and simple technique that can be used to detect TICV in different plant species (Li et al., 1998). In this study, a fragment of CP gene of TICV could be amplied from symptomatic tomato, C. album and C. murale plants by RTPCR. The identity of the amplied fragment was conrmed by sequence analysis. It is important to mention that large numbers of weed species were distributed in most tomato elds surveyed in this study. Some of these weeds, like C. album and C. murale, showed virus-like symptoms, while others were symptomless. TICV could be detected only in symptomatic C. album and C. murale but not in asymptomatic weeds. Similarly, Font et al. (2004) were able to detect TICV in C. album and C. murale in tomato elds in Murcia, Spain. These weed species are widely distributed in Jordan Valley and they can serve as reservoir for TICV. In the presence of the insect vector, Trialeurodes vaporariorum (Westwood), the disease can spread rapidly to other elds and cause large yield losses. Therefore, farmers have to eliminate all weeds in the eld and to include this practice in an integrated programme together with chemical control of the insect vector to reduce the effect of the disease. Several studies have reported on the occurrence of TICV in many countries in the Mediterranean basin such as Spain, Greece, Italy and Portugal (Font et al., 2002; Vaira et al., 2002), however, to our knowledge, this is the rst report on the occurrence of TICV in Jordan. Recently, two studies reported the occurrence of ToCV in neighbouring countries, including Israel and Lebanon (Segcv et al., 2004; Abou-Jawdah et al., 2006). Since the geographical distance between tomato farms in Israel and in Jordan is very short and because B. tabaci, the

Fig. 2 Agarose gel electrophoresis of fragments amplied from plants infected with Tomato infectious chlorosis virus. Lane M, 1 kb DNA marker; lane 1, healthy tomato plant; lanes 2, 3 and 4, TICV-infected tomato, C. album and C. murale, respectively. The arrow indicates the location of the amplied CP fragment (416 bp).

Table 1 Detection of Tomato infectious chlorosis virus in Jordan Valley by dot blot hybridization No. of Fields 2 4 3 5 14 No. of positive samples/ No. of tested samples 12/12 11/15 8/10 9/11 40/48 Infection rate (%) 100 74 80 82

Location Dir Alla South Shoneh North Shouneh Wadi Shoaib Total

when extracts from healthy tomatoes were used. All attempts to detect ToCV in eld-collected samples by RT-PCR failed.
Detection of TICV by dot blot hybridization

Data presented in Table 1 show that dot blot hybridization has successfully been used to detect TICV in tomato samples collected from four regions in Jordan Valley. The highest disease incidence was recorded in Dir Alla where all tested samples were infected with the virus. On the other hand, the lowest disease incidence (74%) was reported in South Shoneh.
Sequencing and alignment analysis

Data of sequence analysis conrmed the identity of the RT-PCR amplied product obtained from symptomatic tomato plants. Results of the alignment analysis (Fig. 3) indicated that the Jordanian isolate of TICV shared high degree of nucleotide identity (> 98%) with TICV from Japan (AB085603) and France (DQ355217.1).

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Fig. 3 Multiple alignment of coat protein gene sequences of Tomato infectious chlorosis virus from Jordan (TICV-Jordan), isolate Gunma from Japan (AB085603.1), and isolate 17 from France (DQ355217.1). Dashed line indicates an identical base at a given position. Sequences of TICV-F and TICV-R primers are underlined.

insect vector of ToCV, is usually abundant in the Jordan Valley during tomato cultivation, attempts were made to detect ToCV by RT-PCR. However, all samples tested for ToCV were negative. This might be due to the low number of plants tested. The incidence of TICV in four regions of the Jordan Valley was investigated by dot blot hybridization. The disease incidence in all surveyed areas was high ranging, from 74 to 100%. This is in accordance with the fact that T. vaporariorum is an efcient vector in transmitting the virus. Since this is the rst report on the occurrence of TICV in Jordan more efforts have to be done to investigate the susceptibility of tomato hybrids commonly grown in Jordan to the infection with TICV. In addition, attempts have to be done to nd sources of resistance or tolerance and breeding them into cultivated tomato will be the most effective way to cope with this disease in the future.

Acknowledgements
This research was supported in part by the Middle East Research and Cooperation (MERC) project M21-037.

Prsence du Tomato infectious chlorosis virus (TICV) en Jordanie

Une nouvelle maladie de la tomate (Lycopersicon esculentum L.) due au Tomato infectious chlorosis virus (TICV) a t dtecte pour la premire fois en Jordanie. Les symptmes de la maladie consistent en des zones de jaunissement internervaire sur les feuilles les plus ges suivies par un jaunissement gnralis. En utilisant des amorces spciques, le Tomato infectious chlorosis virus a t dtect dans des plantes symptomatiques par RT-PCR. Le fragment ampli

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(416 pb) a t clon et squenc. Les rsultats de lanalyse de squence montre que lisolat jordanien du TICV partage une importante similarit de nuclotides (>98%) avec deux autres isolats du Japon et de France. La rpartition du TICV a t tudie dans quatre rgions dans la valle du Jourdain grce une hybridation dot blot non-radioactive. Les donnes de cette tude montrent une forte incidence de la maladie dans toutes les rgions prospectes. En outre, la taille attendue du gne de la capside protique du TICV a pu tre amplie partir de deux espces adventices symptomatiques, Chenopodium album et Chenopodium murale, ce qui indique que ces adventices sont des htes naturels du virus.

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2007 The Authors. Journal compilation 2007 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 37, 186190