Department of Biotechnology, Faculty of Agricultural Technology, Al-BalqaÕ Applied University, Al-Salt, Jordan Molecular Characterization of Jordanian Isolates of Cucurbit yellow stunting disorder virus M. M. Sweiss Sweiss 1 , G. G. Anfoka Anfoka 1 and and Y. Y. Abou-Jawdah Abou-Jawdah 2 AuthorsÕ addresses: 1 Department of Biotechnology, Faculty of Agricultural Technology, Al-BalqaÕ Applied University, Al-Salt, Jordan; 2 Plant Sciences Department, Faculty of Agricultural and Food Sciences, American University of Beirut, Riad El Solh, Beirut 11072020, Lebanon (correspondence to G. Anfoka. E-mail: anfoka@wanadoo.jo) Received February 8, 2007; accepted March 16, 2007 Keywords: single-strand conformation polymorphism, sequence analysis, cucurbits Abstract Cucurbit yellow stunting disorder virus (CYSDV) causes high yield losses to cucurbits in many parts of the world. In 1995, it was detected for the first time in Jordan but Jordanian isolates have never been charac- terized at the molecular level. In 2005 and 2006, leaf samples (2344) from symptomatic plants were collected from Jordan Valley, Ma’daba, Al-Mafraq, Al-Karak, Jarash, Al-Tafila, Al-BalqaÕ, Al-ZarqaÕ, Amman and Irbid. Detection by tissue blot immunoassay (TBIA) showed that the infection rate of CYSDV in collected samples was 58.5%. The coat protein (CP) gene of CYSDV was amplified from 36 selected samples by IC-RT-PCR. The amplicons (753 bp) from 16 isolates, representing all surveyed regions, were cloned and used for the single-strand conformation polymorphism (SSCP) analysis. Seven different SSCP patterns (P1– P7) were observed for the analysed Jordanian isolates. For further characterization, the CP genes from iso- lates with different SSCP patterns were sequenced, and the sequences were deposited in the GenBank under the accession numbers DQ903105–DQ903111. Sequence alignment and phylogenetic analysis revealed that all CYSDV isolates investigated in this study were most closely related to isolates previously reported to belong to the Western group of CYSDV. Introduction Virus diseases are among the most important factors that limit the production of cucurbits wherever they are grown. Since the late 1970s, yellowing diseases of greenhouse- and open field-grown cucurbits incited by whitefly-transmitted closteroviruses caused significant yield losses in many parts of the world. These viruses include Beet pseudo-yellows virus (BPYV), Cucurbit yel- low stunting disorder virus (CYSDV) and Lettuce infec- tious yellows virus (LIYV) (Duffus, 1965; Van Dorst et al., 1980; Duffus et al., 1986; Brown and Bird, 1992; Ce´ lix et al., 1996; Livieratos et al., 1998). CYSDV belongs to the genus Crinivirus of the family Closte- roviridae (Livieratos et al., 1999; Martelli et al., 2002). The virus has a bipartite genome consisting of two single-stranded, plus sense RNA segments estimated at 9123 nt (RNA1) and 7976 nt (RNA2) encapsulated separately (Aguilar et al., 2003). In nature, CYSDV is transmitted in a semi-persistent, non-circulative man- ner by the whiteflies Bemisia tabaci (Gennadius) and B. argentifolii (Bellows& Perring) (Ce´lix et al., 1996; Martelli et al., 2002). The first detection of CYSDV was made in the United Arab Emirates in 1982 (Hassan and Duffus, 1991); since then, it has spread throughout the Mediterranean region, Europe and North America (Duffus, 1995; Ce´lix et al., 1996; Abou-Jawdah et al., 2000; Desbiez et al., 2000; Louro et al., 2000; Kuo et al., 2007). Disease symptoms induced by CYSDV consist of severe interveinal chlorosis and green spots on the old- est leaves. Spots appear between 14 and 22 days post- inoculation; clear symptoms are visible after 30 days (Sese et al., 1994; Ce´lix et al., 1996). Severe symptoms include complete leaf lamina yellowing, leaf-rolling and brittleness (Ce´lix et al., 1996). Although CYSDV has been detected in Jordan many years ago (Duffus, 1995), molecular characterization of Jordanian CYSDV isolates was not reported. Therefore, this work reports on the molecular variability observed in the coat protein (CP) gene between Jordanian isolates of CYSDV using single-strand conformation poly- morphism (SSCP) and sequence analysis. Materials and Methods Sample collection During the period 2005 and 2006, severe yellowing symptoms resembling those caused by CYSDV were observed on cucurbit crops in several cucurbit-growing regions in Jordan. These crops included cucumber (Cucumis sativus), melon (Cucumis melo var. reticulates www.blackwell-synergy.com J. Phytopathology 155, 557–562 (2007) Ó 2007 The Authors Journal compilation Ó 2007 Blackwell Verlag, Berlin doi: 10.1111/j.1439-0434.2007.01280.x