Pomelo, a resistance variety to Citrus tristeza virus in peninsular Malaysia Kavous Ayazpour, Kamaruzaman Sijam, Ganesan Vadamalai, Hawa Jaafar Plant Protection Department University Putra Malaysia Serdang, Malaysia Email: kayazpour@yahoo.com Abstract- Citrus tristeza virus (CTV) is the most important viral disease of Citrus and is distributed worldwide. Results of ELISA and PCR methods showed that all Citrus varieties including Fortunella sp., Citrofortunella microcarpa and Citromelo in major citrus growing areas of Malaysia were infected with CTV in a high rate. In most areas, pomelo however was free of infection, but in Cameron Highlands, we found some strains of CTV that were severe to Citromelo and pomelo. Phylogeny studies revealed that these strains were familiar to CTV isolates from China and Japan and were very differ from CTV isolates of USA and New Zealand. Keywords- Citrus tristeza virus; Phylogeny analysis; Strain; CP gene; Malaysia I. INTRODUCTION Citrus tristeza virus (CTV) is a member of the genus Closterovirus (family Closteroviridae), which is distributed worldwide and causes one of the most economically important diseases of Citrus [1-6]. CTV particles are flexuous, threadlike, and 2000×10-12 nm in size [7-9]. They contain a positive-sense, single-stranded genomic RNA about 20 kb [8-11] with a molecular weight of 6.3-6.9×10 6 [12]. The RNA contains 12 open reading frames [1, 5, 11, 13] and encodes at least 19 proteins [1, 6, 8, 11]. Two of these are capsid proteins of 25 and 27 kDa, which coat respectively about 95% and 5% of the virus length [4, 7, 9, 14]. Members of Closteroviridae are unusual in their size, genomic composition and have a complex replication strategy [11]. CTV is phloem-limited and is transmitted in a semi-persistent manner by aphids such as Aphis gossypii [4, 15-17], Toxoptera citricida [4, 14-16, 18], Aphis spiraecola [15] and Toxoptera aurantii [15, 16]. Toxoptera citricida and Aphis gossypii are the most efficient vectors of CTV [4, 14- 16, 19]. The virus is genetically and biologically diverse. Virus isolate, Citrus cultivar, rootstocks, time of infection, and environmental conditions can affect the symptoms [1, 8]. A complex range of symptoms is produced under field conditions. There are three economically devastating field symptoms caused by CTV. The first is decline and death of trees grafted onto sour orange rootstock. The second is stem pitting of scions, regardless of rootstock [1, 17, 18, 20, 21]. Trees affected with CTV stem pitting strains, have reduced fruit production and quality. A third type of symptoms can cause losses in tree nurseries and is called ‘seedling yellows’. Symptoms of seedling yellows are leaf chlorosis and stunting of sour orange, grapefruit and lemon seedlings[9]. There were some reports on occurrences of CTV in Peninsular Malaysia that needs attention, as information on CTV in Malaysia is limited; Hence a research was carried out to determine distribution and host range of CTV in peninsular Malaysia. II. MATERIALS AND METHODS A. Plant materials Field samples were obtained randomly from 340 Citrus trees, including Citrus aurantifolia, C. sinensis, C. maxima, C. reticulata, C. hystrix, Citrofortunella microcarpa, Fortunella sp., seven Poncirus sp. and 10 weeds including Pasiflora foetida, Melothria pendula and Mikania micrantha growing in Selangor, Pahang, Johor, Terengganu, Perak and Kedah states in Malaysia. Mature shoots and leaves of Citrus plants were gathered from all sides of the trees pointing east, west, south and northward and then samples mixed for the test. Petioles, midrib of leaves and bark of shoots were used to prepare extractions for ELISA test and extraction of total RNA for RT-PCR. B. ELISA determination To diagnose Citrus trees infected by CTV, direct double antibody sandwich (DAS) ELISA was performed [22]. In this study one polyclonal antiserum (Bioreba) was used. Extractions were prepared from 0.5 g of shoot barks, midribs and petioles in 5 ml 0f 1× PBST buffer (0.15 M NaCl; 0.015 M NaH 2 PO 4 ; 0.05% Tween 20, pH 7.0). Positive reactions were defined as an OD 405nm two times higher than negative control. C. Nucleic acid extraction from Citrus tissues Total RNA (tRNA) was extracted from 0.2g of shoot barks, midribs and petioles. First tissues were pulverized with liquid nitrogen by pestle and mortar and then collected in 1.5 ml sterile eppendorf tube. Each sample was suspended in 400 µl TES buffer (100 mM Tris-HCl pH 8.0; 2mM EDTA; 2% w/v SDS) and 400 µl phenol/chloroform/isopropanol (25/24/1) and shook vigorously for 10 min. After centrifugation (14000 rpm) for 10 min, the supernatant (400 µl) was treated with 200 µl ethanol (99.8%) and used in RNeasy mini kit and tRNA extracted according to the manufacturer’s instructions and was used as template for the amplification of the coat protein (CP) gene of CTV. For amplification of the complete CP cistron (672bp) of CTV two primers were used based on Jiang et al. (2008) report [7]. The sense primer was CP1: 5′-ATG-GAC-GAC- 96 2011 2nd International Conference on Biotechnology and Food Science IPCBEE vol.7 (2011) © (2011) IACSIT Press, Singapore