African Journal of Biotechnology Vol. 11(80), pp. 14553-14560, 4 October, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB12.2121 ISSN 1684-5315 ©2012 Academic Journals Full Length Research Paper Identification of Turnip mosaic virus isolated from canola in northeast area of Iran Sabokkhiz M. A.1*, Jafarpour B.1, Shahriari Ahmadi F.2 and Tarighi S.1 1Department of Plant Pathology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. 2Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. Accepted 15 August, 2012 During March and April of 2011, 436 samples showing viral disease symptoms were collected from canola fields in the Khorasan Razavi province. The samples were tested by double-antibody sandwich (DAS)-enzyme linked immunosorbent assay (ELISA) for the presence of Turnip mosaic virus (TuMV). Among the 436 samples, 117 samples were found to be infected with TuMV. One of the infected samples from Govareshk region (TuMV-IRN GSK) was selected for biological purification. Total RNA of this isolate were extracted and reverse transcriptase (RT)-PCR was performed with specific primers according to the coat protein gene. PCR products (986 bp) was first purified and then directly sequenced. Phylogenetic analyses based on ClustalW multiple alignments with previously reported 33 isolates indicated 88 to 98% similarity in nucleotide and 94 to 99% in amino acid levels among isolates. TuMV-IRN GSK represented the highest identity to another Iranian isolate (IRN TRa6). Phylogenetic tree clustered all sequences into four groups and IRN GSK fell into the basal-B group. Nucleotide and amino acid distances between IRN GSK and other isolates in the basal-B group showed that this isolate was closely related to another Iranian isolate IRN TRa6, and distinct from other isolates in the basal-B group. These results indicate that TuMV is a common pathogen of canola crops in the Khorasan Razavi province. Key words: Turnip mosaic virus (TuMV), canola, reverse-transcription polymerase chain reaction (RT-PCR), coat protein gene, sequence analysis. INTRODUCTION Nowadays, oilseed rape or canola (Brassica napus L.), a plants by aphids in a non-persistent manner. The most member of the Brassicaceae family (Brassicaceae important vectors have been proposed to be Myzus Burnett, syn. Crusiferae Juss.) is the world’s third-leading persicae, Brevicoryne brassicae, Lipaphis erysimi, Aphis source of both vegetable oil and oil extraction meal, craccivora and Aphis gossypii (Sako, 1980; Wang and behind oil palm and soybean (Vollmann and Rajcan, Pirone, 1999; Dombrovsky et al., 2005; Ghorbani et al., 2010). In Iran, the areas of oilseed rape production are 2007; Haj Kassem and Walsh, 2008). The CP gene is a going to be more distributed, due to an increasing variable region in potyvirus genomes and has been demand for rapeseed oil. This increased production has widely accepted for classification of different isolates in led to prevalence of infectious diseases. Turnip mosaic the genus (Sanches et al., 2003). The complete virus (TuMV) is an important member of the Potyvirus sequences of Iranian, Italian, German, Canadian, genus, in the Potyviridae family. Wide host range of this Japanese, Danish, Russian, Polish, New Zealand and UK virus can infect several plant families including isolates have been determined and numerous partial Brassicaceae. TuMV could be transmitted to healthy sequences, especially of the coat protein (CP) and 3′- UTR, have also been obtained (Nicolas and Laliberte, 1992; Ohshima et al., 1996; Jenner et al., 2000; Kozubek et al., 2007; Chen et al., 2006; Farzadfar et al., 2005; *Corresponding author. E-mail: [email protected]. Jiang et al., 2010; Ohshima et al., 2010; Wang et al., Tel: +98-511-8795620. Fax: +98-511-8787430. 2009; Tomitaka and Ohshima, 2006). 14554 Afr. J. Biotechnol. Figure 1. Map of sampled regions in Khorasan Razavi province. Previous studies (Ohshima et al., 2002, 2007; TuMV has been reported already from different Tomimura et al., 2003, 2004; Farzadfar et al., 2009) have geographic region of Iran on stock plants (Matthiola sp.), shown that TuMV isolates had been biologically classified oilseed rape (Brassica napus), Cheianthus cheiri, into four host-infecting types; host-type [(B)]: isolates that Chrysanthemum sp., Impatiens balsamina, Petunia infected Brassica plants latently and occasionally and did hybrida and Zinnia elegans and four brassicaceae weed not infect Raphanus plants; host-type [B]: isolates that hosts (Bahar et al., 1985; Farzadfar et al., 2004, 2009; infected many Brassica plants systemically with mosaic Ghorbani et al., 2007; Shahraeen et al., 2002, 2003; on uninoculated leaves, but did not infect Raphanus Zahedi Tabarestani et al., 2010). plants; host-type [B(R)]: isolates that infected many In Khorasan Razavi province which is one of the most Brassica plants systemically, with mosaic symptoms on important agricultural areas of Iran, there is no evidence uninoculated leaves, but infected Raphanus plants of the presence and distribution of this virus. In this study, latently and occasionally; and host-type [BR]: isolates we clarified distribution of TuMV in canola crops of that infected both Brassica and Raphanus plants Khorasan Razavi province. Furthermore, complete coat systemically, with mosaic symptoms on uninoculated protein gene of an Iranian isolate of TuMV (IRN GSK) leaves. Moreover, phylogenetic analysis using different was sequenced and compared with others to provide virus isolates collected from around the world has better understanding of their relationship with other revealed four main TuMV genogroups called basal-B isolates of TuMV. (Brassica), basal-BR (Brassica/Raphanus), Asian-BR and world-B. The basal-B cluster of (B) or B host-type isolates was the most variable, was not monophyletic and came MATERIALS AND METHODS from both nonbrassicas and brassicas. The isolates in the basal-BR and Asian-BR groups and Asian isolates in the Sample collection world-B group seem to be more adapted to host plants such as Brassica and Raphanus than those in the During March and April of 2011, a total of 436 infected canola ancestral basal-B group. Most, but not all, of the plants having viral infection symptoms including: mosaic, leaf European isolates belonging to the world-B group are B malformation, vein clearing, chlorosis and stunting were collected from 39 canola fields throughout different regions (Figure 1 and host types, for instance UK 1 is of B host type and Table 1) of the Khorasan Razavi province (northeast of Iran). Each belongs to the world-B group, and, although many sample was collected from a different plant and immediately Brassica plants are susceptible to it, it rarely infects bagged and transported in cold boxes. Collected samples were Raphanus sativus systemically but asymptomatically. stored at 4°C for further analysis. Sabokkhiz et al. 14555 Table 1. Number of fields, samples collected and infected samples. Location (regions of Khorasan Razavi province) Number of fields Number of samples infected/ collected Mashhad 7 33/132 Chenaran 7 12/56 Ghouchan 6 12/38 Bardaskan 6 0/73 Torbat-e-Heydarieh 4 17/37 Torbat-e-Jam 4 18/40 Neyshabour 5 25/60 Enzyme-linked immunosorbent assay (ELISA) upper leaf tissues of TuMV-infected Brassica rapa using the RNeasy Plant Mini Kit (Qiagen, Valensia CA. USA) according to the Collected samples were tested for the presence of TuMV by manufacturers’ instruction. double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) as described previously by Clark and Adams (1977), using specific polyclonal antibodies purchased from DSMZ Inc. Reverse transcription and PCR amplification Braunschwieg, Germany. Briefly, microtitre plates were coated with 1:1000 TuMV-IgG in carbonate buffer (pH 9.6). Leaves were The Govareshk isolate were subjected to RT-PCR using TuMV ground in plastic bags by phosphate buffered saline (10 mM specific primers (forward primer TuMVF 8705-8726: 5’- potassium phosphate, 150 mM sodium chloride), pH 7.4, containing CAAGCAATCTTTGAGGATTAT-3’ and reverse primer TuMVR 0.5 ml/l of Tween 20 and 20 g/L of polyvinyl pyrrolidone (1:5 wt/vol), 9690-9669: 5’-TATTTCCCATAAGCGAGAATA-3’) (Sanchez et al., and leaf extracts were then added to the plates in duplicate wells 2003), obtaining the expected amplification product of 986 bp from and incubated overnight at 4°C. The presence of TuMV in the all samples. Reverse transcription and PCR amplification was samples was detected by TuMV-specific antibody conjugated to performed using AccuPower RT PreMix Kit and AccuPower PCR alkaline phosphatase using p-nitrophenyl phosphate (pNPP) PreMix (Bioneer Inc. Korea), respectively according to the substrate. Optical density (OD) at 405 nm was measured by ELISA manufacturers’ instructions. For cDNA synthesis, 1 µg of total RNA Reader (Stat Fax 2100, AWARNESS Tech. Inc., USA) 45 min to 1 h and 10 pmol of reverse primer was mixed in a sterile tube and after incubation with the substrate pNPP (1 mg/ml, pH 9.8). Tissue incubated at 70°C for 5 min and immediately placed on ice. The samples from healthy and infected plants grown in greenhouse incubated mixture was transferred to an AccuPower RT PreMix were used as negative and positive controls, respectively. Infection tube and then the reaction was filled up with DEPC-DW to a final of regenerated plants was determined according to the formula; R = volume of 20 µl, vortex and briefly spin down. The cDNA synthesis X + 2SD where, which X is the average OD of negative controls and reaction was performed for 60 min at 42°C followed by 5 min at SD is the standard deviation of this mean relative to its individual 94°C. PCR was conducted using the Biometra (T-personal) wells. thermocycler. The program consisted of a denaturation step of 4 min at 95°C, 35 cycles of 20 s at 94°C, 30 s at 55.5°C and 1 min at 72°C, and a final elongation step of 72°C for 5 min.