First Report on the Occurrence of Tobacco Streak Virus in Sunflower in Iran

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Journal of Plant Pathology (2012), 94 (3), 585-589 Edizioni ETS Pisa, 2012 585

FIRST REPORT ON THE OCCURRENCE OF TOBACCO STREAK VIRUS IN SUNFLOWER IN IRAN

S. Hosseini1, M. Koohi Habibi2, G. Mosahebi2, M. Motamedi2 and S. Winter3

1 Department of Plant Protection, College of Agriculture, Vali-e-Asr University of Rafsanjan, Iran 2 Department of Plant Protection, College of Agriculture, University of Tehran, Karaj, Iran 3 DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany

SUMMARY in the Netherlands (Dijkstra, 1983). Generally, TSV does not become epidemic but in sunflowers there are Sunflower (Helianthus annuus), an important oilseed exceptions reported from India and Australia (Prasada crop grown in Iran and many other countries, is a fre- Rao et al., 2003). The major method of transmission is quent host of Tobacco streak virus (TSV, genus by infected pollen, which can be spread by wind or car- Ilarvirus), which infects a wide range of crops and ried by thrips (Greber et al., 1991). In the present study, weeds. To study the occurrence and distribution of TSV the status of TSV in sunflower plants was determined in in Iran, 1,272 samples were collected from sunflower Iran based on visual symptoms and ELISA testing, and fields in Kerman, Golestan, Isfahan, Mazandaran, Qom, further confirmed by RT-PCR. Two viral isolates from Azarbayejan-Gharbi, Markazi, Hamedan and Tehran two separate Iranian regions were characterized. provinces during 2006 to 2008. TSV was detected by DAS-ELISA in 20.9% of the samples. The characterization of two Iranian virus isolates 43R (from Tehran) and MATERIALS AND METHODS IR (from Isfahan) showed that both have isometric particles 26 to 35 nm in diameter, and coat protein (CP) Survey. A total of 1,272 samples from field-grown subunits with a molecular weight of 30.9 kDa. In me- sunflowers in the Iranian provinces of Kerman, Tehran, chanical inoculation assays, the Iranian TSV isolate in- Golestan, Hamedan, Markazi, Azarbayejan-Gharbi, duced local lesions on a number of plant species of the Qom, Isfahan and Mazandaran (Fig. 1) were collected families Chenopodiaceae and Solanaceae. Vicia faba and during the 2006, 2007 and 2008 growing seasons. In- sunflower reacted with systemic symptoms. A fragment fected plants showed yellowing, deformation, chlorotic of 747 nt in length from the CP gene of each isolate was or necrotic lesions and mottling of the leaves, and stunt- amplified, cloned and sequenced. BLAST analysis of ing. Young leaves from some symptomatic plants were the CP sequences showed that both isolates are similar sampled, kept separate in plastic bags and stored at 4oC to Sudanese TSV isolates and belong to the same group. until analyzed. This is the first report on the detection of TSV in Iran. Virus identification. The samples were tested by Key words: SDS-PAGE, Western blotting, Coat pro- DAS-ELISA (Clark and Adams, 1977) using a specific tein, DAS-ELISA, RT-PCR. polyclonal antibody to TSV (AS-906, DSMZ, Ger- many). Absorbance at 405 nm was measured using a Flow ELISA microplate reader. The reaction was con- INTRODUCTION sidered positive if absorbance was at least twice higher than the mean of healthy control samples. Sunflower (Helianthus annuus) is an important Collection and maintenance of virus isolates. Two oilseed crop grown in Iran and many countries is sus- TSV isolates from Tehran (43R) and Isfahan (IR) ceptible to different diseases, also induced by viruses, provinces were transferred to and maintained on Vicia which represent major constraint to production and can faba by mechanical inoculation of tissue extracts in lead to significant reductions in yield and seed quality. 0.05M phosphate buffer, pH 7, containing 1mM EDTA, Tobacco streak virus (TSV), the type member of the 5mM Na-DIECA and 5mM thioglycolic acid. genus Ilarvirus, was first detected in sunflower in 1980 Experimental host range. Host reactions to the two viral isolates were determined using 25 plant species be- longing to six botanical families (Table 2). Mechanically Corresponding author: S. Hosseini Fax: +98.39143202041 inoculated plants were maintained at 23-25°C in a E-mail: [email protected] greenhouse. 010_JPP1080RP(Hosseini)_585 20-11-2012 11:46 Pagina 586

586 TSV in sunflower in Iran Journal of Plant Pathology (2012), 94 (3), 585-589

Fig. 1. Map of Iran showing the regions where field-grown sunflower crops were surveyed for To- bacco streak virus during the 2006, 2007 and 2008 growing seasons.

Immuno-electron microscopy. Immuno-electron mi- RT-PCR and sequencing. Total RNA from V. faba croscopy (IEM) was used on leaf dips from sunflower experimentally infected with each of the viral isolates plants infected by both viral isolates (Milne and (43R and IR) was extracted using commercially avail- Luisoni, 1977). able kits [RNeasy Plant Mini Kit (Qiagen, Germany) and Peq Gold RNA pure TM solution (Peqlab Biotech- Protein electrophoresis and Western blot analysis. nologie Germany)] following the manufacture’s recom- The molecular weight of the viral coat protein (CP) was mendations One-step RT-PCR was performed using the estimated by SDS-PAGE on a 4% stacking gel and a SuperscriptTM III One-Step PlatinumR Taq HiFi RT- 15% resolving gel. After electrophoresis for 1.5-2 h at PCR kit (Invitrogen, USA) in a 50 µl reaction mix con- 100 V at room temperature, proteins were stained with sisting of 1 µl total RNA extract, 1 µl reverse primer (10 Coomassie brilliant blue and their mol. wt was estimat- µM), 1 µl forward primer (10 µM), 1 µl RT Platinum ed by comparing their relative mobility with standard Taq HiFi enzyme mix, 25 µl reaction mix buffer and 21

markers (Laemmli, 1970; Sambrook, 2001). Separated µl deionized H2O. The specific primer pair used was: proteins were electro-transferred onto nitrocellulose TSV CP RNA-3 set (5’-AGG TAG CAG GAG ATA membrane at 300-400 mA for 1.5-2h (Towbin et al., TAA CAA TGA ATA CTT TGA TCC AAG G-3’ and 1979) which, after blocking with 2% skim milk, was in- 5’-TCG ACT CTA GAA ACT AGT CTT GAT TCA cubated in TSV IgGs (2 µg/ml) followed by immune CCA GAA AAT CTT C-3’). This primer set, designed detection of the bound IgGs with a goat-anti-rabbit al- on the CP sequence of a TSV sunflower isolate from In- kaline phosphatase conjugate (DAKO) diluted to dia, amplified a cDNA fragment of 747 bp. RT-PCR re- 1:10000 (V/V). actions were performed with an initial RT reaction at 50°C for 30 min, followed by a denaturation step at 010_JPP1080RP(Hosseini)_585 20-11-2012 11:46 Pagina 587

Journal of Plant Pathology (2012), 94 (3), 585-589 Hosseini et al. 587

Table 1. Detection of TSV in sunflower by DAS-ELISA dur- (pDrive; Qiagen, Germany). Isolates 43R and IR were ing 2006-2008. custom-sequenced by MWG Biotech (Germany). Phy- logenetic analyses of amino acid sequences of the viral No. analyzed / No. of positive samples Provinces isolates were performed using DNAMAN program, ver- 2006 2007 2008 Kerman 82/1 0/0 82/48 sion 4.02. (Lynnon Biosoft, Canada). Tehran 18/0 130/37 60/30 Hamedan 0/0 115/36 0/0 Markazi 0/0 30/0 0/0 Mazandaran 94/11 0/0 120/18 RESULTS Golestan 116/5 0/0 0/0 Qom 40/2 55/8 0/0 Results of these surveys are summarized in Table 1. Azarbayjan-Gharbi 0/0 55/31 140/26 Isfahan 0/0 135/12 0/0 Virus-like symptoms were observed in several fields and Total 350/20 520/124 402/122 TSV was found in all surveyed provinces except for Markazi. Of the 350, 520 and 402 samples collected in 2006, 2007 and 2008 respectively, 20, 124 and 122 were 95°C for 3 min, then by 30 cycles at 94°C for 1 min, infected with TSV. 60°C for 1 min and 68°C for 2 min, and a final elonga- tion step at 68°C for 10 min. In this test positive and Mechanical transmission and host range. Both viral negative controls were used, the latter being distilled isolates were efficiently transmitted to certain plants by water. Ten microliters of each PCR product were ana- sap-inoculation and their host range was determined lyzed by electrophoresis in a 1% agarose gel and visual- (Table 2). Severe symptoms were induced in V. faba, ized by ethidium bromide staining, then excised from mosaic symptoms in sunflower and chlorotic or necrotic the gel, purified and cloned into a plasmid vector local lesions in some of the Chenopodium species.

Table 2. Reaction of some diagnostic species to infection by two Iranian TSV isolates. gp y

Family Species Local lesion/ systemic symptoms 43R IR Aizoaceae Tetragonia tetragonoides (Pallas) Kuntze. -/- -/-

Chenopodiaceae Chenopodium quinoa Willd. CLL/- CLL/CL C. amaranticolor Coste & Reyn. CLL/- CLL/- C. murale L. CLL/- -/- C. foliosum (Moench) Asch. CLL/- -/-

Compositae Lactuca sativa L. -/- -/- Helianthus annuus L. -/M -/M,W

Cucurbitaceae Cucurbita pepo Duch. -/- -/- Cucumis melo L. -/- -/-

Leguminosae Pisum sativum L. -/- -/- Vicia faba L. NLL/M NLL/M,W Phaseolus vulgaris L. -/- -/-

Solanaceae Datura metel L. NLL/- NLL/- D. stramonium L. NLL/- NLL/- Lycopersicum esculentum Mill. -/- -/- Nicotianan. benthamiana Domin. -/L -/L N. clevelandii A. Gray -/- -/- N. debneyi Domin. -/L -/L N. glutinosa L. -/L -/L N. hesperis N.T. Burb. NLL/L -/L N. rustica L. -/- -/- N. occidentalis H.M. Wheeler -/L -/L N. tabacum L. cv. Samsun -/L -/L N.tabacum L. cv. Xanthi -/L -/L N. tabacum L. cv. White Burley -/- -/- Physalis floridana Rydb. NLL/- NLL/- Solanum demissum Lindl. NLL/- NLL/- CLL: chlorotic local lesion; NLL: necrotic local lesion; M: mosaic; L: latent; W: wilt 010_JPP1080RP(Hosseini)_585 20-11-2012 11:46 Pagina 588

588 TSV in sunflower in Iran Journal of Plant Pathology (2012), 94 (3), 585-589

Molecular variability of TSV isolates. The CP amino acid sequence of the two TSV isolates were used with other published sequences to construct a phylogenetic tree (Fig. 4). Iranian and Sudanese isolates clustered to- gether and were divergent from other isolates.

DISCUSSION

This study showed that sunflower plants in several ar- eas of Iran, including the provinces of Kerman, Golestan, Mazandaran, Azarbayjan-Gharbi, Isfahan, Hamedan, Qom and Tehran, are infected with TSV. The symptoms include stunting, deformation, chlorotic and necrotic lesions, yellowing and mosaic. The infection rates in 2006, 2007 and 2008 were 20.9% and showed an increase Fig. 2. Negatively stained preparation of the virus showing trend in the Tehran province during three years of the spherical virions decorated by antibodies to TSV (Bar=50 nm). survey and in Kerman, Mazandaran and Qom provinces during two years, while the percentage of infection de- Symptoms caused by the IR isolate in sunflower and V. creased during two years in Azarbayjan-Gharbi. faba were more severe than those caused by isolate 43R. Host ranges for TSV isolates from Tehran (43R) and In a number of Nicotiana species, the virus became sys- Isfahan (IR) were similar, but symptoms of the IR iso- temic without symptom induction. late on V. faba and sunflower were more severe than those of the isolate 43R. The symptoms were similar to Immuno-electron microscopy. Electron microscopy those described by Ravi et al. (2001) for an Indian TSV analyses of infected leaves showed spherical virus parti- isolate from sunflower but the damages caused by Indi- cles, with diameters ranging from 26 to 35 nm (Fig. 2). an isolates were more severe than those caused by Iran- ian isolates. Coat protein analysis. Both isolates gave a single pro- The mol. wt of the CP of the Iranian isolates was tein band with an estimated mol. wt of 30.9 kDa in 30.9 kDa, in accordance with previous estimates (Ali et SDS-PAGE (Fig. 3A). A single band was also observed al., 2009; Almeida et al., 2005). when a membrane containing the TSV capsid protein Phylogenetic analysis with the CP amino acid se- was probed with a TSV polyclonal antiserum in West- quences of TSV isolates revealed that all formed a ern blot (Fig. 3B). monophyletic group. Australian and Brazilian isolates

Fig. 3. Panel A. SDS-PAGE of proteins from dissociated TSV particles on polyacrylamide gel. Panel B. Western blot of TSV capsid protein, probed with a polyclonal antiserum to TSV. Lanes 1A and 1B, healthy plant; lanes 2A and 2B, TSV-43R-infected plant; lanes 3A, 3B, molecular weight markers (BenchMarkTM); lanes 4A, 4B, healthy plant; lanes 5A and 5B, TSV-IR- infected plant. Asterisks indicate the position of virus capsid protein. 010_JPP1080RP(Hosseini)_585 20-11-2012 11:46 Pagina 589

Journal of Plant Pathology (2012), 94 (3), 585-589 Hosseini et al. 589

clustered in group 1, the closely related Iranian and Su- isolates were in group 3. Iranian isolates in group 2 had danese isolates clustered in group 2 while the Indian 91% and 85% identity with isolates of groups 1 and 3, respectively. To the best of our knowledge this is the first report on the occurrence of TSV in sunflower in Iran.

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