pcr detection and partial molecular characterization of ... - SIPaV

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Journal of Plant Pathology (2008), 90 (2), 247-251

Edizioni ETS Pisa, 2008

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PCR DETECTION AND PARTIAL MOLECULAR CHARACTERIZATION OF CHICKPEA CHLOROTIC DWARF VIRUS IN NATURALLY INFECTED SUGAR BEET PLANTS IN IRAN Sh. Farzadfar1, R. Pourrahim1, A.R. Golnaraghi2 and A. Ahoonmanesh3 1 Department

of Plant Virology, Plant Pests and Diseases Research Institute, P.O. Box 19395-1454, Tehran, Iran of Plant Protection, College of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, P.O. Box 14515-775, Tehran, Iran 3 Department of Plant Pathology, College of Agriculture, Esfahan University of Technology, Esfahan, Iran

2 Department

Key words: Sugar beet, Chickpea chlorotic dwarf virus, detection, PCR.

conditions, some of which occur worldwide and substantially reduce crop yield and quality (Brunt et al., 1995; Whitney and Duffus, 1998). In 2001, a field survey was conducted in the main sugar beet growing areas of Iran in order to assess the presence of virus diseases in the crop. Plants with viruslike symptoms were brought to the Department of Plant Virology of Plant Pest and Disease Research Institute (PPDRI) for diagnosis. One or more of the following viruses were detected in the symptomatic plants when tested by tissue-blot immunoassay (TBIA): Alfalfa mosaic virus (AlMV), Beet curly top virus (BCTV), Beet mosaic virus (BtMV), Beet western yellows virus (BWYV), Beet yellows virus (BYV), Cucumber mosaic virus (CMV) and Turnip mosaic virus (TuMV). However, mild chlorosis and stunting symptoms were observed on some sugar beet samples which were negative in TBIA tests to these viruses. For these samples, electron microscopy of negatively stained preparations of crude sap extracted from diseased leaves revealed the presence of geminate particles, typical of members of the family Geminiviridae, measuring ca. 25 nm in size. Among geminiviruses, serologically related curtoviruses of BCTV, Beet mild curly top virus (BMCTV) and Beet severe curly top virus (BSCTV) can infect sugar beet plants under natural conditions (Fauquet et al., 2005). The aim of the present study was to identify and characterize some biological, serological and molecular properties of the geminivirus present in sugar beet in Iran.

INTRODUCTION

MATERIALS AND METHODS

Sugar beet (Beta vulgaris L.) is one of the major sources of sugar in Iran, covering an area of ca. 180,000 ha, for a production of 4,900,000 tons (Faostat, 2004). This crop is cultivated in many Iranian regions mainly in autumn, except for Khuzestan where it is grown in spring. Sugar beet is affected by more than 100 different viruses and virus strains under natural or experimental

Virus source. A sugar beet plant showing mild chlorosis and stunting symptoms was used in this study. The plant came from a field in Songhor (Kermanshah province).

SUMMARY

The causal agent of mild chlorosis and stunting of sugar beet in Iran was identified as Chickpea chlorotic dwarf virus (CpCDV). The virus reacted positively with CpCDV-specific antibodies in ELISA, but not with antibodies specific to other viruses of the family Geminiviridae (Beet curly top virus, Bean golden mosaic virus and Tomato yellow leaf curl virus). The virus isolate under study (SK-11) was transmitted by grafting and by the leafhopper Orosius orientalis Matsumara to a range of plant species in the Chenopodiaceae, Fabaceae and Solanaceae, and induced symptoms like those reported for CpCDV. By using a pair of degenerate primers, a short viral genome fragment was amplified by PCR, cloned and sequenced, revealing a close homology to Bean yellow dwarf virus (84.9%-85.3% identity), and a more distant relationship to Tobacco yellow dwarf virus (58.8%). In the present study, two specific primers were designed for detecting CpCDV which allowed the amplification of a DNA fragment of 0.64 kbp. Using these primers, the virus was successfully detected in symptomatic sugar beet field samples collected from the main sugar beet production provinces of Iran. This study reports for the first time information on the genome sequence of CpCDV.

Corresponding author: Sh. Farzadfar Fax: +98 21 22403691 E-mail: [email protected]

Serological assays. DAS-ELISA (Clark and Adams, 1977) was used to identify the virus, using different polyclonal and monoclonal antibodies against different geminiviruses. M. Bahar (College of Agriculture, Esfahan University of Technology, Esfahan, Iran) and S. Winter (DSMZ, Braunschweig, Germany) kindly provided spe-

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CpCDV in naturally infected sugar beet

cific antibodies to BCTV and Bean golden mosaic virus (BGMV), respectively. Unfractionated antiserum to Chickpea chlorotic dwarf virus (CpCDV) was kindly provided by H. J. Vetten (DSMZ); the IgG and IgGconjugated antibodies of this virus were subsequently prepared according the methods described previously (Ball et al., 1990; MacKenzie, 1990). Polyclonal antibodies of Tomato yellow leaf curl virus (TYLCV) were purchased from Bioreba (Reinach, Switzerland). Positive controls for BCTV, CpCDV and TYLCV were provided by S. Jalali (Agricultural Research Center, Esfahan, Iran), K.M. Makkouk (ICARDA, Syria) and V. Alavi (Agricultural Research Center, Sari, Iran), respectively. Mechanical inoculation. Symptomatic sugar beet leaves were macerated in 0.1 M sodium phosphate buffer (pH 7.0) containing 0.1% 2-mercaptoethanol. The extract was used to inoculate carborundum-dusted leaves of Beta vulgaris, Chenopodium amaranticolor and C. quinoa (Chenopodiaceae), Brassica rapa and Raphanus sativus (Brassicaceae), Cucumis sativus (Cucurbitaceae), Datura metel, D. stramoniun, Lycopersicon esculentum, Nicotiana glutinosa, N. rustica, N. tabacum cvs Samsun and White Burley (Solanaceae), Gomphrena globosa (Amaranthaceae), Phaseolus vulgaris cv. Talash, Pisum sativum, Vicia faba and Vigna unguiculata cv. Mashad (Fabaceae). Inoculation tests were repeated twice. Plants were kept under greenhouse conditions for 4 to 6 weeks after inoculation, to observe appearance of symptoms. Graft transmission. Infected sugar beet leaves were grafted to 15-20 indicator plants, including D. stramoniun, L. esculentum, N. rustica, N. tabacum cv. Samsun, N. tabacum cv. White Burley and V. faba. Insect transmission. Colonies of non-viruliferous Circulifer tenellus Baker and C. opacipennis Lethierry leafhoppers were maintained on sugar beet plants. A colony of Orosius orientalis Matsumara (also named O. albicinctus Distant) was maintained on sesame (Sesamum indicum) and sugar beet. Colonies of the whitefly Bemisia tabaci Gennadius was maintained on cotton (Gossypium hirsutum) leaves. After a two-day acquisi-


tion access period on graft-infected N. tabacum cvs Samsun or White Burley plants, the leafhoppers and whiteflies were kept for one week on pea (Pisum sativum) and sugar beet (B. vulgaris cv. IC1) plants, using 10-15 insects per test plant. Inoculated plants were then sprayed with the insecticide imidacloprid (Bayer, Germany) and kept in a greenhouse for six weeks for symptom observation. PCR. Total nucleic acids were extracted from symptomatic sugar beets as described by Noris et al. (1996). A pair of degenerate primers was designed on the basis of the nucleotide sequences of Bean yellow dwarf virus (BeYDV; accession No. DQ458791 and Y11023) and Tobacco yellow dwarf virus (TYDV; accession No. M81103), two monopartite dicot-infecting geminiviruses in the genus Mastrevirus (Fauquet et al., 2005). The degenerate primers F1 and R1 (Table 1) were used as upstream and downstream primers, respectively, to amplify a fragment of approximately 1.45 kbp. PCR amplifications were carried out using Pfu DNA polymerase (CinnaGen, Iran) and a Primus (MWG-Biotech, Germany) thermal cycler. The PCR program was as follows: 94°C for 1 min, 30 cycles of 94°C for 30 sec, 47°C for 1 min, 72°C for 1 min, and finally 72°C for 10 min. All primers used in this study were synthesized by MWGBiotech (Germany). PCR products and DNA ladder (Fermentas, Lithuania) were fractionated by electrophoresis on a 1% agarose gel in the presence of 1 µg/ml ethidium bromide using 1X Tris-Borate EDTA (TBE) buffer (Sambrook et al., 1989). Gels were visualized and photographed with UV-illumination (Imago, The Netherlands). Cloning, sequencing and sequence analysis. The DNA fragment amplified using F1 and R1 primers, was excised from the gel, cleaned by Nucleospin (MachereyNagel, Germany) and cloned into pGEM-T Easy vector (Promega, USA) as suggested by the suppliers. Sequences from both strands of the cloned DNA were determined for three independent clones by the custom sequencing service of MWG Biotech Co. (Germany) using standard M13 primer pairs. The nucleotide se-

Table 1. Degenerate primers for PCR-amplification of the sugar beet mastrevirus (SK-11). Primer

Sequence

Length

F1

5’-GAAAA(G/C)GTG(T/A)(G/A)AGTTT(C/A)TCCCAG-3’

23 nt

R1

5’-TTTTATTGATT(G/T)CC(A/C)ACTGA(C/T)T-3’

22 nt

Position BYD-1: 2280-2302 BYD-2: 2278-2300 TYD-1: 2451-2473 BYD-1: 1138-1159 BYD-2: 1138-1159 TYD-1: 1306-1327

BYD-1, BeYDV (DQ458791); BYD-2, BeYDV (Y11023); TYD-1, TYDV (M81103); F1 and R1 are upstream and downstream primers, respectively.

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quence obtained was compared with other sequences available in GenBank using BLAST (Altschul et al., 1997) at the National Center for Biotechnology Information. Multiple sequence alignments were generated by Clustal W (Thompson et al., 1994) using MegAlign v 5.00 (DNASTAR, USA) (Burland, 2000). Design of specific primers. For the specific detection of the virus, a pair of primers for the amplification of a DNA fragment of approximately 0.64 kbp, was designed on the basis of the sequenced genome fragment obtained using the degenerate primers. Primer F2 (5’CAGAAGGCATGTTGTTGTGACTCC-3’) was used as an upstream primer and primer R2 (5’-AACCGCTACGCTTCCGCTTCTTA-3’) was used as a downstream primer. Thermocycling was as described above. These primers were also used for virus detection in 241 sugarbeet leaf samples with CpCDV-like symptoms collected in summer 2006 in Esfahan, Kermanshah and Khorasan provinces (78, 55 and 108 samples, respectively) in 34 sugar beet fields (9, 5 and 20 fields, respectively).

RESULTS

Serological assays. Symptomatic sugar beet samples clearly reacted with the CpCDV antiserum but not with antisera to BCTV, BGMV and TYLCV. Transmission. All grafted or mechanically-inoculated plants were tested by ELISA using CpCDV-specific antibodies. Mechanical inoculations were not successful, but graft transmission elicited chlorotic symptoms in D. stramoniun and N. tabacum cv. Samsun, and leaf rolling in N. tabacum cv. White Burley. The virus infected symptomlessly L. esculentum and V. faba, but not N. rustica. It was transmitted by O. orientalis from tobacco to pea plant, in which it induced chlorosis and rolling of the leaves and stunting, but not by B. tabaci, C. tenellus and C. opacipennis. When healthy sugar beet plants (Beta vulgaris cv. IC1) were inoculated by O. orientalis, chlorosis with mild stunting symptoms developed, resembling those observed in the field. PCR and sequencing. Degenerate primers F1 and R1, designed on nucleotide sequences of dicot-infecting mastreviruses (BeYDV and TYDV), amplified a DNA fragment of approximately 1.45 kbp (Fig. 1), which was cloned and sequenced (GenBank accession No. EU034169). BLAST search results showed the highest identity with BeYDV. The 1401 nucleotide fragment contained the complete sequence of open reading frame (ORF) V1 and the partial sequences of ORFs V2, C1 and C1:C2. Multiple nucleotide sequence alignment using the Clustal W program showed 84.9% and 85.3%

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identity with BeYDV (accession No. DQ458791 and Y11023) and 58.8% identity with TYDV (accession No. M81103) (Fig. 2). When the primers F2 and R2 were used for the detection of isolate SK-11, an amplicon of the expected size (ca. 0.64 kbp) was obtained (Fig. 3, lane 2). These primers failed to amplify visible products from total nucleic acids extracted from healthy sugar beets (Fig. 3, lanes 7 and 8). Using these primers, CpCDV infections were found in 48 symptomatic samples (19.9%) collected from 26 fields (Fig. 3, lane 3-6). The highest infection rate was recorded in Khorasan (28.7%), followed by Kermanshah (20.0%) and Esfahan (7.7%).

DISCUSSION

In the present work, a geminivirus isolate (SK-11) causing mild chlorosis and stunting symptoms on sugar beets has been studied. SK-11 proved to be serologically unrelated to BGMV and TYLCV, two whitefly-transmitted viruses of the genus Begomovirus (Fauquet et al., 2005), and to BCTV, a leafhopper-transmitted member of the genus Curtovirus (Bennett, 1971; Thomas, 1979). However, SK-11 was clearly detected by antibodies to CpCDV, a monopartite dicot-infecting virus tentatively assigned to the genus Mastrevirus (Horn et al., 1993; Fauquet et al., 2005). In experimental trials, SK-11 was transmitted by the leafhopper O. orientalis, a known vector for CpCDV (Horn et al., 1993) and by grafting to a range of species of the Chenopodiaceae, Fabaceae and Solanaceae families, inducing symptoms resembling those described for CpCDV (Horn et al., 1993). Healthy sugar beet plants which were inoculated by viruliferous leafhoppers showed chlorosis and mild stunting resembling symptoms observed in the field, and those previously described on sugar beet experimentally infected with CpCDV (Horn et al., 1993). In this study, the partial sequence of SK-11 was determined and compared with those of other dicot-infecting mastreviruses. The ca. 1.45 kbp SK-11 genome fragment amplified by PCR was phylogenetically related

Fig. 1. Agarose gel electrophoresis of PCR products using F1/R1 degenerate primers designed to amplify a DNA fragment of ca. 1.45 kbp. Lane 1, 1-kb DNA ladder (Fermentas, Lithuania); lanes 2 and 3, healthy plant extracts; lane 4, sugar beet isolate SK-11.

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Fig. 2. Sequence alignment of the sugar beet isolate SK-11 (EU034169) with dicot-infecting mastreviruses BeYDV (BYD-1, DQ458791; BYD-2, Y11023) and TYDV (TYD-1; M81103). Conserved sequences are boxed. Gaps (.) have been introduced to maximize alignment.

to two BeYDV isolates (84.9%-85.3% identity) and, more distantly, to TYDV (58.8% identity). Based on serological and host range responses and

Fig. 3. Agarose gel electrophoresis of PCR products using the sugar beet mastrevirus-specific primers F2/R2 to amplify a DNA fragment of ca. 0.64 kbp. Lane 1, 1 kb DNA ladder (Fermentas, Lithuania). Lane 2, sugar beet isolate SK-11. Lanes 3-6, sugar beet samples that gave a positive ELISA reaction to CpCDV, collected from different Iranian fields. Lanes 7 and 8, healthy plant extracts.

vector transmission SK-11 can be regarded as an isolate of CpCDV (Horn et al., 1993), a virus previously reported from Iran (Makkouk et al., 2002). This virus, however, remains to be confirmed as a distinct mastrevirus species and its classification should await the determination of its complete nucleotide sequence. Interestingly, the symptoms caused by CpCDV on sugar beet are similar to those produced by beet yellowing viruses (Whitney and Duffus, 1998). However, the primers F2 and R2, presently designed for the specific detection of CpCDV can be used for differentiating this virus from other sugar beet yellowing disease agents, as shown by the results of the present survey. The widespread presence of CpCDV in sugar beet fields of three Iranian provinces may reflect the activity of leafhopper vectors, which will need to be studied in the future.

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Journal of Plant Pathology (2008), 90 (2), 247-251 ACKNOWLEDGEMENTS

This work was supported by the Plant Virology Department of PPDRI (Tehran). The authors are greatly indebted to Drs. S. Winter, H.J. Vetten, K.M. Makkouk, M. Bahar and V. Alavi, and S. Jalali for their generous gifts of antisera and positive samples that were used in this study. We also wish to thank Dr. J. Stanley (Department of Disease and Stress Biology, John Innes Centre, Colney Lane, Norwich, UK) and Dr. M. Digiaro (Istituto Agronomico Mediterraneo, Valenzano, Italy) for their kind help in this study.

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Received November 14, 2007 Accepted March 19, 2008

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