Journal of Virological Methods 202 (2014) 34–38
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions S. Majumder 1 , V.K. Baranwal ∗ Plant Virology Unit, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, India
a b s t r a c t Article history: Received 19 July 2013 Received in revised form 16 February 2014 Accepted 23 February 2014 Available online 2 March 2014 Keywords: Multiplex RT-PCR Garlic viruses OYDV GarCLV SLV Allexivirus
Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Garlic (Allium sativum L.) is one of the most important culinary herbs of the world with an annual production of 22.28 million tons planted globally (FAO 20011) and it is used widely (Keusgen, 2002). Garlic stocks worldwide are infected with a number of viruses often designated as ‘garlic viral complex’ (Walkey and Antill, 1989; Van Dijk, 1994). The complex may include potyviruses; Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV), carlaviruses; Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and Allexiviruses; Garlic virus X (GVX), Garlic virus A,B,D,E, Garlic mite borne filamentous virus, and Shallot Virus X (ShVX). OYDV, SLV, GarCLV and allexiviruses (GVX) have been recorded in garlic cultivars from India (Arya et al., 2006, 2009; Majumder and Baranwal, 2009; Baranwal et al., 2011). These viruses may not kill the plant but can reduce yield up to 50% during successive cultivation (Lot et al., 1998; Conci et al., 2003; Cafrune et al., 2006). Serological methods such as enzyme-linked immunosorbent assay (ELISA), direct tissue blotting immunoassay and Dot ELISA are often employed for indexing of viruses associated with garlic (Tsuneyoshi and Sumi, 1996; Takaichi et al., 2001; Conci et al., 2003; MeloFilho et al., 2004). But antibodies to all the viruses are not
always easily available and their production is a complex process due to multiple viral infections in the garlic. Reverse transcription polymerase chain reaction (RT-PCR) has been proved to be many times more sensitive and results are obtained faster compared to serological assays like ELISA (Dovas et al., 2001; Shiboleth et al., 2001; Du et al., 2006). RT-PCR is regularly used for detecting individual garlic viruses such as OYDV (Dovas et al., 2001; Takaichi et al., 2001; Arya et al., 2006), SLV (Tsuneyoshi et al., 1998; Arya et al., 2009) and Allexivirus (Dovas et al., 2001; Baranwal et al., 2011). Duplex RT-PCR has been demonstrated for simultaneous detection of OYDV and SLV in Indian garlic and for OYDV and Allexivirus in Indian onion (Majumder et al., 2008; Kumar et al., 2010). However, as mixed infection of OYDV, SLV, GarCLV and allexiviruses have been recorded in different garlic accessions of India (Baranwal et al., 2011), it became imperative to develop a multiplex PCR which can be used for simultaneous detection of these viruses. We developed a standardized multiplex RT-PCR for detection of these viruses and used to investigate the occurrence and distribution of these viruses in garlic accessions from geographically diverse garlic growing areas of India. 2. Materials and methods 2.1. Source of plant material from different regions of India
∗ Corresponding author. Tel.: +91 9818756899; fax: +91 11 25843113. E-mail address: [email protected] (V.K. Baranwal). 1 Present address: Department of Biotechnology, Sharda University, Greater Noida 201306, UP, India. http://dx.doi.org/10.1016/j.jviromet.2014.02.019 0166-0934/© 2014 Elsevier B.V. All rights reserved.
Five garlic accessions viz., Selection 17 (Sel-17; Northern India), Pusa Accession 10 (Northern India), G-1 (Western India), Phull (Central India) and RAUG-5 (Eastern India), were initially used to
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Table 1 Details of primers used for multiplex RT- PCR for simultaneous detection of four viruses in garlic. Virus OYDV F 10214 OYDV R-10539 Allexi 1 F 8623 Allexi 2 R 8813 SLV5F 2124 SLVNABR2710 GarCLVCP1045 GarCLV R 1494
Primer sequence
5 -CGTTTGTTTGGCCTGGATGGTAACG-3 5 -GTCTCYGTAATTCACGC-3 5 -CYGCTAAGCTATATGCTGAARGG-3 5 -TGTTRCAARGTAAGTTTAGYAATATCAACA-3 5 -GTTCTGGGAYATWTGCATGTA-3 5 -CCCGCACTTTCACACACTAT-3 5 -AAATGTTAATCGCTAAACGACC-3 5 -CWRCCATTAAAACGTAGCAGC-3
Genes used
TM ◦ C
Best at
Size of product
CP 3 UTR NB 3 UTR CP NB CP NB
61 56 62 60 57 60 60 56
58
318
60
180–190
60
587
60
451
CP: coat protein; NB: nucleic acid binding protein; UTR: untranslated region.
standardize and validate individual RT-PCR for OYDV, SLV, GarCLV and Allexivirus. Since these viruses were observed in Sel-17, this accession was selected for standardizing multiplex RT- PCR. Half the cloves from a mother bulb were planted for future use and the other half were used in the experiments. Apical meristem of a clove from Sel-17 was micropropagated (Dantu and Bhojwani, 1992) and virus free plants were used as healthy negative control. 2.2. Nucleic acid extraction Total RNA was extracted from 50 mg tissues of infected leaves/cloves and healthy leaves of garlic, using RNeasy Plant Mini Kit (Qiagen, Germany) as per manufacturer’s protocol. The yield and the quality of the RNA were analyzed in a UV visible double beam spectro-photometer (Shimadzu-1800, Japan). 2.3. Primer design and synthesis For detection of allexiviruses previously published primer set producing an amplicon of ∼200 bp was used (Dovas et al., 2001). Primers for detection of OYDV, SLV and GarCLV were designed manually (Table 1) using gene sequences available at NCBI GenBank. Bioedit (7.0.7.1) (Hall, 1999) was used to align sequences and locate conserved regions. Primers for each OYDV, SLV and GarCLV were designed to amplify different sizes of amplicons in the size range of 300–600 bp with annealing temperatures in the range of 56–60 ◦ C. The primers were synthesized by Sigma Aldrich, Bangalore, India. 2.4. Standardization of RT-PCR for individual virus First strand cDNA was synthesized separately for all the four viruses using 4 l of total RNA and reverse transcription (RT) mixture using reverse primers of OYDV/SLV/GarCLV or allexiviruses (final concentration of 0.2 M), 20 U M-MuLV reverse trancriptase (Fermentas, USA), 0.3 mM dNTPs in the total reaction mixture of 20 l and incubated at 42 ◦ C for 45 min. The reaction was stopped by heating at 70 ◦ C for 10 min. To standardize the annealing temperature for primer pairs, gradient PCR (temperature range of 54–60 ◦ C) was performed for each virus in a thermocycler (Eppendorf Mastercycler Gradient, USA). PCR was performed in a 50 l reaction volume containing 5 l of cDNA, 5 l of 10× reaction buffer, 1.5 mM MgCl2 , primer pairs at 0.2 M each, 0.2 mM dNTPs and 5 U of Taq DNA polymerase (Promega, USA). The reaction profile consisted of an initial denaturation step at 94 ◦ C for 5 min, 30 cycles of 45 s at 94 ◦ C, 20 s at annealing temperature (54, 55, 56, 57, 58, 59 and 60 ◦ C) and 1 min extension at 72 ◦ C and a final extension step at 72 ◦ C for 10 min. The reactions were repeated 3–4 times with primer sets for each virus and the best combination of primer concentration and annealing temperature (Table 1) was chosen for further work. Ten microlitres of amplified product were separated by electrophoresis in a 1.2% agarose gel containing ethidium bromide and photographed under UV illumination with an imaging system
(Biorad XR documentation system). To confirm the identity of the amplified DNA fragments, the purified PCR products from garlic Sel-17 were cloned and sequenced. PCR products were purified using PCR Purification kit (Qiagen, Germany), ligated into pGEMT Easy vector (Promega, USA) and transformed Escherichia coli, DH5␣ (Sambrook et al., 1989). Recombinant clones were identified by colony PCR and sequenced at Chromous Biotech, Bangalore, India. The nucleotide sequences obtained were analyzed by BLAST to verify the sequence identity. 2.5. Optimization of multiplex RT-PCR To standardize multiplex RTPCR, cDNAs for OYDV, SLV, GarCLV and allexiviruses were prepared simultaneously in a single tube. The 25 l reaction mixture contained 6 l of total RNA, the optimized reverse primer for OYDV, SLV, and GarCLV at a concentration of 0.3 M and allexiviruses at a concentration of 0.2 M, 20 unitsMuLV reverse trancriptase (Fermentas, USA), 5 l of 5× reaction buffer and 0.3 mM dNTPs. The reaction mixture was incubated at 42 ◦ C for 45 min and the reaction was stopped by heating at 70 ◦ C for 10 min. The multiplex PCR was performed using a reaction mixture containing 5 l of reverse transcription (RT) mixture, 5 U of Taq DNA polymerase (Promega, Madison, USA), 5 l of 10× reaction buffer, 1.5 mM MgCl2 , primer pairs at a concentration of 0.2 M and 0.2 mM dNTPs. The temperature profile consisted of a denaturation step at 94 ◦ C for 5 min, 30 cycles of 45 s at 94 ◦ C, 20 s annealing temperature of 56/57/58 ◦ C and 1 min at 72 ◦ C and one final extension step at 72 ◦ C for 10 min on a gradient PCR. As a control, standard RTPCR was performed for each of the viruses using 5 l of the same RT reaction (cocktail) with 56 ◦ C as annealing temperature. To confirm the identity, four PCR products obtained were clone sequenced. 2.6. Sensitivity level of multiplex RT-PCR To determine the sensitivity limit of multiplex RT-PCR, different dilutions of RNA ranging from 101 (1 l of 40 l RNA isolated from 50 mg tissue) to 10−4 , corresponding to 0.318–0.0003 g of RNA (extracted from both leaves as well as cloves of diseased sample) were used for preparing individual as well as mixed cDNA. Reaction conditions for cDNA preparation were same as described in the previous section. Individual as well as multiplex RT-PCR was performed using these cDNA. To evaluate the performance of standardized multiplex PCR, it was initially performed on four garlic accessions mentioned earlier. Tissue culture raised plants were used as negative control. Individual RT-PCR was performed for all the four viruses to cross check the results. 2.7. Validation of multiplex RT-PCR The optimized multiplex RT-PCR was validated by performing it on twenty-one accessions of garlic from different geographical regions of India (Table 3). The samples were collected from the
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Table 2 Size of the RT-PCR product and their sequence identity. Virus
Accessions used for Sequencing
OYDV
Sel-17 RauG-5 G-1 G-288
SLV
Size of the product
% Identity with sequences available at NCBI Nucleotide
Position in genome
∼318 bp
100–96%
CP
Sel-17 G-1 G-282 AVTG-4
∼587 bp
83–85%
CP and NB
GarCLV
Sel 9 Sel 17 Sel 34 PJS-14
∼451 bp
85–90%
CP
Allexivirus
Sel 17 G-282 Sel 34 Acsession-10
∼190–200 bp
86–93%
NB and UTR
Division of Vegetable Science, IARI. Random leaf samples were collected for each selection line and tested at least twice by RT-PCR. The mixture of the viruses present in garlic accessions was reconfirmed by individual RT-PCR for each virus.
3. Results 3.1. Uniplex RT-PCR standardization The primer sets chosen for each virus amplified the target cDNA of the virus with specificity proving their polyvalence for different virus isolate and garlic accessions. The best amplifications were observed over the temperature range of 58–60 ◦ C when gradient RT-PCR were performed for individual viruses with the chosen set of primers (Table 1).
3.2. Confirmation of RT-PCR products by sequencing Sequencing of the cloned PCR products from garlic accession Sel-17 confirmed the identity of each product and produced 318 bp, 587 bp, 451 bp and 185 bp long nucleotide sequences for OYDV, SLV, GarCLV and Allexivirus respectively (Table 2). BLAST analysis of the nucleotide sequence showed that OYDV fragment had a maximum sequence identity of 99% with CP region of another OYDV sequence (Karnal isolate) from India (Acc. No. DQ519034). SLV fragment has highest sequence similarity of 85% with NB and CP region of SLV sequence from China (Acc. No. AJ292228). Nucleotide sequences of GarCLV and Allexivirus fragments showed 83% and 81% sequence identity with sequences of GarCLV from China (HQ873855) and Garlic Virus X from Zhejiang, China (AJ292229) respectively.
3.3. Optimization of multiplex RT-PCR The multiplex RT-PCR protocol standardized in this study simultaneously amplified target genes of all the four viruses present in the accession Sel-17 (Fig. 1). Annealing temperature was set at 56/57/58 ◦ C while standardizing the multiplex RT-PCR with the optimized set of primers. Amplification of all the four viruses was observed at all the three annealing temperatures but the best amplification was observed at 56 ◦ C (data not shown) and therefore, this annealing temperature was used in optimized multiplex RT-PCR condition.
3.4. Sensitivity level Multiplex RT-PCR with RNA extracted from leaves and cloves of infected garlic amplified OYDV and Allexiviruses up to a dilution of 10−4 from both cloves and leaves while SLV and GarCLV was detected up to a dilution of 10−3 in leaves and 10−4 in cloves (result not shown). Differences in the sensitivity level were not observed between individual RT-PCR and the multiplex RT-PCR. The multiplex RT-PCR protocol was initially validated on four garlic accessions i.e., Accession 10 (Northern India), G-1 (Western India), Phull (Central India), RAUG-5 (Eastern India) and the standardized reaction efficiently detected all viruses present in the garlic samples (Fig. 1). The results were reconfirmed by RT-PCR for individual virus. Amplifications were not observed with tissue culture raised healthy plants. Standardized multiplex RT-PCR successfully detected mixed infection of different viruses in twenty-one garlic accessions from diverse geographical regions in India. OYDV and Allexivirus were detected in all the accessions, SLV in eight accessions and GarCLV in eleven accessions (Table 3) and results were highly reproducible. However, results obtained with twelve accession lines are presented (Fig. 2). The results show the composition of the viral complex in garlic was distinct in different parts of India. OYDV and Allexivirus were found to be present in all the accessions, while carlaviruses i.e., SLV and GarCLV were absent in accession RAU5 from Bihar and two accessions, JNDG-213 and JG-03-263, from Gujarat.
4. Discussion PCR is a routinely used diagnostic technique to test for presence of plant pathogens particularly plant viruses. It is preferred over other methods for its accuracy and rapidity. Use of individual PCR reactions gets limited due to its cost, labor intensive nature and paucity of test sample availability specially samples with multiple infections. Multiplex PCR overcomes these constraints and has the potential to produce accurate results with less effort and resources. It has been successfully used for plant virus diagnostics in different crops (Bariana et al., 1994; Bertolini et al., 2001; Elnifro et al., 2000; Nie and Singh, 2000, 2001, 2002; Hassan et al., 2006; Tao et al., 2012; Xie et al., 2009). In this study a multiplex PCR was standardized for simultaneous detection and identification of four important garlic viruses i.e., OYDV, SLV, GarCLV and Allexivirus. This highly sensitive multiplex
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Fig. 1. Simultaneous detection of OYDV, SLV, GarCLV and Allexivirus in garlic, by multiplex RT-PCR Lanes: M – 100 bp Ladder, Lanes 1 – SLV positive control; 2 – GarCLV positive control; 3 – OYDV positive control; 4 – Allexivirus positive control; multiplex RT-PCR in garlic 5 – G1; 6 – Accession 10; 7 – Phull; 8 – RAUG-5; 9 – Healthy control M – 100 bp Ladder.
Table 3 Occurrence of Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and Allexivirus in Indian garlic accessions from different geographical regions of India. Sl. No
Accession
Place of origin/state
Geographical distribution
No of samples (Clove/leaf) analyzed
OYDV
SLV
GarCLV
Allexivirus
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Sel 9 Sel 17 Sel 34 Accession 10 G-282 G-1 NRCRG-1 AVTG-1 AVTG-4 AVTG-6 AVTG-7 AVTG-10 IETG- 10 IETG-14 IETG-16 JG-03-263 JNDG-213 PJS-14 RAUG-5 Phull Kanpur local
I.A.R.I./Delhi I.A.R.I./Delhi I.A.R.I./Delhi I.A.R.I./Delhi Nasik/Maharashtra Nasik/Maharashtra Pune/Maharashtra Maharashtra Maharashtra Maharashtra Maharashtra Maharashtra Maharashtra Maharashtra Maharashtra Junagad/Gujrat Junagad/Gujrat Punjab Bihar Chattisgarh U.P.
Northern India Northern India Northern India Northern India Western India Western India Western India Western India Western India Western India Western India Western India Western India Western India Western India Western India Western India Northern India Eastern India Central India Northern India
10 20 11 8 15 9 7 5 5 6 5 5 7 5 6 10 11 10 9 7 7
+ + + + + + + + + + + + + + + + + + + + +
+ + + + + + − − + − − − + − − − − − − − −
− + + − + + + + − − − + − + − − − + − + +
+ + + + + + + + + + + + + + + + + + + + +
RT-PCR was able to efficiently and simultaneously detect presence of four garlic viruses by single RT-PCR reaction even at a total RNA dilutions dilution of 10−4 equivalent of 0.0003 microgram RNA. The protocol proved to be equally effective on RNA extracted from cloves as well from the leaves. Further, the standardized multiplex RT-PCR was successfully used to detect the regional distribution of these viruses in garlic accessions from geographically diverse garlic growing areas. Factors that can limit the efficiency of multiplex PCR are poorly designed primers, suboptimal buffer constituents and annealing temperature (Elnifro et al., 2000; Ge et al., 2013; Xie et al., 2009). The presence of more than one primer pair in the multiplex PCR increases the chance of mis-pairing and specificity and/or preferential amplification of certain specific targets (Brownie et al., 1997). Additionally, primer design and prediction of the performance
of a selected primer pair is not possible till the actual reaction is performed (Henegariu et al., 1997). This is even more acute for multiplex PCR because of multiple targets and primers. Keeping these factors in mind, primer pairs were designed with near identical annealing temperatures, function independently without non-specific pairing and produce target amplicons which could be easily resolved. In this study several sets of primers for each virus were empirically evaluated for their sensitivity and specificity by individual RT-PCR. The combinations of primers for multiplex RTPCR were chosen based on their efficacy to perform in groups. The primer combination optimized in our study depending on annealing temperature, amplicon size, buffer conditions and polyvalence for different isolates to successfully amplify from respective virus specific RNA optimally under multiplex condition from all geographically distinct garlic accessions.
Fig. 2. Simultaneous detection of OYDV, SLV, GarCLV and Allexivirus by multiplex RT-PCR in field samples of garlic from different geographical areas Lanes: M – 100 bp Ladder; Lanes 1 – Accessions 10; 2 – Sel 17; 3 – NRCRG-1; 4 – G282; 5 – Sel 9; 6 – Sel34; 7 – JNDG-213; 8 – RAU-5; 9 – Kanpur Local; 10 – AVTG-4; 11 – Phull; 12 – PGS-14; M – 100 bp Ladder.
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In this study it was found that the composition, identity and distribution of garlic viruses varied in various garlic accessions from different geographical regions of India. OYDV and allexiviruses were uniformly distributed in all the accessions, whereas carlaviruses; SLV, GarCLV were absent in the accessions of some geographic areas such as state of Bihar and Gujarat. Of the two carlaviruses, GarCLV was more prevalent than SLV in garlic accessions under trial from the states of Punjab, Chhattisgarh and Uttar Pradesh. Since some of the garlic accessions are not infected by all four viruses, care should be taken to prevent the introduction of these to newer germplasm of the agroclimatic zone. Thus the knowledge of distribution of viruses should be taken into account during movement of material or trafficking garlic seed bulbs. In conclusion, the accuracy and rapidity of multiplex RT-PCR to detect multiple viruses by single reaction will save the resources in large scale studies and its efficiency in using any part of the plant will not only help virus indexing during in vitro virus elimination procedures but will also facilitate the epidemiological studies, breeding for resistance and quarantine applications. Acknowledgements The authors would like to thank Department of Science and Technology, Government of India, for the financial assistance and Dr. R.K. Jain Head, Division of Plant Pathology IARI, New Delhi, for his valuable suggestions. References Arya, M., Baranwal, V.K., Ahlawat, Y.S., Singh, L., 2006. Rt-PCR detection and molecular characterization of Onion yellow dwarf virus associated with garlic and onion. Curr. Sci. 91, 1230–1234. Arya, M., Majumder, S., Baranwal, V.K., 2009. Partial characterisation of coat protein gene of Shallot latent virus associated with garlic in India. Indian J. Virol. 20, 9–11. Baranwal, V.K., Singh, P., Jain, R.K., 2011. First report of Garlic virus X infecting garlic in India. Plant Dis. 95, 1197. Bariana, H.S., Shannon, A.L., Chu, P.W.G., Waterhouse, P.M., 1994. Detection of five seedborne legume viruses in one sensitive multiplex polymerase chain reaction test. Phytopathol. 84, 1201–1205. Bertolini, E., Olmos, A., Martinez, M.C., Gorris, M.T., Cambra, M., 2001. Single step multiplex RT-PCR for simultaneous and colourimetric detection of six RNA viruses in olive trees. J. Virol. Methods 96, 33–41. Brownie, J., Shawcross, S., Theaker, J., Whitcombe, D., Ferrie, R., Newton, C., Little, S., 1997. The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res. 25, 3235–3241. Cafrune, E.E., Perotto, M.C., Conci, V.C., 2006. Effect of two Allexivirus isolates on garlic yield. Plant Dis. 90, 898–904. Conci, V.C., Canavelli, A., Lunello, P., 2003. Yield losses associated with virus-infected garlic plants during five successive years. Plant Dis. 87, 1411–1415. Dantu, P.K., Bhojwani, S.S., 1992. In vitro propagation of gladiolus: optimisation of conditions for shoot multiplication. J. Plant Biochem. Biotechnol. 1, 115–118. Dovas, C.I., Hatzibukas, E., Salomon, R., Barg, E., Shiboleth, Y.M., Katis, N.I., 2001. Comparison of methods for virus detection in Allium spp. J. Phytopathol. 149, 731–737.
Du, Z., Chen, J., Hiruki, C., 2006. Optimization and application of a multiplex RT-PCR system for simultaneous detection of five potato viruses using 18S rRNA as an internal control. Plant Dis. 90, 185–189. Elnifro, E.M., Ashshi, A.M., Cooper, R.J., Klapper, P.E., 2000. Multiplex PCR: optimization and application in diagnostic virology. Clin. Microbiol. Rev. 13, 559–570. Ge, B., Li, Q., Liu, G., Lu, M., Li, S., Wang, H., 2013. Simultaneous detection and identification of four virusesinfectingpepino by multiplex RT-PCR. Arch. Virol. 158, 1181–1187. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucl Acids Symp Ser. 41, 95–98. Hassan, M., Myrta, A., Polak, J., 2006. Simultaneous detection and identification of four pome fruit viruses by one-tube pentaplex RT-PCR. J. Virol. Methods 133, 124–129. Henegariu, O., Heerema, N.A., Dlouhy, S.R., Vance, G.H., Vogt, P.H., 1997. Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23, 504–511. Keusgen, M., 2002. Health and alliums. In: Rabinowitch, H.D., Currah, L. (Eds.), Allium Crop Science: Recent Advances. CAB International, UK. Kumar, S., Baranwal, V.K., Joshi, S., Arya, M., Majumder, S., 2010. Simultaneous detection of mixed infection of Onion yellow dwarf virus and an Allexivirus in RT-PCR for ensuring virus free onion bulbs. Indian J. Virol. 21, 64–68. Lot, H., Chovelon, V., Souche, S., Delecolle, B., 1998. Effects of Onion yellow dwarf and Leek yellow stripe viruses on symptomatology and yield loss of three French garlic cultivars. Plant Dis. 82, 1381–1385. Majumder, S., Baranwal, V.K., Joshi, S., 2008. Simultaneous detection of Onion yellow dwarf virus and Garlic latent virus in infected leaves and cloves of garlic by duplex RT-PCR. J. Plant Pathol. 90, 369–372. Majumder, S., Baranwal, V.K., 2009. First report of Garlic common latent virus in garlic from India. Plant Dis. 93, 106. MeloFilho, P., de, A., Nagata, T., Dusi, A.N., Buso, J.A., Torres, A.C., Eiras, M., Resende, R., de, O., 2004. Detection of three Allexivirus species infecting garlic in Brazil. Pesqui. Agropecu. Bras. 39, 735–740. Nie, X., Singh, R.P., 2000. Detection of multiple potato viruses using an oligo(dT) as a common cDNA primer in multiplex RT-PCR. J. Virol. Methods 86, 179–185. Nie, X., Singh, R.P., 2001. A novel usage of random primers for multiplex RT-PCR detection of virus and viroid in aphids, leaves, and tubers. J. Virol. Methods 91, 37–49. Nie, X., Singh, R.P., 2002. A new approach for the simultaneous differentiation of biological and geographical strains of potato virus Y by uniplex and multiplex RT-PCR. J. Virol. Methods 104, 41–54. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York, NY, USA. Shiboleth, Y.M., Gal-On, a., Koch, M., Rabinowitch, H.D., Salomon, R., 2001. Molecular characterisation of Onion yellow dwarf virus (OYDV) infecting garlic (Allium sativum L.) in Israel: thermotherapy inhibits virus elimination by meristem tip culture. Ann. Appl. Biol. 138, 187–195. Takaichi, M., Yamamoto, M., Nagakubo, T., Oeda, K., 2001. Mixed virus infections of garlic determined by a multivalent polyclonal antiserum and virus effects on disease symptoms. Plant Dis. 85, 71–75. Tao, Y., Man, J.Y., Wu, Y.F., 2012. Development of a multiplex polymerase chain reaction for simultaneous detection of wheat viruses and a phytoplasma in China. Arch. Virol. 157, 1261–1267. Tsuneyoshi, T., Sumi, S., 1996. Differentiation among garlic viruses in mixed infections based on RT-PCR procedures and direct tissue blotting immunoassays. Phytopathol. 86, 253–259. Tsuneyoshi, T., Matsumi, T., Natsuaki, K.T., Sumi, S., 1998. Nucleotide sequence analysis of virus isolates indicates the presence of three potyvirus species in Allium plants. Arch. Virol. 143, 97–113. Van Dijk, P., 1994. Virus diseases of Allium species and prospects for their control. Acta Hortic. 358, 299–306. Walkey, D.G.A., Antill, D.N., 1989. Agronomic evaluation of virus-free and virus infected garlic (Allium sativum L.). J. Hortic. Sci. 64, 53–60. Xie, Y., Wang, M., Xu, D., Li, R., Zhoua, G., 2009. Simultaneous detection and identification of four sugarcane viruses by one-step RT-PCR. J. Virol. Method 162, 64–68.
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions S. Majumder 1 , V.K. Baranwal ∗ Plant Virology Unit, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, India
a b s t r a c t Article history: Received 19 July 2013 Received in revised form 16 February 2014 Accepted 23 February 2014 Available online 2 March 2014 Keywords: Multiplex RT-PCR Garlic viruses OYDV GarCLV SLV Allexivirus
Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Garlic (Allium sativum L.) is one of the most important culinary herbs of the world with an annual production of 22.28 million tons planted globally (FAO 20011) and it is used widely (Keusgen, 2002). Garlic stocks worldwide are infected with a number of viruses often designated as ‘garlic viral complex’ (Walkey and Antill, 1989; Van Dijk, 1994). The complex may include potyviruses; Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV), carlaviruses; Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and Allexiviruses; Garlic virus X (GVX), Garlic virus A,B,D,E, Garlic mite borne filamentous virus, and Shallot Virus X (ShVX). OYDV, SLV, GarCLV and allexiviruses (GVX) have been recorded in garlic cultivars from India (Arya et al., 2006, 2009; Majumder and Baranwal, 2009; Baranwal et al., 2011). These viruses may not kill the plant but can reduce yield up to 50% during successive cultivation (Lot et al., 1998; Conci et al., 2003; Cafrune et al., 2006). Serological methods such as enzyme-linked immunosorbent assay (ELISA), direct tissue blotting immunoassay and Dot ELISA are often employed for indexing of viruses associated with garlic (Tsuneyoshi and Sumi, 1996; Takaichi et al., 2001; Conci et al., 2003; MeloFilho et al., 2004). But antibodies to all the viruses are not
always easily available and their production is a complex process due to multiple viral infections in the garlic. Reverse transcription polymerase chain reaction (RT-PCR) has been proved to be many times more sensitive and results are obtained faster compared to serological assays like ELISA (Dovas et al., 2001; Shiboleth et al., 2001; Du et al., 2006). RT-PCR is regularly used for detecting individual garlic viruses such as OYDV (Dovas et al., 2001; Takaichi et al., 2001; Arya et al., 2006), SLV (Tsuneyoshi et al., 1998; Arya et al., 2009) and Allexivirus (Dovas et al., 2001; Baranwal et al., 2011). Duplex RT-PCR has been demonstrated for simultaneous detection of OYDV and SLV in Indian garlic and for OYDV and Allexivirus in Indian onion (Majumder et al., 2008; Kumar et al., 2010). However, as mixed infection of OYDV, SLV, GarCLV and allexiviruses have been recorded in different garlic accessions of India (Baranwal et al., 2011), it became imperative to develop a multiplex PCR which can be used for simultaneous detection of these viruses. We developed a standardized multiplex RT-PCR for detection of these viruses and used to investigate the occurrence and distribution of these viruses in garlic accessions from geographically diverse garlic growing areas of India. 2. Materials and methods 2.1. Source of plant material from different regions of India
∗ Corresponding author. Tel.: +91 9818756899; fax: +91 11 25843113. E-mail address: [email protected] (V.K. Baranwal). 1 Present address: Department of Biotechnology, Sharda University, Greater Noida 201306, UP, India. http://dx.doi.org/10.1016/j.jviromet.2014.02.019 0166-0934/© 2014 Elsevier B.V. All rights reserved.
Five garlic accessions viz., Selection 17 (Sel-17; Northern India), Pusa Accession 10 (Northern India), G-1 (Western India), Phull (Central India) and RAUG-5 (Eastern India), were initially used to
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Table 1 Details of primers used for multiplex RT- PCR for simultaneous detection of four viruses in garlic. Virus OYDV F 10214 OYDV R-10539 Allexi 1 F 8623 Allexi 2 R 8813 SLV5F 2124 SLVNABR2710 GarCLVCP1045 GarCLV R 1494
Primer sequence
5 -CGTTTGTTTGGCCTGGATGGTAACG-3 5 -GTCTCYGTAATTCACGC-3 5 -CYGCTAAGCTATATGCTGAARGG-3 5 -TGTTRCAARGTAAGTTTAGYAATATCAACA-3 5 -GTTCTGGGAYATWTGCATGTA-3 5 -CCCGCACTTTCACACACTAT-3 5 -AAATGTTAATCGCTAAACGACC-3 5 -CWRCCATTAAAACGTAGCAGC-3
Genes used
TM ◦ C
Best at
Size of product
CP 3 UTR NB 3 UTR CP NB CP NB
61 56 62 60 57 60 60 56
58
318
60
180–190
60
587
60
451
CP: coat protein; NB: nucleic acid binding protein; UTR: untranslated region.
standardize and validate individual RT-PCR for OYDV, SLV, GarCLV and Allexivirus. Since these viruses were observed in Sel-17, this accession was selected for standardizing multiplex RT- PCR. Half the cloves from a mother bulb were planted for future use and the other half were used in the experiments. Apical meristem of a clove from Sel-17 was micropropagated (Dantu and Bhojwani, 1992) and virus free plants were used as healthy negative control. 2.2. Nucleic acid extraction Total RNA was extracted from 50 mg tissues of infected leaves/cloves and healthy leaves of garlic, using RNeasy Plant Mini Kit (Qiagen, Germany) as per manufacturer’s protocol. The yield and the quality of the RNA were analyzed in a UV visible double beam spectro-photometer (Shimadzu-1800, Japan). 2.3. Primer design and synthesis For detection of allexiviruses previously published primer set producing an amplicon of ∼200 bp was used (Dovas et al., 2001). Primers for detection of OYDV, SLV and GarCLV were designed manually (Table 1) using gene sequences available at NCBI GenBank. Bioedit (7.0.7.1) (Hall, 1999) was used to align sequences and locate conserved regions. Primers for each OYDV, SLV and GarCLV were designed to amplify different sizes of amplicons in the size range of 300–600 bp with annealing temperatures in the range of 56–60 ◦ C. The primers were synthesized by Sigma Aldrich, Bangalore, India. 2.4. Standardization of RT-PCR for individual virus First strand cDNA was synthesized separately for all the four viruses using 4 l of total RNA and reverse transcription (RT) mixture using reverse primers of OYDV/SLV/GarCLV or allexiviruses (final concentration of 0.2 M), 20 U M-MuLV reverse trancriptase (Fermentas, USA), 0.3 mM dNTPs in the total reaction mixture of 20 l and incubated at 42 ◦ C for 45 min. The reaction was stopped by heating at 70 ◦ C for 10 min. To standardize the annealing temperature for primer pairs, gradient PCR (temperature range of 54–60 ◦ C) was performed for each virus in a thermocycler (Eppendorf Mastercycler Gradient, USA). PCR was performed in a 50 l reaction volume containing 5 l of cDNA, 5 l of 10× reaction buffer, 1.5 mM MgCl2 , primer pairs at 0.2 M each, 0.2 mM dNTPs and 5 U of Taq DNA polymerase (Promega, USA). The reaction profile consisted of an initial denaturation step at 94 ◦ C for 5 min, 30 cycles of 45 s at 94 ◦ C, 20 s at annealing temperature (54, 55, 56, 57, 58, 59 and 60 ◦ C) and 1 min extension at 72 ◦ C and a final extension step at 72 ◦ C for 10 min. The reactions were repeated 3–4 times with primer sets for each virus and the best combination of primer concentration and annealing temperature (Table 1) was chosen for further work. Ten microlitres of amplified product were separated by electrophoresis in a 1.2% agarose gel containing ethidium bromide and photographed under UV illumination with an imaging system
(Biorad XR documentation system). To confirm the identity of the amplified DNA fragments, the purified PCR products from garlic Sel-17 were cloned and sequenced. PCR products were purified using PCR Purification kit (Qiagen, Germany), ligated into pGEMT Easy vector (Promega, USA) and transformed Escherichia coli, DH5␣ (Sambrook et al., 1989). Recombinant clones were identified by colony PCR and sequenced at Chromous Biotech, Bangalore, India. The nucleotide sequences obtained were analyzed by BLAST to verify the sequence identity. 2.5. Optimization of multiplex RT-PCR To standardize multiplex RTPCR, cDNAs for OYDV, SLV, GarCLV and allexiviruses were prepared simultaneously in a single tube. The 25 l reaction mixture contained 6 l of total RNA, the optimized reverse primer for OYDV, SLV, and GarCLV at a concentration of 0.3 M and allexiviruses at a concentration of 0.2 M, 20 unitsMuLV reverse trancriptase (Fermentas, USA), 5 l of 5× reaction buffer and 0.3 mM dNTPs. The reaction mixture was incubated at 42 ◦ C for 45 min and the reaction was stopped by heating at 70 ◦ C for 10 min. The multiplex PCR was performed using a reaction mixture containing 5 l of reverse transcription (RT) mixture, 5 U of Taq DNA polymerase (Promega, Madison, USA), 5 l of 10× reaction buffer, 1.5 mM MgCl2 , primer pairs at a concentration of 0.2 M and 0.2 mM dNTPs. The temperature profile consisted of a denaturation step at 94 ◦ C for 5 min, 30 cycles of 45 s at 94 ◦ C, 20 s annealing temperature of 56/57/58 ◦ C and 1 min at 72 ◦ C and one final extension step at 72 ◦ C for 10 min on a gradient PCR. As a control, standard RTPCR was performed for each of the viruses using 5 l of the same RT reaction (cocktail) with 56 ◦ C as annealing temperature. To confirm the identity, four PCR products obtained were clone sequenced. 2.6. Sensitivity level of multiplex RT-PCR To determine the sensitivity limit of multiplex RT-PCR, different dilutions of RNA ranging from 101 (1 l of 40 l RNA isolated from 50 mg tissue) to 10−4 , corresponding to 0.318–0.0003 g of RNA (extracted from both leaves as well as cloves of diseased sample) were used for preparing individual as well as mixed cDNA. Reaction conditions for cDNA preparation were same as described in the previous section. Individual as well as multiplex RT-PCR was performed using these cDNA. To evaluate the performance of standardized multiplex PCR, it was initially performed on four garlic accessions mentioned earlier. Tissue culture raised plants were used as negative control. Individual RT-PCR was performed for all the four viruses to cross check the results. 2.7. Validation of multiplex RT-PCR The optimized multiplex RT-PCR was validated by performing it on twenty-one accessions of garlic from different geographical regions of India (Table 3). The samples were collected from the
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Table 2 Size of the RT-PCR product and their sequence identity. Virus
Accessions used for Sequencing
OYDV
Sel-17 RauG-5 G-1 G-288
SLV
Size of the product
% Identity with sequences available at NCBI Nucleotide
Position in genome
∼318 bp
100–96%
CP
Sel-17 G-1 G-282 AVTG-4
∼587 bp
83–85%
CP and NB
GarCLV
Sel 9 Sel 17 Sel 34 PJS-14
∼451 bp
85–90%
CP
Allexivirus
Sel 17 G-282 Sel 34 Acsession-10
∼190–200 bp
86–93%
NB and UTR
Division of Vegetable Science, IARI. Random leaf samples were collected for each selection line and tested at least twice by RT-PCR. The mixture of the viruses present in garlic accessions was reconfirmed by individual RT-PCR for each virus.
3. Results 3.1. Uniplex RT-PCR standardization The primer sets chosen for each virus amplified the target cDNA of the virus with specificity proving their polyvalence for different virus isolate and garlic accessions. The best amplifications were observed over the temperature range of 58–60 ◦ C when gradient RT-PCR were performed for individual viruses with the chosen set of primers (Table 1).
3.2. Confirmation of RT-PCR products by sequencing Sequencing of the cloned PCR products from garlic accession Sel-17 confirmed the identity of each product and produced 318 bp, 587 bp, 451 bp and 185 bp long nucleotide sequences for OYDV, SLV, GarCLV and Allexivirus respectively (Table 2). BLAST analysis of the nucleotide sequence showed that OYDV fragment had a maximum sequence identity of 99% with CP region of another OYDV sequence (Karnal isolate) from India (Acc. No. DQ519034). SLV fragment has highest sequence similarity of 85% with NB and CP region of SLV sequence from China (Acc. No. AJ292228). Nucleotide sequences of GarCLV and Allexivirus fragments showed 83% and 81% sequence identity with sequences of GarCLV from China (HQ873855) and Garlic Virus X from Zhejiang, China (AJ292229) respectively.
3.3. Optimization of multiplex RT-PCR The multiplex RT-PCR protocol standardized in this study simultaneously amplified target genes of all the four viruses present in the accession Sel-17 (Fig. 1). Annealing temperature was set at 56/57/58 ◦ C while standardizing the multiplex RT-PCR with the optimized set of primers. Amplification of all the four viruses was observed at all the three annealing temperatures but the best amplification was observed at 56 ◦ C (data not shown) and therefore, this annealing temperature was used in optimized multiplex RT-PCR condition.
3.4. Sensitivity level Multiplex RT-PCR with RNA extracted from leaves and cloves of infected garlic amplified OYDV and Allexiviruses up to a dilution of 10−4 from both cloves and leaves while SLV and GarCLV was detected up to a dilution of 10−3 in leaves and 10−4 in cloves (result not shown). Differences in the sensitivity level were not observed between individual RT-PCR and the multiplex RT-PCR. The multiplex RT-PCR protocol was initially validated on four garlic accessions i.e., Accession 10 (Northern India), G-1 (Western India), Phull (Central India), RAUG-5 (Eastern India) and the standardized reaction efficiently detected all viruses present in the garlic samples (Fig. 1). The results were reconfirmed by RT-PCR for individual virus. Amplifications were not observed with tissue culture raised healthy plants. Standardized multiplex RT-PCR successfully detected mixed infection of different viruses in twenty-one garlic accessions from diverse geographical regions in India. OYDV and Allexivirus were detected in all the accessions, SLV in eight accessions and GarCLV in eleven accessions (Table 3) and results were highly reproducible. However, results obtained with twelve accession lines are presented (Fig. 2). The results show the composition of the viral complex in garlic was distinct in different parts of India. OYDV and Allexivirus were found to be present in all the accessions, while carlaviruses i.e., SLV and GarCLV were absent in accession RAU5 from Bihar and two accessions, JNDG-213 and JG-03-263, from Gujarat.
4. Discussion PCR is a routinely used diagnostic technique to test for presence of plant pathogens particularly plant viruses. It is preferred over other methods for its accuracy and rapidity. Use of individual PCR reactions gets limited due to its cost, labor intensive nature and paucity of test sample availability specially samples with multiple infections. Multiplex PCR overcomes these constraints and has the potential to produce accurate results with less effort and resources. It has been successfully used for plant virus diagnostics in different crops (Bariana et al., 1994; Bertolini et al., 2001; Elnifro et al., 2000; Nie and Singh, 2000, 2001, 2002; Hassan et al., 2006; Tao et al., 2012; Xie et al., 2009). In this study a multiplex PCR was standardized for simultaneous detection and identification of four important garlic viruses i.e., OYDV, SLV, GarCLV and Allexivirus. This highly sensitive multiplex
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Fig. 1. Simultaneous detection of OYDV, SLV, GarCLV and Allexivirus in garlic, by multiplex RT-PCR Lanes: M – 100 bp Ladder, Lanes 1 – SLV positive control; 2 – GarCLV positive control; 3 – OYDV positive control; 4 – Allexivirus positive control; multiplex RT-PCR in garlic 5 – G1; 6 – Accession 10; 7 – Phull; 8 – RAUG-5; 9 – Healthy control M – 100 bp Ladder.
Table 3 Occurrence of Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and Allexivirus in Indian garlic accessions from different geographical regions of India. Sl. No
Accession
Place of origin/state
Geographical distribution
No of samples (Clove/leaf) analyzed
OYDV
SLV
GarCLV
Allexivirus
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Sel 9 Sel 17 Sel 34 Accession 10 G-282 G-1 NRCRG-1 AVTG-1 AVTG-4 AVTG-6 AVTG-7 AVTG-10 IETG- 10 IETG-14 IETG-16 JG-03-263 JNDG-213 PJS-14 RAUG-5 Phull Kanpur local
I.A.R.I./Delhi I.A.R.I./Delhi I.A.R.I./Delhi I.A.R.I./Delhi Nasik/Maharashtra Nasik/Maharashtra Pune/Maharashtra Maharashtra Maharashtra Maharashtra Maharashtra Maharashtra Maharashtra Maharashtra Maharashtra Junagad/Gujrat Junagad/Gujrat Punjab Bihar Chattisgarh U.P.
Northern India Northern India Northern India Northern India Western India Western India Western India Western India Western India Western India Western India Western India Western India Western India Western India Western India Western India Northern India Eastern India Central India Northern India
10 20 11 8 15 9 7 5 5 6 5 5 7 5 6 10 11 10 9 7 7
+ + + + + + + + + + + + + + + + + + + + +
+ + + + + + − − + − − − + − − − − − − − −
− + + − + + + + − − − + − + − − − + − + +
+ + + + + + + + + + + + + + + + + + + + +
RT-PCR was able to efficiently and simultaneously detect presence of four garlic viruses by single RT-PCR reaction even at a total RNA dilutions dilution of 10−4 equivalent of 0.0003 microgram RNA. The protocol proved to be equally effective on RNA extracted from cloves as well from the leaves. Further, the standardized multiplex RT-PCR was successfully used to detect the regional distribution of these viruses in garlic accessions from geographically diverse garlic growing areas. Factors that can limit the efficiency of multiplex PCR are poorly designed primers, suboptimal buffer constituents and annealing temperature (Elnifro et al., 2000; Ge et al., 2013; Xie et al., 2009). The presence of more than one primer pair in the multiplex PCR increases the chance of mis-pairing and specificity and/or preferential amplification of certain specific targets (Brownie et al., 1997). Additionally, primer design and prediction of the performance
of a selected primer pair is not possible till the actual reaction is performed (Henegariu et al., 1997). This is even more acute for multiplex PCR because of multiple targets and primers. Keeping these factors in mind, primer pairs were designed with near identical annealing temperatures, function independently without non-specific pairing and produce target amplicons which could be easily resolved. In this study several sets of primers for each virus were empirically evaluated for their sensitivity and specificity by individual RT-PCR. The combinations of primers for multiplex RTPCR were chosen based on their efficacy to perform in groups. The primer combination optimized in our study depending on annealing temperature, amplicon size, buffer conditions and polyvalence for different isolates to successfully amplify from respective virus specific RNA optimally under multiplex condition from all geographically distinct garlic accessions.
Fig. 2. Simultaneous detection of OYDV, SLV, GarCLV and Allexivirus by multiplex RT-PCR in field samples of garlic from different geographical areas Lanes: M – 100 bp Ladder; Lanes 1 – Accessions 10; 2 – Sel 17; 3 – NRCRG-1; 4 – G282; 5 – Sel 9; 6 – Sel34; 7 – JNDG-213; 8 – RAU-5; 9 – Kanpur Local; 10 – AVTG-4; 11 – Phull; 12 – PGS-14; M – 100 bp Ladder.
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In this study it was found that the composition, identity and distribution of garlic viruses varied in various garlic accessions from different geographical regions of India. OYDV and allexiviruses were uniformly distributed in all the accessions, whereas carlaviruses; SLV, GarCLV were absent in the accessions of some geographic areas such as state of Bihar and Gujarat. Of the two carlaviruses, GarCLV was more prevalent than SLV in garlic accessions under trial from the states of Punjab, Chhattisgarh and Uttar Pradesh. Since some of the garlic accessions are not infected by all four viruses, care should be taken to prevent the introduction of these to newer germplasm of the agroclimatic zone. Thus the knowledge of distribution of viruses should be taken into account during movement of material or trafficking garlic seed bulbs. In conclusion, the accuracy and rapidity of multiplex RT-PCR to detect multiple viruses by single reaction will save the resources in large scale studies and its efficiency in using any part of the plant will not only help virus indexing during in vitro virus elimination procedures but will also facilitate the epidemiological studies, breeding for resistance and quarantine applications. Acknowledgements The authors would like to thank Department of Science and Technology, Government of India, for the financial assistance and Dr. R.K. Jain Head, Division of Plant Pathology IARI, New Delhi, for his valuable suggestions. References Arya, M., Baranwal, V.K., Ahlawat, Y.S., Singh, L., 2006. Rt-PCR detection and molecular characterization of Onion yellow dwarf virus associated with garlic and onion. Curr. Sci. 91, 1230–1234. Arya, M., Majumder, S., Baranwal, V.K., 2009. Partial characterisation of coat protein gene of Shallot latent virus associated with garlic in India. Indian J. Virol. 20, 9–11. Baranwal, V.K., Singh, P., Jain, R.K., 2011. First report of Garlic virus X infecting garlic in India. Plant Dis. 95, 1197. Bariana, H.S., Shannon, A.L., Chu, P.W.G., Waterhouse, P.M., 1994. Detection of five seedborne legume viruses in one sensitive multiplex polymerase chain reaction test. Phytopathol. 84, 1201–1205. Bertolini, E., Olmos, A., Martinez, M.C., Gorris, M.T., Cambra, M., 2001. Single step multiplex RT-PCR for simultaneous and colourimetric detection of six RNA viruses in olive trees. J. Virol. Methods 96, 33–41. Brownie, J., Shawcross, S., Theaker, J., Whitcombe, D., Ferrie, R., Newton, C., Little, S., 1997. The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res. 25, 3235–3241. Cafrune, E.E., Perotto, M.C., Conci, V.C., 2006. Effect of two Allexivirus isolates on garlic yield. Plant Dis. 90, 898–904. Conci, V.C., Canavelli, A., Lunello, P., 2003. Yield losses associated with virus-infected garlic plants during five successive years. Plant Dis. 87, 1411–1415. Dantu, P.K., Bhojwani, S.S., 1992. In vitro propagation of gladiolus: optimisation of conditions for shoot multiplication. J. Plant Biochem. Biotechnol. 1, 115–118. Dovas, C.I., Hatzibukas, E., Salomon, R., Barg, E., Shiboleth, Y.M., Katis, N.I., 2001. Comparison of methods for virus detection in Allium spp. J. Phytopathol. 149, 731–737.
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