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Journal of Virological Methods 159 (2009) 34–39

Contents lists available at ScienceDirect

Journal of Virological Methods

journa l homepage: www.e lsev ier .com/ locate / jv i romet

n improved method of DNA isolation suitable for PCR-based detection ofegomoviruses from jute and other mucilaginous plants

aju Ghosh, Sujay Paul, Subrata Kumar Ghosh, Anirban Roy ∗

lant Virus Laboratory and Biotechnology Unit, Division of Crop Protection, Central Research Institute for Jute and Allied Fibres,arrackpore, Kolkata 700120, West Bengal, India

rticle history:eceived 23 November 2008eceived in revised form 12 February 2009ccepted 19 February 2009vailable online 3 March 2009

a b s t r a c t

A relatively quick and inexpensive modified cetyl trimethylammonium bromide method for extraction ofDNA from leaf materials containing large quantities of mucilage is described. The modification includinguse of more volume of extraction buffer and dissolving crude nucleic acid pellet in 1 M NaCl, reducedmarkedly the viscosity of the mucilage and thus in the final purification step yielded a larger quan-tity of mucilage-free DNA suitable for subsequent PCR-based detection of begomoviruses. The method

eywords:egomovirusesetasatellitesNA extractionucilage

ute

was standardized with jute samples with yellow mosaic disease and validated with different othermucilaginous-hosts with low titre of begomoviruses. DNA isolated using this method showed consis-tency in yield and compatibility with PCR for detection of begomoviruses from different mucilaginousplant species. The method was compared for efficacy with other reported methods and it was found tobe superior over the existing methods described for isolation of DNA from mucilaginous hosts. Thus themethod described could be used on a wider scale for reliable and consistent detection of begomoviruses

for ch

esta from mucilaginous hosts

. Introduction

In tropical and subtropical regions of the world, begomoviruesransmitted by whitefly and belonging to the family Geminiviri-ae are emerging currently as a major threat to a number of cropsmportant economically (Polston and Anderson, 1997; Faria and

axwell, 1999). Furthermore, evolution of newer begomovirusesith their variants and the occurrence of mixed infection withifferent begomoviruses have aggravated the disease spectrum.he ability of adaptation to new hosts by these begomovirusomplexes in association with satellite molecules (betasatellites)volved newly posed an additional threat to the cultivation ofrops (Varma and Malathi, 2003). To provide more efficient croprotection strategies, a better understanding of the variability ofhe begomoviruses and their geographical distribution are neededhich essentially calls for a rapid method for detection of these

iruses. DNA-based diagnostic approaches such as amplificationf targeted viral genome or genomic component(s) by the poly-

erase chain reaction (PCR) and DNA sequencing has replaced

he use of serological techniques for detection and identificationf begomoviruses (Briddon and Markham, 1994; Jose and Usha,000; Sharma et al., 2005). To accommodate the need for PCR-

∗ Corresponding author. Tel.: +91 3325356124; fax: +91 3325350415.E-mail address: anirbanroy75@yahoo.com (A. Roy).

166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2009.02.020

aracterization and variability study.© 2009 Elsevier B.V. All rights reserved.

based detection and characterization, good quality DNA with largequantity is a prerequisite since the concentration of the genomiccomponents of the begomoviruses in infected plant tissues wasoften found to be very low (Swanson et al., 1992). Hence, rapid,simple and reliable DNA isolation method is the most importantinitial step to progress further.

DNA extraction from plant tissues, unlike DNA isolation frommammalian tissues, remains difficult due to the presence of arigid cell wall surrounding the plant cells. Plants such as bhendi(Abelmoschus esculentus), jute (Corchorus sp.), mesta (Hibiscuscannabinus), sida (Sida sp.), which serve as natural hosts for dif-ferent begomoviruses (Jose and Usha, 2003; Ha et al., 2008; Ghoshet al., 2008; Chatterjee and Ghosh, 2007a; Das et al., 2008a,b; Roy etal., 2009; Hofer et al., 1997), possess large amount of foliar mucilagethat hinders DNA isolation. Mucilage is highly viscous secondarymetabolite, exopolysaccharide in nature and is composed of polarpolymer of glycoprotein. Many malvaceous plants contain largeamounts of mucilage and the removal of the mucilaginous sub-stances during DNA isolation from such plants has been reported tobe very difficult (Bayer et al., 1999). Mucilage often binds to othersecondary metabolites such as phenolics, tannins and alkaloidsand co-precipitates with DNA during isolation. Contamination of

mucilage and other secondary metabolites not only makes the DNAunmanageable during pipetting but also hinders further down-stream applications such as the polymerase chain reaction (PCR)or other enzymatic reactions since they inhibit Taq polymeraseactivity (Fang et al., 1992) and interfere directly or indirectly with

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he enzymatic reactions (Weishing et al., 1995). Such a problemould be overcome by extracting the viral DNA from virus-infectedon-mucilaginous permissive hosts such as tobacco (Ikegami et al.,981; Hamilton et al., 1982). However, for the characterization ofhe genomic components of begomoviruses it is desirable to haveiral DNA extracted from the natural hosts (Saunders et al., 2000).ue to the chemical complexity and variation in the rheologicalroperties, the concentration and viscosity of mucilage found variesrom one plant species to other or even among different cultivarsnd germplasm of one species (Muralikrishna et al., 1989; Bhat andharanathan, 1987; Kawamura et al., 2000; Axel Diederichsen et al.,006). Hence, the method for mucilage-free DNA isolation for onelant species may not be suitable for the other species.

Recently, full and partial genome sequences of different bego-oviruses and betasatellites infecting two important bast fibre

rops, mesta and jute, which are rich in mucilage content wereeported (Chatterjee and Ghosh, 2007a,b; Das et al., 2008a,b; Pault al., 2008; Ghosh et al., 2008; Roy et al., 2009). Mention was madehat the DNA isolated by the standard cetyl trimethylammoniumromide (CTAB) method (Doyle and Doyle, 1987) either did notespond to PCR or gave inconsistent amplification due to contami-ation of such mucilaginous substances and thus the isolated DNAas purified further through DNeasy mini spin column (QIAGEN,mbH, Hilden, Germany). This additional purification step involvedot only more time and cost but also the quantity of the isolatedNA was lower and was not reproducible by PCR. The present studyddresses such problems and describes an improved, rapid andnexpensive, modified cetyl trimethylammonium bromide (CTAB)

ethod for DNA isolation suitable for further downstream amplifi-ation of full or partial genome of begomoviruses and betasatellites.he method described in this study has been standardized withute plants infected with a begomovirus, Corchorus golden mosaicirus (CoGMV), and was validated further with different diseaseducilaginous crops with low titre of different begomoviruses.

. Materials and methods

.1. Plant samples for DNA isolation

Initially, for standardization of the method, CoGMV infectedhite jute (Corchorus capsularis) leaves with yellow mosaic

ymptoms were chosen as the target plant samples. After stan-ardization, the method was validated with different mucilaginouslants with typical symptoms of begomovirus infection. Plant sam-les used for this purpose were yellow mosaic disease affectedorchorus olitorius, yellow vein mosaic and leaf curl disease affectedesta (Hibiscus cannabinus), yellow vein mosaic disease affected

orchorus pseudo-capsularis, Congo-jute (Urena lobata), bhendiAbelmoschus esculentus), Sida sp., and Malachra sp. Symptomaticeaves from these plants were collected from different states suchs Uttar Pradesh, West Bengal and Andhra Pradesh of India.

.2. DNA isolation protocol

The extraction buffer used for this method was composed of00 mM Tris–HCl pH 8, 10 mM EDTA pH 8, 1.4 M NaCl, 2% CTABnd 0.2% �-marcaptoethanol. Fresh or frozen leaf tissue (100 mg)as ground to a fine powder with liquid nitrogen using pre-chilledortar and pestle. The samples were then transferred to 30 ml

olypropylene tubes and 10 ml of pre-warmed (65 ◦C) extraction

uffer was added to the samples and incubated at 65 ◦C for 30 min.uring incubation, the contents were mixed three to four timesy inverting the tubes gently. For removing the organic contami-ants, 0.6 volume of chloroform:iso-amylalcohol (24:1) was addednd mixed thoroughly for 5 min, to form an emulsion. The samples

l Methods 159 (2009) 34–39 35

were then centrifuged at 16,500 × g for 10 min at room temperatureand the upper aqueous phase was transferred to a fresh tube. Abouttwo-thirds of volume of chilled iso-propanol was added and mixedwell by inverting the tubes. The samples were then centrifuged at14,000 × g for 10 min to collect the nucleic acid precipitate. Thecrude nucleic acid pellet was air-dried and resuspended in 1 mlof 1 M NaCl instead of dissolving it in Tris–EDTA (TE) buffer. Theentire solution was transferred to a 2 ml microcentrifuge tube andtreated with RNase at 37 ◦C for 30 min. RNase contamination wasremoved by adding an equal volume of phenol:chloroform and theaqueous phase was collected in a fresh microfuge tube after cen-trifugation at 14,000 × g for 5 min at room temperature. Again, anequal volume of chloroform:iso-amylalcohol (24:1) was added andmixed thoroughly to form an emulsion. The samples were cen-trifuged at 16,500 × g for 5 min at room temperature and the topaqueous phase was transferred to a fresh tube. To precipitate theDNA, a double volume of absolute ethanol was added into the col-lected aqueous phase and was mixed gently by inverting the tube.After centrifugation at 14,000 × g for 10 min at 4 ◦C, the DNA pel-let was washed with 70% ethanol, air-dried and finally the purifiedDNA pellet was dissolved in 100 �l of TE buffer. After spectropho-tometric estimation, 50–100 ng of DNA was used for amplificationby PCR.

The method used in the study differed basically from the stan-dard cetyl trimethylammonium bromide (CTAB) method (Doyle andDoyle, 1987) in four major ways: (i) the ratio of volume of extrac-tion buffer added to amount of leaf tissue extracted (v/w) is 100:1,which diluted the viscosity of the mucilage; (ii) instead of Tris–EDTAbuffer, the crude nucleic acid pellet was dissolved in 1 M NaCl,which ensured further reduction of viscosity of the mucilaginoussubstances; (iii) RNase treatment was employed under 1 M NaClfor increasing the specificity of RNase; (iv) two additional steps ofphenol:chloroform and chloroform:iso-amylalcohol were includedto remove RNase contamination.

To compare the efficiency of the current protocol, total DNAwas isolated from CoGMV infected C. capsularis samples obtainedfrom eastern India (District-North 24 Parganas, West Bengal state)and northern India (District-Bahraich, Uttar Pradesh state) usingfollowing methods, viz.: (i) cetyl trimethylammonium bromide(CTAB) method (Doyle and Doyle, 1987); (ii) modified Dellaportamethod with silica (Echevarria-Machado et al., 2005); (iii) Gem-CTAB method (Rouhibakhsh et al., 2008); (iv) extraction with citratebuffer and alkali lysis, PEG precipitation (Jose and Usha, 2000); (v)extraction using DNeasy plant mini kit (QIAGEN, GmbH, Hilden,Germany) following the recommendations of the manufacturer.The present method (CTAB-Mucilage-free) was evaluated and com-pared with other methods in respect of: (i) the yield of the DNA,and (ii) the quality of the DNA as judged by their reproducibilityby PCR. For the reproducibility study, DNA was isolated by all thesefive methods along with the present method from 25 different jutesamples infected with CoGMV.

2.3. PCR amplification

The DNA isolated by each method was used as template in PCRfor amplification of the DNA-A and DNA-B genomic componentsof CoGMV. For PCR, two abutting primer sets: (i) JM FL-A F: 5′-CGTACGAATGCTGGGACCTCC-3′/JM FL-A R: 5′-CCATGGGGCCTCAC-GTTTCATC-3′ and (ii) JM FL-B F: 5′-GTTCTGCAGTTGATGGGA-ATTGCAG-3′/JM FL-B R: 5′-CAACTGCAGAACAATGGGGTGCAAC-3′,specific for the DNA-A and DNA-B components, respectively, were

designed from the accession No. DQ641688 and DQ641689.

For application of the present method on a wider scale, DNAwas isolated from different mucilaginous crops as mentionedabove and different primers were used for amplification of eitherthe full-length or partial DNA-A of different begomoviruses and

36 R. Ghosh et al. / Journal of Virological Methods 159 (2009) 34–39

Table 1Evaluation of different DNA extraction protocols for amplification of DNA-A and DNA-B components of CoGMV from Corchorus capsularis plants.

Method DNA yield (�g/100 mg) No. of PCR positive samples/no. of samples tested

DNA-A specific primers DNA-B specific primers

CTAB method 35–40 9/25 9/25Modified Dellaporta method with silica 30–40 12/25 11/25GEEC

blyprbmpbe5u(prttAtwic

FD61

em-CTAB 25–30xtraction with citrate buffer and alkali lysis 45–50xtraction using DNeasy plant mini kit 10–15TAB-Mucilage-free (presented method) 75–80

etasatellites. For amplification of the full-length DNA-A homo-ogue of different monopartite begomoviruses associated withellow vein mosaic and leaf curl disease affected mesta plants,rimers used by Das et al. (2008a), and Paul et al. (2008),espectively, were used. For amplifying partial DNA-A of differentegomoviruses infecting mucilaginous crops other than jute andesta, coat protein gene specific primers (Jose and Usha, 2000) and

artial DNA-A specific primers (Rojas et al., 1993) were used. Foretasatellite amplification universal betasatellite primers (Briddont al., 2002) were used. All the amplifications were performed in0 �l reaction mixture containing 50–100 ng of template DNA, 1-nit of Taq polymerase (Fermentas, Inc., MD, USA), 25 mM MgCl23 �l), 10 mM dNTP (1 �l), 100 ng of each of the forward and reverserimers (1 �l), 10× reaction buffer (5 �l). The amplification was car-ied out using a thermal cycler (Bio-Rad, CA, USA). Before addinghe Taq polymerase, the other reagents were mixed and the mix-ure was subjected to 94 ◦C for 5 min for an initial denaturation.

fter such hot-start, Taq polymerase was added to the reaction mix-

ure and amplification was carried out for 30 cycles. The PCR cyclesere programmed as 30 s each for denaturation at 94 ◦C, anneal-

ng at 52–55 ◦C, and chain-extension at 72 ◦C. A final extensionycle at 72 ◦C for 7 min was carried out to ensure the completion of

ig. 1. Comparison of different DNA isolation methods based on reproducibility of PCR aNA-A (a) and DNA-B (b) genomic components of CoGMV. DNA used as template were ext–10); Gem-CTAB mehod (lanes 11–15); citrate buffer and alkali lysis method (lanes 16–2kb DNA ladder.

16/25 14/258/25 6/254/25 2/2524/25 23/25

amplification of all the target templates. Amplified products werevisualized by agarose gel electrophoresis in the presence of ethid-ium bromide. Such amplifications were carried out with 25 samplesof each diseased plant.

2.4. Cloning and sequencing of virus DNA

To determine the authenticity of the PCR amplification, ampli-cons with respect to DNA-A and DNA-B of CoGMV (one isolateeach from Uttar Pradesh and West Bengal state) were purified fromagarose gel using the gel extraction kit (QIAGEN, GmbH, Hilden,Germany) and cloned into pJET1.2 positive selection vector usingGeneJETTM PCR Cloning Kit (Fermentas, Inc., MD, USA) follow-ing the manufacturer’s protocol. Competent Escherichia coli cells(strain DH5-�) were transformed by following standard molecu-lar biology procedures (Sambrook and Russell, 2001). Applying thesimilar methodology, size-specific amplicons with respect to DNA-

A homologue, coat protein genes, partial DNA-A fragments andbetasatellites obtained from begomovirus infected samples of H.cannabinus, C. olitorius, bhendi, Sida, Urena and Malachra were alsocloned. The authenticity of the cloned inserts were confirmed uponrestriction release of those fragments from the cloning vector using

mplified products from a representative batch of five jute samples with respect toracted by: CTAB method (lanes 1–5); modified Dellaporta method with silica (lanes0); DNeasy plant mini kit (lanes 21–25), present method (lanes 26–30). M denotes

R. Ghosh et al. / Journal of Virological Methods 159 (2009) 34–39 37

Table 2PCR-based detection of begomoviral DNA components and betasatellites in different mucilage and polysaccharide rich diseased host plants.

Disease sample Collection region Target begomovirus/betasatellite Target genomic component/region with expected size

No. of PCR positive samples/no. of sample tested

Yellow vein mosaic diseaseaffected mesta (Hibiscuscannabinus)

West Bengal Mesta yellow vein mosaic virus DNA-A homologue, 2.7 kb 23/25

Cotton leaf curl Multanbetasatellite

Betasatellite, 1.3 kb 21/25

Uttar Pradesh Mesta yellow vein mosaicBahraich virus

DNA-A homologue, 2.7 kb 22/25

Ludwigia leaf distortionbetasatellite

Betasatellite, 1.3 kb 24/25

Leaf curl disease affected mesta(Hibiscus cannabinus)

Andhra Pradesh Tomato leaf curl Joydebpur virus DNA-A homologue, 2.7 kb 23/25

Tomato leaf curl Joydebpurbetasatellite

Betasatellite, 1.3 kb 23/25

Uttar Pradesh Kenaf leaf curl virus DNA-A homologue, 2.7 kb 24/25Cotton leaf curl Multanbetasatellite

Betasatellite, 1.3 kb 23/25

Yellow vein mosaic diseaseaffected Corchoruspseudo-capsularis

West Bengal Yet to be established Partial DNA-A, 1.2 kb 24/25

Betasatellite, 1.3 kb 21/25

Yellow mosaic disease affectedCorchorus Olitorius

Uttar predesh Yet to be established Partial DNA-A, 1.2 kb 22/25

Yellow vein mosaic diseaseaffected Congo-jute (Urenalobata)

West Bengal Yet to be established Coat Protein gene, 0.7 kb 25/25

Betasatellite, 1.3 kb 25/25

Yellow vein mosaic diseaseaffected bhendi (Abelmoschusesculentus)

West Bengal Bhendi yellow vein mosaic virus Coat Protein gene, 0.7 kb 24/25

Bhendi yellow vein mosaicbetasatellite

Betasatellite, 1.3 kb 25/25

Yellow vein mosaic diseaseaffected Sida sp.

West Bengal Yet to be established for thisIndian isolate

Coat Protein gene, 0.7 kb 21/25

Betasatellite, 1.3 kb 24/25

Y

XdG

3

3s

jtmmttlTioicm9

ellow vein mosaic diseaseaffected Malachra sp.

West Bengal Yet to be established

hoI and XbaI digestion and have been sequenced entirely in bothirections using automated DNA sequencing service of Bangaloreenei Pvt. Ltd., India.

. Results

.1. Evaluation of DNA extraction from CoGMV infected juteamples

The amount of DNA obtained from 100 mg of CoGMV infectedute leaves by various extraction methods was determined spec-rophotometrically and the average of 25 samples for each of these

ethods is summarized in Table 1. It was observed that the presentethod yielded comparably more DNA (75–80 �g/100 mg) than

he other methods using the same amount of leaf tissue. Amonghe other methods, DNA obtained by a commercial kit yielded theeast amount of DNA due to clogging of the column with mucilage.o compare the efficiency of the current protocol, total DNA wassolated from CoGMV infected C. capsularis samples (25 samples)

btained from eastern and northern India using six different DNAsolation protocols and PCR amplification was carried out with spe-ific primers as mentioned above. It was found that the presentethod gave highest reproducibility (96% with DNA-A primers and

2% with DNA-B primers) than others (Table 1). PCR amplification

Coat Protein gene, 0.7 kb 23/25

Betasatellite, 1.3 kb 21/25

from a batch of five samples with all the methods is presented inFig. 1.

3.2. Validation of the present method by PCR-based detection ofbegomoviruses and betasatellites from different mucilaginoushosts

DNA was isolated from different mucilaginous plant samplesinfected with begomoviruses by the present method. Details ofthese hosts with the corresponding diseases associated bego-moviruses and betasatellites wherever known are shown in Table 2.Among these plants, full-genome-length begomovirus size-specificamplicons and betasatellite amplicons were obtained from themesta plants showing yellow vein mosaic symptoms (obtainedfrom West Bengal and Uttar Pradesh states) and leaf curl symp-toms (obtained from Uttar Pradesh and Andhra Pradesh states). Outof the 25 DNA samples with respect to each of these diseased mestasamples, 84–96% samples gave the expected amplicons with theircorresponding primer sets having an average of 90.6% (Table 2). Themethod was also found to be suitable for amplification of partial

DNA or coat protein gene and betasatellites from other plant sam-ples and in each case more than 80% reproducibility was observed(Table 2).

To check the authenticity of these PCR amplicons, some of theamplicons were cloned, sequenced and the accession numbers are

38 R. Ghosh et al. / Journal of Virologica

Table 3Accession numbers of genomic components of different begomoviruses andbetasatellites characterized in the study.

Disease sample Cloned fragment Accession No.

Yellow mosaic disease affectedCorchorus capsularis

DNA-A EU636712

FJ463902

DNA-B FJ455448FJ463901

Leaf curl disease affected Hibiscuscannabinus

DNA-A EU366903

EU822321EU822322

Betasatellite EF620566EU825205EU825206

Yellow mosaic disease affectedCorchorus Olitorius

Partial DNA-A EU047706

Yellow vein mosaic disease affectedCongo-jute (Urena lobata)

Coat Protein gene EU184017

Betasatellite EU188922

Yellow vein mosaic disease affectedbhendi (Abelmoschus esculentus)

Coat Protein gene EF417918

Betasatellite EF417919

Yellow vein mosaic disease affectedMalachra sp.

Coat Protein gene EU285590

Betasatellite EU285589

Y

sno

4

icTawitmlr(irltc2h1

bDpas

ellow vein mosaic disease affectedSida sp.

Coat Protein gene EU188921

Betasatellite EU184016

hown in Table 3. Sequence analysis indicated the viral specificature of those PCR amplicons and thus confirmed the accuracyf the new protocol.

. Discussion

A simple, modified cetyl trimethylammonium bromide methods described for isolation of DNA suitable for downstream amplifi-ation of begomoviruses from jute and other mucilaginous crops.he method was found to be easy, inexpensive and involved smallermount of leaf tissue. The major modifications made in this methodere an increase in the volume of the extraction buffer and dissolv-

ng the crude nucleic acid pellet in 1 M NaCl followed by RNasereatment and these modifications helped to remove the gluey

ucilage which usually co-precipitates along with the DNA pel-et. Increase in the volume of extraction buffer upto 10:1 for theemoval of phenolics from leguminous crops was described earlierRouhibakhsh et al., 2008). The use of high-salt buffer for dissolv-ng crude nucleic acid pellet followed by ethanol precipitation waseported by Fang et al. (1992). Dissolving the crude nucleic acid pel-et in 1 M NaCl reduced the viscosity of the mucilage and increaseshe specificity of RNase. Such a role of NaCl in the reduction of vis-osity of mucilage has been described previously (Chen and Chen,004). It was also reported that at NaCl concentration of 0.3 M origher, RNase A cleaves specifically single-stranded RNA (Struhl,989).

Characterization of begomoviruses by PCR is adopted widely

ecause they replicate within the host via a circular double strandedNA replicative-intermediate which can be used readily as a tem-late for amplification. Various modified CTAB-based methods formplification of begomoviral DNA have been recommended byeveral investigators (Briddon and Markham, 1994; Deng et al.,

l Methods 159 (2009) 34–39

1994; Mansoor et al., 1999; Rothenstein et al., 2005) but thesemethods are not suitable for isolation of DNA from the host con-taining high amount of foliar mucilage. Contamination of mucilagewith DNA inhibited PCR and gave inconsistent amplification andthus hinders the detection of viruses. There have been numerousreports of extraction of begomoviral DNA from a wide range of plantspecies rich in mucilage and polysaccharides including bhendi (Joseand Usha, 2000), Bixa (Echevarria-Machado et al., 2005), Sedum(Barnwell et al., 1998), cotton (Mansoor et al., 1999), forest plants(Scott and Playford, 1996) and legumes (Rouhibakhsh et al., 2008).Besides their specificity towards a particular plant species, most ofthese protocols are either time consuming or involve some addi-tional reagents such as polyethylene glycol and silica which makethese methods expensive and cumbersome. Some of these methodshave been employed to isolate the DNA from jute samples infectedwith CoGMV and compared with the present method. The resultsindicated that the present method of DNA extraction is advanta-geous over the other methods with respect to the yield of the DNAand reproducibility by PCR. The method described not only showedreproducibility for isolation of begomovirus DNA from jute sam-ples but also from a wide range of mucilaginous plants with a lowtitre of begomoviruses and is thus better than other methods. Thenew method could also detect the presence of unidentified bego-moviruses from the host such as C. pseudo-capsularis, Urena sp. andMalachra sp. and thus opens the possibility of characterization ofunidentified begomoviruses. The new method, thus, could be usedon a wider scale as a simple and cheaper method of DNA isola-tion suitable for characterization of begomoviruses infecting manyother mucilaginous hosts.

Acknowledgements

The authors are thankful to the Director, Central Research Insti-tute for Jute and Allied Fibres for providing the infrastructuralsupport during the present investigation. The authors are also grate-ful to the Indian Council of Agricultural Research, New Delhi forproviding financial support in the form of project grants.

References

Axel Diederichsen, J., Philip, R., Duguid, S.D., 2006. Variation of mucilage in flaxseed and its relationship with other seed characters. Crop. Sci. 46, 365–371.

Barnwell, P., Blanchard, A.N., Bryant, J., Smirnoff, N., Weir, A.F., 1998. Isolation ofDNA from the highly mucilagenous succulent plant shape sedum telephium.Plant Mol. Biol. Rep. 16, 133.

Bayer, C., Fay, M.F., De Bruijn, A.Y., 1999. Support for an expanded family conceptof Malvaceae within a recircumscribed order Malvales: a combined analysis ofplastid atpB and rbcL DNA sequence. Bot. J. Linnean Soc. 129, 267–303.

Bhat, U.R., Tharanathan, R.N., 1987. Functional properties of okra (Hibiscus esculentus)mucilage. Starch 39, 165–167.

Briddon, R.W., Bull, S.E., Mansoor, S., Amin, I., Markham, P.G., 2002. Universal primersfor the PCR-mediated amplification of DNA beta: a molecule associated withsome monopartite begomoviruses. Mol. Biotechnol. 20, 315–318.

Briddon, R.W., Markham, P.G., 1994. Universal primers for the PCR amplification ofdicot-infecting geminiviruses. Mol. Biotechnol. 1, 202–205.

Chatterjee, A., Ghosh, S.K., 2007a. A new monopartite begomovirus isolated fromHibiscus cannabinus L. in India. Arch. Virol. 152, 2113–2118.

Chatterjee, A., Ghosh, S.K., 2007b. Association of a satellite DNA � molecule withmesta yellow vein mosaic disease. Virus Genes 35, 835–844.

Chen, R.H., Chen, W.Y., 2004. Rheological properties of the water-soluble mucilageof a green laver, Monostroma nitidium. J. Appl. Phycol. 13, 481–488.

Das, S., Ghosh, R., Paul, S., Roy, A., Ghosh, S.K., 2008a. Complete nucleotide sequenceof a monopartite begomovirus associated with yellow vein mosaic disease ofmesta from north India. Arch. Virol. 153, 1791–1796.

Das, S., Roy, A., Ghosh, R., Paul, S., Acharyya, S., Ghosh, S.K., 2008b. Sequence variabil-ity and phylogenetic relationship of betasatellite isolates associated with yellowvein mosaic disease of mesta in India. Virus Genes 37, 414–424.

Deng, D., McGrath, P.F., Robinson, D.J., Harrison, B.D., 1994. Detection and differ-entiation of whitefly transmitted geminiviruses in plants and vector insectsby polymerase chain reaction with degenerate primer. Ann. Appl. Biol. 125,327–336.

Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities offresh leaf tissue. Phytochem. Bull. 19, 11–15.

logica

E

F

F

G

H

H

H

I

J

J

K

S

M

M

R. Ghosh et al. / Journal of Viro

chevarria-Machado, I., Lucila, A., Sanchez, C., Hernandez-Zepeda, C., Rivera-Madrid,R., Moreno-velenzuela, O.A., 2005. A simple and efficient method for isolationof dna in high mucilaginous plant tissues. Mol. Biotechnol. 31, 129–135.

ang, G., Hammer, S., Grumet, R., 1992. A quick and inexpensive method for removingpolysaccharides from plant genomic DNA. Bio Techniques 13, 52–56.

aria, J.C., Maxwell, D.P., 1999. Variability in geminivirus isolates associated withPhaseolus spp. in Brazil. Phytopathology 89, 262–268.

hosh, R., Paul, S., Das, S., Palit, P., Acharyya, S., Das, A., Mir, J.I., Ghosh, S.K., Roy, A.,2008. Molecular evidence for existence of a New World begomovirus associatedwith yellow mosaic disease of Corchorus capsularis in India. Aust. Plant Dis. Notes3, 59–62.

a, C., Coombs, S., Revill, P., Harding, R., Vu, M., Dale, J., 2008. Molecular characteri-zation of begomoviruses and DNA satellites from Vietnam: additional evidencethat the New World geminiviruses were present in the Old World prior to con-tinental separation. J. Gen. Virol. 89, 312–326.

amilton, W.D.O., Bisaro, D.M., Buck, K.W., 1982. Identification of novel DNA formsin tomato golden mosaic virus infected tissue. Evidence for a two componentviral genome. Nucl. Acids Res. 10, 4901–4912.

ofer, P., Engel, M., Jeske, H., Frischmuth, T., 1997. Nucleotide sequence of a newbipartite geminivirus isolated from the common weed Sida rhombifolia in CostaRica. J. Gen. Virol. 78, 1785–1790.

kegami, M., Haber, S., Goodman, R.M., 1981. Isolation and characterization of virusspecific double-stranded DNA from tissues infected by bean golden mosaic virus.Proc. Natl. Acad. Sci. U.S.A. 78, 4102–4106.

ose, J., Usha, R., 2000. Extraction of geminiviral DNA from a highly mucilaginousplant (Abelmoschus esculentus). Plant Mol. Biol. Rep. 18, 349–355.

ose, J., Usha, R., 2003. Bhendi yellow vein mosaic disease in India is caused byassociation of a DNA beta satellite with a begomovirus. Virology 305, 310–317.

awamura, T., Hisata, Y., Okuda, K., Ren, J., Noro, Y., Yamaguchi, S., Tanaka, T., 2000.Diversity of alkaloid and mucilage in phellodendron barks from different habi-tats. Nat. Med. 54, 193–195.

cott, K.D., Playford, J., 1996. DNA extraction technique for PCR in rain forest plantspecies. Bio Techniques 20, 974–978.

ansoor, S., Bashir, A., Khan, S.H., Hussain, M., Saeed, M., Zafar, Y., Markham, P.G.,

Malik, K.A., 1999. Rapid multiplex PCR for the specific detection of two whitefly-transmitted geminivirus species associated with cotton leaf curl disease inPakistan. Pak. J. Bot. 31, 115–123.

uralikrishna, G., Bhat, U.R., Tharanathan, R.N., 1989. Functional characteristics ofthe mucilaginous polysaccharides derived from cowpea (Vigna sinensis), blackgram (Phaseolus mungo) and linseed (Linum usitatissimum). Starch 39, 107–109.

l Methods 159 (2009) 34–39 39

Paul, S., Ghosh, R., Das, S., Palit, P., Acharyya, S., Das, A., Mir, J.I., Chaudhuri, S., Ghosh,S.K., Roy, A., 2008. First report of Tomato leaf curl Joydebpur virus and associatedbetasatellite in kenaf (Hibiscus cannabinus) plants showing leaf curl symptomsfrom southern India. Plant Path. doi:10.1111/j.1365-3059.2008.01929.x.

Polston, J.E., Anderson, P.K., 1997. The emergence of whiefly-transmitted gem-iniviruses in tomato in the western hemisphere. Plant Dis. 81, 1358–1369.

Rojas, M.R., Gilbertson, R.L., Russell, D.R., Maxwell, D.P., 1993. Use of degenerateprimers in the polymerase chain reaction to detect whitefly-transmitted gemi-niviruses. Plant Dis. 77, 340–347.

Rothenstein, D., Haible, S., Dasgupta, I., Dutt, N., Patil, B.L., Jeske, H., 2005. Biodiver-sity and recombination of cassava-infecting begomoviruses from southern India.Arch. Virol. 151, 55–69.

Rouhibakhsh, A., Priya, J., Periasamy, M., Haq, Q.M.I., Malathi, V.G., 2008. Animproved DNA isolation method and PCR protocol for efficient detectionof multicomponents of begomovirus in legumes. J. Virol. Methods 147,37–42.

Roy, A., Acharyya, S., Das, S., Ghosh, R., Paul, S., Srivastava, R.K., Ghosh, S.K., 2009.Distribution, epidemiology and molecular variability of the begomovirus com-plexes associated with yellow vein mosaic disease of mesta in India. Virus Res.141, 237–246.

Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual. Coldspring Harbour Laboratory Press, New York.

Saunders, K., Bedford, I.D., Briddon, R.W., Markham, P.G., Wong, S.M., Stanley, J., 2000.A unique virus complex causes Ageratum yellow vein disease. Proc. Natl. Acad.Sci. U.S.A. 97, 6890–6895.

Sharma, P., Rishi, N., Malathi, V.G., 2005. Molecular cloning of coat protein gene of anIndian Cotton leaf curl virus (CLCuV-hs2) isolate and its phylogenetic relationshipwith others members of Geminiviridae. Virus Genes 30, 85–91.

Struhl, K., 1989. Ribonucleases. In: Ausubel, M.A., Brent, R., Kingston, R., Moore, D.D.,Seidman, J.G., Smith, J.A., Struhl, K. (Eds.), Current Protocol in Molecular Biology,vol. 3.13.3, John Wiley and Sons, Inc., USA.

Swanson, M.M., Varma, A., Muniyappa, V., Harrison, B.D., 1992. Comparative epitopeprofiles of the particle proteins of whitefly-transmitted geminiviruses from nine

crop legumes in India. Ann. Appl. Biol. 120, 425–433.

Varma, A., Malathi, V.G., 2003. Emerging geminivirus problems: a serious threat tocrop production. Ann. Appl. Biol. 142, 145–164.

Weishing, K., Nybom, H., Wolff, K., Meyer, W., 1995. DNA isolation and purifica-tion. In: DNA Fingerprinting in Plants and Fungi. CRC Press, Boca Raton, FL,pp. 44–59.