Detection of Geminiviruses in Sweetpotato by Polymerase Chain Reaction

Ruhui Li, Sarbagh Salih, and Suzanne Hurtt, United States Department of Agriculture–Agricultural Research Service, Fruit Laboratory/Plant Germplasm Quarantine Office, Beltsville, MD 20705

and the sensitivity of the PCR was com- ABSTRACT pared with that of the grafting assay. Li, R., Salih, S., and Hurtt, S. 2004. Detection of geminiviruses in sweetpotato by polymerase chain reaction. Plant Dis. 88:1347-1351. MATERIALS AND METHODS isolates. Twenty sweetpotato ac- Geminivirus infection of sweetpotato (Ipomoea spp.) germplasm acquired from foreign regions cessions naturally infected with is common. Graft inoculation of the indicator host, Ipomoea setosa, is the accepted detection from Brazil, China, Guatemala, Guyana, method for these viruses, but the assay is laborious and requires up to 8 weeks. When infected Jamaica, Korea, Mexico, Puerto Rico, sweetpotato is subjected to meristem tip culture to eliminate these viruses, the eradication rate is Taiwan, and Vietnam were maintained in low. In this study, a polymerase chain reaction (PCR) detection assay was developed for the detection of geminiviruses in a variety of sweetpotato cultivars. Different methods were evalu- the Plant Germplasm Quarantine Office ated to extract nucleic acids suitable for PCR from Ipomoea spp., and a reliable and simple ex- (PGQO) quarantine facilities. Most acces- traction method was developed for large-scale sample preparation. PCR products of the expected sions were cultivars, and several acces- sizes were amplified from infected plants using degenerate and virus-specific primers, but not sions from Taiwan were hybrids from from noninoculated indicator plants. PCR assays using three primer pairs detected nine unchar- crosses of I. batatas and I. trifida. These acterized isolates of the geminiviruses in sweetpotato from Asia and America. However, the best accessions were infected by either gemi- PCR result was obtained with degenerate primers SPG1/SPG2, which detected a Taiwan isolate nivirus or geminivirus and other virus of of Sweet potato leaf curl virus (SPLCV-Taiwan) in a sample diluted to 10–9. Viral identities of sweetpotato based on development of three amplicons from SPLCV-Taiwan were confirmed by sequencing. The degenerate primers symptoms when grafted onto I. setosa and had a broader detection range than virus-specific primers; therefore, they were used to detect on immunoblotting assays using antisera geminiviruses in in vitro plantlets and greenhouse-grown sweetpotato plants, and in several against several sweetpotato viruses. These Ipomoea hosts. PCR was shown to be as reliable for virus detection as grafting. isolates were maintained in greenhouse- grown plants and in in vitro plantlets Additional keywords: , DNA extraction, Ipomoea leaf curl virus, Ipomoea yellow propagated from the infected sweetpotato vein virus, malate dehydrogenase accessions. Noninoculated indicator plants and sweetpotato accessions with negative

results from grafting and immunoblotting Geminiviruses (family ) quarantine programs because infected assays were used as negative controls. are plant viruses that have a circular, sin- plants are essentially symptomless (4,14), DNA extracts of Bean golden mosaic vi- gle-stranded DNA genome encapsidated and recombination or reassortment among rus (BGMV), Cabbage leaf curl virus- within twinned isometric particles (8). species and strains could lead to occur- Florida (CaLCuV), yellow leaf They are grouped into four genera based rence of more virulent strains or species curl virus (TYLCV), and Tomato mottle on insect vector, host range, and genome (19). To detect these viruses, vine seg- virus (ToMoV) were kindly provided by E. organization (8). Members of the genus ments from sweetpotato are graft in- Hiebert (University of Florida). DNA ex- are transmitted by , oculated onto an indicator host, I. setosa, tracts of Beet curly top virus (BCTV), have single or bipartite component ge- which develops symptoms if the source Cotton leaf crumple virus (CLCrV), and nomes, and infect dicotyledonous plants. material was infected (5,12,14,15). To Squash leaf curl virus (SLCV) were kindly Three geminiviruses, Sweet potato leaf ensure accuracy, the grafting assay has to provided by H.-Y. Liu (United States De- curl virus (SPLCV, AF104036), Ipomoea be done twice. Infected materials undergo partment of Agriculture–Agricultural Re- crinkle leaf curl virus (ICLCV), and Ipo- meristem tip culture with or without search Service, Salinas, CA) and ILCV moea leaf curl virus (ILCV, AF326775) therapeutic treatments for virus elimina- was kindly provided by R. A. Valverde have been reported to infect sweetpotato tion, but the rate of eradication is low. To (Louisiana State University). (Ipomoea batatas) (3,5,14), whereas Ipo- identify a virus-free clone, many in vitro In vitro plantlets. Shoots 5 cm in moea yellow vein virus (IYVV, plantlets have to be grown in the green- length were taken from infected accessions NC_003879), has been isolated from I. house, and then tested two times by the and micropropagated in vitro on Mura- indica (1). Occurrence of geminiviruses in grafting assay. shige and Skoog (MS) basal salts (17) sweetpotato is widespread (1,3–5,9,10,12– Nucleic acid-based techniques, includ- supplemented with MS vitamins, thiamine 16), and they commonly have been found ing polymerase chain reaction (PCR), offer at 0.3 mg/liter, sucrose at 30 mg/liter, and in imported germplasm by virus indexing the potential of great savings in time, gelrite (Sigma-Aldrich, St. Louis) at 1.5 in quarantine (unpublished data). Gemi- greenhouse space, efficiency, and cost. The g/liter. Plant growth regulators were not niviruses are of particular significance to genomic sequences of three geminiviruses needed for micropropagation or rooting. that infect Ipomoea spp., and those for Most accessions grew vigorously and in- many other geminiviruses, are available duced roots easily in the same medium. Corresponding author: R. Li and can be utilized for designing primers Plantlets were maintained at ambient tem- E-mail: [email protected] for detection of geminiviruses in sweetpo- perature with 16 h of cool white fluores- 2 –1 Accepted for publication 2 July 2004. tato by PCR. In this article, the use of PCR cent light (40 µmol m s ) per day. Meris- to detect SPLCV and several other gemi- tem tip cultures were prepared from niviruses of sweetpotato is reported. The infected shoots directly or after different Publication no. D-2004-0913-02R PCR assay developed was used to test in therapeutic treatments. Sixty-two in vitro This article is in the public domain and not copy- vitro plantlets generated from infected plantlets generated from seven infected rightable. It may be freely reprinted with custom- ary crediting of the source. The American Phyto- sweetpotato, greenhouse-grown sweetpo- sweetpotato accessions then were tested by pathological Society, 2004. tato plants, and grafted indicator plants, PCR at lease twice for presence of gemi-

Plant Disease / December 2004 1347 nivirus, and virus-free clones were se- PW285-1/PW285-2 (14) were used. Incon- s; and 72°C for 10 min. PCR products lected. sistent results were obtained when the were assessed by electrophoresis in 1 or DNA extraction. Shoots or leaves (100 same plantlets were assayed by PCR two 1.2% agarose gels in Tris-acetate (TAE) mg) were collected from in vitro plantlets, times in 2 months. To increase PCR sensi- buffer (40 mM Tris-acetate, 1 mM EDTA, greenhouse-grown sweetpotato plants, and tivity and detection range, two additional pH 8.0), stained with ethidium bromide, grafted I. setosa plants. In the initial tests, pairs of primers, degenerate primers and viewed under ultraviolet light. nucleic acids were extracted from plant SPG1/SPG2 and specific primers Cloning and sequencing. The PCR tissues using plant DNAzol buffer (Invi- SPG3/SPG4, were designed and tested in products (1,148, 912, and 514 bp) ampli- trogen, Carlsbad, CA) according to the PCR assays (Table 1). To design degener- fied from extracted DNA of SPLCV-Tai- method described by Lotrakul et al. (12). ate primers, the genomic sequences of the wan were purified using the QIAquick Fresh shoot and leaf tissue was ground to following 11 Begomovirus were obtained PCR Purification Kit (QIAGEN Inc., Va- fine powder in liquid nitrogen with a mor- from GenBank (National Center for Bio- lencia, CA) according to the manufac- tar and pestle and either used for nucleic technology Information): SPLCV-US turer’s instructions. Eluted DNA was acid extraction or stored at –30°C for fu- (AF104036), ILCV (AF326775), IYVV cloned into pGEM-T Easy vector ture use. Other frozen, powdered leaf tis- (NC_003879), Chili leaf curl virus (Promega Corp., Madison, WI) according sues, stored at –30°C for about 1 year, also (AF314531), leaf curl virus to the manufacturer’s instructions. The were used. To simplify the extraction (NC_004147), Cotton leaf curl virus nucleotide sequence was determined for method and avoid contamination between (CLCVAJ455), SLCV (SLE420319), Ag- both directions of the clones using an samples, a semi-automatic homogenizer, eratum yellow vein virus (AF327902), automated DNA sequencer (Auburn Ge- FastPrep Instrument (Savant Instruments Tobacco leaf curl virus (TLE319674), nomics and Sequencing Lab, Auburn Uni- Inc., Holbrook, NY), which holds 2-ml Soybean crinkle leaf virus (AB050781), versity, AL). Sequences were compared microtubes containing plant tissues, ex- and Tomato leaf curl virus (AF327436). with those available for SPLCV, ILCV, or traction buffer, and two steel beads, was The sequences of each genome were IYVV using Sequence Analysis (South- used for nucleic acid extraction. aligned directly using CLUSTALW ampton Bioinformatics Data Server, Uni- Two nucleic acid extraction protocols, (EMBL-EBI, UK). Based on alignments, versity of Southampton, UK). one using plant DNAzol buffer and another degenerate primers SPG1/SPG2 were de- Comparison of PCR and grafting as- using cetyltrimethylammonium bromide signed to bind to conserved regions in says. To determine the reliability of the (CTAB) buffer (2% CTAB, 2% poly- open reading frame (ORF) AC2 and ORF PCR, 16 plantlets derived from two in- vinylpyrrolidone [PVP], 100 mM Tris- AC1 of these viruses. Specific primers fected accessions were assayed by grafting HCl, pH 8.0, 1.4 M NaCl, 20 mM EDTA, SPG3/SPG4 were designed to anneal to the onto I. setosa at the PGQO greenhouse. and 0.2% 2-mecaptoethanol) (7), were nucleotide sequences in the coat protein These plantlets were tested three times by tested in the FastPrep Instrument. The gene (AV1) and ORF AC2 of SPLCV and PCR within 2 months before grafting as- method using CTAB then was selected for ILCV. Primers MDH-H968 and MDH- say, and both positive and negative plant- large-scale processing of samples. Plant C1163, designed for detection of the lets from each accession were included. tissues (100 mg) cooled in a –30°C freezer malate dehydrogenase (MDH) gene of host Plantlets were planted in soil pots and were processed in 1 ml of CTAB at a speed plants (18), were used to check the quality maintained in the greenhouse after a 14- set of 4.5 for 60 s in the FastPrep Instru- of the nucleic acids extracted in PCR as- day acclimation period in a growth cham- ment. The homogenate was incubated at says. ber. Approximately 6 weeks later, two to 65°C for 15 min and centrifuged at 10,000 PCR. The volume of the DNA extract, three vine segments (two to four nodes) × g for 10 min. The supernatant (650 µl) primer concentration, and PCR tempera- were taken from each plant and wedge was transferred to a 1.5-ml microcentrifuge ture conditions were optimized on a Hy- grafted onto 4- to 7-week-old indicator tube and mixed with an equal volume of baid PCR Express thermocycler (Thermo plants, which were observed periodically chloroform:isoamyl alcohol (24:1). The Hybaid US, Franklin, MA). The optimized for symptom development for 8 weeks. mixture was centrifuged at 12,000 × g for amplification was performed in 30-µl reac- The grafted plants typically showed symp- 10 min and the supernatant (500 µl) was tion volumes containing 1 µl of the DNA toms 3 to 4 weeks after inoculation if the transferred to a 1.5-ml microcentrifuge tube extract, 0.6 µl of each primer (10 µM), 0.6 source material was infected. before adding 350 µl of isopropanol. The µl of 10 mM dNTP mix, 3 µl of 10× Taq mixture was incubated on ice for 10 min DNA polymerase reaction buffer, 2 µl of RESULTS and centrifuged at 12,000 × g for 10 min. 50 mM MgCl2, 0.2 µl (1 U) of Taq DNA Sample preparation. In initial experi- The pellet was washed with 70% ethanol polymerase (Invitrogen, Carlsbad, CA), ments, PCR for DNA samples extracted by and centrifuged at 12,000 × g for 5 min. The and 22 µl of water. Touchdown PCR condi- grinding plant tissues in liquid nitrogen pellet was air dried and dissolved in 100 µl tions used were as follows: 11 cycles of with mortars and pestles in plant DNAzol of 20 mM Tris-HCl, pH 8.0. 94°C for 40 s, (72-n)°C for 40 s (n equals buffer always resulted in the expected Oligonucleotide primers. In initial cycle number), 72°C for 90 s; 24 cycles of fragments (Figs. 1A, 2, and 3). PCR assays PCR tests, the SPLCV-specific primers 94°C for 40 s, 60°C for 40 s, 72°C for 90 failed to detect the viruses in the nucleic

Table 1. Primer pairs used for the detection of geminiviruses in sweetpotato and respective amplicon produced by polymerase chain reaction (PCR) Name Orientation Sequence Locationa Product size (bp) PW285-1b Sense 5′-TAATTCGAACTGCAGTTCCGTATTTCAGTT-3′ 1795–1828 514 PW285-2b Antisense 5′-GCTAGAGGAGGCCTGCAGACTGCTAACGACG-3′ 2308–2278 … SPG1 Sense 5′-CCCCKGTGCGWRAATCCAT-3′ 1490–1508 912 SPG2 Antisense 5′-ATCCVAAYWTYCAGGGAGCTAA-3′ 2412–2391 … SPG3 Sense 5′-ACTTCGAGACAGCTATCGTGCC-3′ 363–385 1,148 SPG4 Antisense 5′-AGCATGGATTCACGCACAGG-3′ 1511–1492 … MDH-H968c Sense 5′-GCATCTGTGGTTCTTGCAGG-3′ … 196 MDH-C1163c Antisense 5′-CCTTTGAGTCCACAAGCCAA-3′ … … a Numbers indicate the locations of different primers on the sequence of the U.S. isolate of Sweet potato leaf curl virus (12). b Primers used by Lotrakul et al. (1998). c Internal primers that anneal to the sequences of the malate dehydrogenase gene of the host plants (18).

1348 Plant Disease / Vol. 88 No. 12 acids extracted using the FastPrep Instru- ment if microcentrifuge tubes containing plant tissues in DNAzol buffer were not cooled in a –30°C freezer before process- ing (data not shown). Virus was detected in only 4 of 14 positive samples in a set of 42 plantlets when plant tissues in DNAzol buffer were cooled before processing in the FastPrep Instrument. However, when CTAB was used instead of DNAzol buffer, DNA extracts obtained from cooled plant tissues using the FastPrep Instrument gave the same results in the PCR assay as those Fig. 1. Comparison of two DNA extraction methods by the polymerase chain reaction for detection of obtained by grinding tissue in liquid nitro- the geminiviruses in indicator host, Ipomoea setosa, and greenhouse-grown sweetpotato plants. The gen (Fig. 1; data not shown), indicating DNA extracts were obtained by either A, grinding plant tissue in liquid nitrogen with mortar and pestle that this extraction method was reliable. and then mixing with plant DNAzol buffer or B, grinding plant tissue cooled at –30°C in cetyl- Host primers MDH-H968 and MDH- trimethylammonium bromide buffer with FastPrep Instrument. The degenerate primers SPG1/SPG2 C1163 were used to confirm that the DNA amplified a 912-bp product from the infected plants, and the internal primers MDH-H968/MDH- C1063 amplified a 400-bp fragment from host plants. Lane 1, 1-kb DNA ladder; lanes 2–11, the graft- extracts were suitable for PCR. PCR am- inoculated indicator plants; and lanes 12–18, greenhouse-grown sweetpotato plants. plification with these primers yielded a 400-bp DNA fragment from extracts of several Ipomoea spp. Two expected prod- ucts, one of virus origin (912 bp) and the other of host origin (400 bp), were co- amplified by PCR when both viral and MDH primers were used with infected samples (Fig. 1). False negative results were identified by the absence of the 400- bp amplicon (Fig 1A, lanes 16 and 18). Sensitivity of primer pairs. Initial PCR assays using primers PW285-1/PW285-2, developed by Lotrakul and Valverde (14), for SPLCV-Taiwan yielded a major ampli- con of 514 bp (Fig. 2A). No PCR ampli- cons were produced using the DNA ex- tracts of noninoculated plants. However, these primers gave inconsistent results when the same in vitro plantlets were Fig. 2. Polymerase chain reaction (PCR) products amplified from dilutions of the DNA extracted from tested two times within 2 months (data not a plantlet infected with a Taiwan isolate of Sweet potato leaf curl virus. Lane 1, 1-kb DNA ladder; lane shown). To increase the sensitivity and 2, healthy plantlet; lane 3, undiluted DNA extract from infected plantlet; lanes 4–12, 10-fold serial detection range, degenerate primers dilutions: lane 4, 10–1 (200 µg of plant tissue); lane 5, 10–2 (20 µg); lane 6, 10–3 (2 µg); lane 7, 10–4 SPG1/SPG2 and specific primers (200 ng); lane 8, 10–5 (20 ng); lane 9, 10–6 (2 ng); lane 10, 10–7 (200 pg); lane 11, 10–8 (20 pg); and –9 SPG3/SPG4 were compared with primers lane 12, 10 (2 pg). Primers were A, PW285-1/PW285-2, B, SPG1/SPG2, C, and SPG3/SPG4. Ar- PW285-1/PW285-2 in PCR assays and rows indicate the positions of the A, 514-bp, B, 912-bp, and C, 1,148-bp PCR amplicons. found to be highly sensitive (Fig. 2B and C). Three PCR assays were carried out on 10-fold dilutions of DNA extracted from a plantlet infected with SPLCV-Taiwan. PCR results indicated an endpoint at 10–3 (an equivalent of 2 µg of plant tissues) for primers PW285-1/PW285-2, at least 10–9 (2 pg) for degenerate primers SPG1/SPG2 (no endpoint was obtained), and at 10–5 (20 ng) for primers SPG3/SPG4 (Fig. 2). The PCR results for DNA extracted from the plant infected with SPLCV-Taiwan indi- cated a single amplicon of 912 bp for de- generate primers SPG1/SPG2, and a major amplicon of 1,148 bp for primers SPG3/SPG4. The nucleotide sequences for the three PCR clones obtained from the Taiwan isolate shared 86.2 to 96.9% identity with

SPLCV-US, confirming that it is an isolate of SPLCV and that the amplicons were of Fig. 3. Detection of sweetpotato geminivirus isolates by polymerase chain reaction (PCR) using prim- viral origin, as expected. ers A, PW285-1/PW285-2, B, SPG1/SPG2, or C, SPG3/SPG4. Lane 1, 1-kb DNA ladder; lanes 2 and 3, two isolates from China; lane 4, Korean isolate; lanes 5 and 6, two isolates from Taiwan; lane 7, Specificity of primer pairs. To deter- Brazilian isolate; lane 8, Mexican isolate; lanes 9 and 10, two isolates from Puerto Rico; lane 11, mine the range of detection, nine isolates uninoculated Ipomoea setosa; and lanes 12 and 13, potyviruses used as negative controls. Amplicon of geminiviruses infecting sweetpotato sizes are A, 514 bp, B, 912 bp, and C, 1,148 bp. Five times more PCR product was loaded for gel A from five different geographic regions than for gels B and C.

Plant Disease / December 2004 1349 were examined by PCR using three primer SPG3/SPG4 in PCR (Fig. 4A and C). of bleaching of the internal leaf tissue, pairs, PW285-1/PW285-2, SPG1/SPG2, However, DNA fragments of different sizes with or without upward curling of the leaf and SPG3/SPG4 (Fig. 3). There was no were amplified from seven non- margins. The results of the grafting assay discernible difference in the sizes of the sweetpotato geminiviruses by PCR using for 16 plantlets correlated very well with amplicons obtained from all nine isolates, the degenerate primers SPG1/SPG2. All those of the PCR assay using primers although nonspecific bands were observed PCR amplicons except that of BCTV were SPG1/SPG2 (Table 2). Several in vitro when primers PW285-1/PW285-2 or smaller than those for sweetpotato gemi- clones that were negative by PCR and SPG3/SPG4 were used. niviruses (Fig. 4B). When in vitro plantlets grafting assays were propagated and re- To determine if these three pairs of were tested by PCR, geminiviruses were leased as virus-free materials to customers. primers could amplify similar-sized prod- detected in 40 of the 62 plantlets using ucts from other geminiviruses, we tested primers SPG1/SPG2, compared with 29 DISCUSSION DNA extracts of two uncharacterized using primers PW285-1/PW285-2 (data Previously, the only reliable test for de- sweetpotato geminivirus isolates, ILCV not shown). Consequently, the SPG1/SPG2 tection of geminiviruses in sweetpotato and seven geminiviruses infecting different primers, together with the MDH primers, germplasm in a quarantine indexing pro- hosts by PCR (Fig. 4). PCR assays using were selected as the best primers for PCR- gram was a grafting assay. Hybridization primers PW285-1/PW285-2, SPG1/SPG2, based detection of the geminiviruses in assays using probes generated from clones and SPG3/SPG4 yielded amplicons of the sweetpotato. of other geminiviruses (i.e., BGMV) have same size (514, 912, and 1,148 bp) for two Comparison of PCR and grafting. been used to detect ICLCV and SPLCV- uncharacterized sweetpotato geminivirus During the summer, I. setosa plants in- US in sweetpotato (5,14). PCR using both isolates and ILCV. Very weak or no ampli- fected with geminiviruses developed leaf degenerate and virus-specific primers also cons were produced for DNA extracts of curl symptoms approximately 3 weeks has been used for virus detection and iden- other non-sweetpotato geminiviruses by after grafting. Under greenhouse condi- tification in indicator plants (1,14). Despite the primers PW285-1/PW285-2 and tions during the fall, symptoms consisted the use of the above techniques for virus detection in experimentally infected plants, they have not been used in the virus- indexing program due to the lack of a comparative evaluation with the grafting assay, the standard detection method. In this study, the successful application of PCR to detect 20 different geminivirus species or isolates in both in vitro plantlets and greenhouse-grown sweetpotato plants, and in Ipomoea spp. indicator plants, was reported. The extraction method using plant DNAzol buffer and liquid nitrogen yielded suitable quality DNA for PCR, but it was laborious and time consuming. Sev- eral DNA extraction kits designed for iso- lation of DNA from plant samples are now available. However, the kits are too expen- Fig. 4. Detection of different geminiviruses by polymerase chain reaction (PCR) using primers A, sive for use in the virus-indexing program PW285-1/PW285-2, B, SPG1/SPG2, or C, SPG3/SPG4. Lane 1, 1-kb DNA ladder; lane 2, uninocu- because many samples have to be tested. lated Ipomoea setosa; lane 3, an uncharacterized Jamaican isolate infecting sweetpotato; lane 4, an The DNA extraction method described by uncharacterized Puerto Rican isolate infecting sweetpotato; lane 5, Ipomoea leaf curl virus; lane 6, Doyle and Doyle (7) was modified and Tomato yellow leaf curl virus; lane 7, Tomato mottle virus; lane 8, Bean golden mosaic virus; lane 9, used for sample preparation in the Fast- Cabbage leaf curl virus; lane 10, Squash leaf curl virus; lane 11, Cotton leaf crumple virus; lane 12, Prep Instrument. This method uses a Beet curl top virus; lane 13, a potyvirus infecting sweetpotato used as negative control, and lane 14, water control. Five times more PCR product was loaded for gel A than for gels B and C. common buffer, CTAB, and a semi- automatic homogenizer for sample prepa- ration, and it allows for fast sample proc- Table 2. Comparisons of the polymerase chain reaction (PCR) assays and grafting for detection of essing and minimizes cross contamination. geminiviruses in sweetpotato plantlets The method has been used successfully to Plantlet no. First PCRa Second PCRb Symptoms in Ipomoea setosac extract nucleic acids suitable for PCR not only from in vitro plantlets and three Ipo- 1 + + + moea indicator hosts, but also from green- 2 + + + house-grown sweetpotato plants. The pro- 3 + + + 4 + + + tocol is easily scalable to large numbers of 5 – + + samples in a virus-indexing or certification 6 – + + program. Both fresh and frozen tissue (at 7 – + + –30°C for 1 year) could be used in DNA 8 – – – extractions without any significant differ- 9 + + + ences in the quality of the extracts. DNA 10 + + + extracts stored at –20°C for at least 1 year 11 + + + 12 + + + resulted in no loss of amplification. 13 + + + Three pairs of primers were evaluated 14 – – – for use in detection of geminiviruses in 15 + + + sweetpotato by PCR assays. All primers 16 – – – amplified PCR products of predicted sizes a Primer pair PW285-1/PW285-2 was used in the PCR. from all geminivirus species and isolates b Primer pair SPG2/SPG2 was used in the PCR. infecting sweetpotato, regardless of geo- c Tested plantlets were grown in a greenhouse and graft inoculated to indicator host I. setosa. graphic origin of the plant material. Thus,

1350 Plant Disease / Vol. 88 No. 12 variation at the primer regions among intensity of the geminivirus-specific bands 7. Doyle, J. J., and Doyle, J. L. 1987. A rapid DNA these isolates is low. Interestingly, degen- did not decrease. isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19:11-19. erate primers SPG1/SPG2 were more sen- This PCR assay offers several advan- 8. Fauquet, C. M., Bisaro, D. M., Briddon, R. W., sitive than the other primer pairs tested in tages over the grafting assay in the sweet- Brown, J. K., Harrison, B. D., Rybicki, E. P., the PCR assays. Degenerate primers have potato indexing program. Sequence infor- Stenger, D. C., and Stanley, J. 2003. Revision been used extensively in identification and mation determined from PCR products of taxonomic criteria for species demarcation detection of geminiviruses (2,6,13,20,21). will be used for identifying uncharacter- in the family Geminiviridae, and an updated list of begomovirus species. Arch. Virol. Sequence analyses revealed that the iden- ized geminiviruses in sweetpotato, exam- 148:405-421. tity of the coat protein (CP) sequences ining genetic variations, and establishing 9. Girardeau, J. H., and Ratcliffe, T. J. 1960. The between three geminiviruses infecting taxonomic relationships. Rapid screening vector-virus relationship of the sweetpotato sweetpotato and other begomoviruses was of in vitro plantlets generated from therapy and a mosaic of sweetpotatoes in less than 49%, lower than those among eliminates the need to grow large numbers South Georgia. Plant Dis. Rep. 44:48-50. 10. Hildebrand, E. M. 1959. A whitefly, Trileu- other begomoviruses (11,13). Therefore, of plants in the greenhouse for grafting rodes abutilonea, an insect vector of sweetpo- degenerate primers based on the CP se- assay. Unlike the grafting assay, PCR can tato feathery mottle in Maryland. Plant Dis. quence for other non-sweet-potato- be carried out year-round, thereby speed- Rep. 43:712-714. infecting geminiviruses may not be suit- ing up dissemination of healthy plant ma- 11. Howarth, A. J., and Vandemark, G. J. 1989. able for detection of target viruses. Degen- terial to the intended users and greatly Phylogeny of geminiviruses. J. Gen. Virol. 70:2717-2727. erate primers SPG1/SPG2 anneal to re- reducing costs associated with elimination 12. Lotrakul, P., and Valverde, R. A. 1999. Cloning gions of ORFs AC2 and AC1 which are of geminiviruses from germplasm. Finally, of a DNA-A like genomic component of sweet highly conserved in geminiviruses infect- the DNA extraction method and the PCR potato leaf curl virus: nucleotide sequence and ing sweetpotato and other begomoviruses. assay developed in this study also may be phylogenetic relationships. Mol. Plant. Pathol. They amplified PCR products not only applicable to detection of geminiviruses in On-line. 13. Lotrakul, P., Valverde, R. A., Clark, C. A., from geminiviruses infecting sweetpotato, other crops that must be tested by PGQO Hurtt, S. S., and Hoy, M. W. 2002. Sweetpo- but also from other geminiviruses tested. or other similar institutions. tato leaf curl virus and related geminiviruses in The high sensitivity and broad detection sweetpotato. Acta Hortic. 583:135-141. range of these primers make them the ACKNOWLEDGMENTS 14. Lotrakul, P., Valverde, R. A., Clark, C. A., Sim, best choice for general use in PCR-based We thank A. Murphy and C. Losckinkohl for J., and De La Torre, R. 1998. Detection of a technical assistance; W. Moskal for recommenda- geminivirus infecting sweet potato in the detection. The broad detection range of tion of the MDH primers in the virus detection United States. Plant Dis. 82:1253-1257. the degenerate primers also may aid in system; and H.-Y. Liu, H.-T. Hsu, and J. Foster for 15. Moyer, J. M., Jackson, G. V. H., and Frison, E. the detection of other uncharacterized review and helpful comments. A., eds. 1989. FAO/IBPGR Technical Guide- geminiviruses in sweetpotato. The lack of lines for the Safe Movement of Sweet Potato effectiveness of primers PW285-1/ LITERATURE CITED Germplasm. Food and Agriculture Organization 1. Banks, G. K., and Bedford, I. D. 1999. A novel of the United Nations, Rome/International PW285-2 can be explained by three mis- geminivirus of Ipomoea indica (Convol- Board for Plant Genetic Resources, Rome. matches and one deletion in PW285-1 vulacae) from southern Spain. Plant Dis. 16. Moyer, J. W., and Salazar, L. F. 1989. Viruses and by a single mismatch in PW285-2 83:486. and viruslike diseases of sweet potato. Plant when compared with the SPLCV-US se- 2. Briddon, R. W., and Markham, P. G. 1994. Dis. 73:451-455. quence. The decreased sensitivity of Universal primers for the PCR amplification of 17. Murashige, T., and Skoog, F. 1962. A revised dicot-infecting geminiviruses. Mol. Biotech- medium for rapid growth and bioassays with to- primers SPG3/SPG4 probably is an arti- nol. 1:202-205. bacco tissue cultures. Physiol. Plant. 15:473-497. fact of PCR in which the longer PCR 3. Chung, M. L., Liao, C. H., Chen, M. J., and 18. Nassuth, A., Pollari, E., Helmeczy, K., Stewart, fragment cannot be replicated as effi- Chiu, R. J. 1985. The isolation, transmission, S., and Kofalvi, S. A. 2000. Improved RNA ex- ciently as the shorter ones. and host range of sweet potato leaf curl agent traction and one-tube RT-PCR assay for simul- The use of primers MDH-H968 and in Taiwan. Plant Prot. Bull. (Taiwan, ROC) taneous detection of control plant RNA plus 27:333-341. several viruses in plant extracts. J. Virol. MDH-C1163 in the PCR allowed for the 4. Clark, C. A., and Moyer, J. W. 1988. Compen- Methods 90:37-49. simultaneous detection of host DNA and dium of Sweet Potato Diseases. The American 19. 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