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Detection of Geminiviruses in Sweet Potato By 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 Virus isolates. Twenty sweetpotato ac- Geminivirus infection of sweetpotato (Ipomoea spp.) germplasm acquired from foreign regions cessions naturally infected with viruses 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: begomoviruses, 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 Geminiviridae) 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), Tomato 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- Begomovirus are transmitted by whiteflies, 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), Papaya 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,
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