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Biological and molecular diagnosis of seedborne viruses in cowpea germplasm of geographically diverse sub-Saharan origins: Seedborne cowpea viruses

A total of 983 cowpea accessions obtained from the University of California, Riverside (UCR) Cowpea Repository were analysed for seedborne viruses. A majority of the accessions originated from 11 countries representing different agroclimatic zones in
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  Biological and molecular diagnosis of seedborne viruses incowpea germplasm of geographically diverse sub-Saharansrcins N. M. Salem a † , J. D. Ehlers b , P. A. Roberts b and J. C. K. Ng a * a Department of Plant Pathology and Microbiology, and Institute for Integrative Genome Biology, University of California, Riverside,CA 92521; and   b Department of Nematology, University of California, Riverside, CA 92521, USA A total of 983 cowpea accessions obtained from the University of California, Riverside (UCR) Cowpea Repository wereanalysed for seedborne viruses. A majority of the accessions srcinated from 11 countries representing different agroclimaticzones in sub-Saharan Africa, and included landraces, local cultivars and breeding lines. Following the initial grow-out tests,69 cowpea accessions, mostly with symptoms of virus infection, were selected for further evaluation using a combination of host range, reverse transcription-polymerase chain reaction (RT-PCR) and sequence analyses. The analyses revealed thatsamplesfrom46(67%)accessionsharbouredoneormoreknownseedbornevirusesofcowpea.Theseincludedseedsamplesof accessions srcinating from Botswana (13 accessions), Ghana (6), Nigeria (6), Mali (1), Kenya (5), Cameroon (7), Niger(4),Co ˆ ted’lvoire(1),Benin(1),India(1)andChina(1).ViruseswereidentifiedbyRT-PCRanalysisoftotalRNAsextractedfrom suspected virus-infected samples using virus species-specific primers, as well as the cloning and sequencing of RT-PCRproducts amplified using virus genus- and family-specific degenerate oligonucleotide primers. The viruses identified included Cowpea aphid-borne mosaic virus  (CABMV),  Cucumber mosaic virus  (CMV) and  Southern bean mosaic virus  (SBMV).Phylogenetic analysis of the deduced coat protein (CP) amino acid sequences of selected CMV isolates recovered from fiveagroclimatically distinct locations confirmed their affiliations as new members of CMV subgroup IB. This is the first timethat seedborne viruses of cowpea accessions in a major collection (UCR) have been identified using RT-PCR and sequencingapproaches. Keywords : biosecurity,  Cucumovirus,  genetic diversity,  Potyviridae,  RT-PCR,  Vigna unguiculata Introduction Cowpea ( Vigna unguiculata ) is a major food crop inmany developing nations in the tropical and subtropicalregions of the world because of its high-quality dietaryprotein content, acceptable palatability and low cost of production (Ehlers & Hall, 1997; Kareem & Taiwo,2007). However, cowpea production is constrained by arange of abiotic and biotic factors, including viral dis-eases, and yields in sub-Saharan Africa are typically only10–20% of the known cowpea yield potential. Over 140viruseshavebeenidentifiedasnaturallyinfectingcowpea(Hampton  et al. , 1992; Shoyinka  et al. , 1997; Hughes &Shoyinka, 2003); of these, at least 15 have been reportedto be seedborne (Hampton, 1983; Mali & Thottappilly,1986; Hampton  et al. , 1997), and they all possess RNAgenomes. These viruseshavebeenwidelydistributedandare established in most cowpea-producing areas of theworldasaresultofthecowpeaseedtradeandmovementofinfectedseedlotsforvarietytrialsorgermplasmcollec-tions.Amajorityofthevirusesthatareseedborneincow-pea are also insect-transmitted. Accordingly, infectedgrown-outplantscanserveasinitialinfectionfociforsec-ondaryvirus spread among field-grown plants (Johansen etal. ,1994).Examples ofsuchvirusesincludethe aphid-transmitted  Cowpea aphid-borne mosaic virus  (CAB-MV),  Bean common mosaic virus  (BCMV),  Cucumbermosaic virus  (CMV) and  Peanut stunt virus  (PSV); andthe beetle-transmitted  Cowpea mosaic virus  (CPMV), Cowpea severe mosaic virus  (CPSMV),  Southern beanmosaic virus  (SBMV) and  Cowpea mottle virus (CPMoV).  Cowpea mild mottle carlavirus  (CPMMV) istheonlycowpeaseedbornevirusreportedtobetransmit-tedbywhiteflies(Brunt&Kenten,1973;Jeyanandarajah&Brunt,1993;Naidu etal. ,1998). *E-mail: † Present address: Industrial Chemistry Centre, Royal ScientificSociety, PO Box 1438, Al-Jubaiha,Amman 11941, Jordan. Published online 14 April 2010 ª  2010 The AuthorsJournal compilation  ª  2010 BSPP  773 Plant Pathology  (2010)  59 , 773–784 Doi: 10.1111/j.1365-3059.2010.02285.x  Efficientandaccuratediagnosisiskeytomitigatingtheconsequencesassociatedwiththeseedbornetransmissionof viruses in cowpea. Currently, the most common diag-nosis method adopted by cowpea seed banks focuses lar-gely on the monitoring of viral-like symptoms on plantsgrown to propagate seed stocks. Several other methodsinclude determining the biological (mechanical or vectortransmission, host-range test   ⁄   infectivity assay, and in vitro  features) and serological (Gillaspie  et al. , 1995;seebelow)propertiesoftheviruses.Frequently,severalof these methods are used in combination for greater effec-tiveness, thereby further intensifying existing efforts andresources devoted to this aspect of cowpea cultivation(Naidu & Hughes, 2003). Thus, it is not surprising thatdespite the importance of seedborne viruses and theircrop-loss potential if left unchecked, only limitedattempts have been made to study the distribution of themajor seedborne viruses from cowpea germplasm acces-sions. In one previous study, using enzyme-linkedimmunosorbent assay (ELISA), Gillaspie  et al.  (1995)detected CABMV, CMV, CPMoV, CPSMV and SBMVeither singly or as mixed infections in 60 cowpea seedsamples submittedfromBotswana, India andKenyathatwere to be deposited in the UCR germplasm collection.Also with ELISA, Bashir & Hampton (1996a) detectedone of three viruses,  Blackeye cowpea mosaic virus (BlCMV; a strain of BCMV), CABMV and CMV, inplants grown from cowpea seed samples of 182 germ-plasm accessions (from Afghanistan, Botswana, Brazil,China, Hungary, India, Iran, Mexico, Nigeria, Pakistan,South Africa and the USA) submitted for inclusion intotheSouthernRegionalPlantIntroductionStationatGrif-fin, Georgia (USA), and in the Plant Genetic ResourcesInstitute,NationalAgriculturalResearchCenter,Islama-bad, Pakistan. The same study by Bashir & Hampton(1996a) also reported that CABMV was the only virusfound in 2930 cowpea germplasm seed samples (fromSenegal and Botswana) obtained from the UCR germ-plasmcollection.Thepresenceofonlyonevirusinsuchalarge quantity of seed samples is remarkable, although itis not known whether the progenitor seeds from theseaccessions had been subjected to virus screening andclean-up, e.g. by visual selection and ELISA, prior tobeingdepositedinthecollection.Recent progress in molecular biology has led to thedevelopment of RT-PCR-based methods that facilitatethe accurate, rapid and less labour-intensive detection of a number of cowpea-infecting viruses (Bariana  et al. ,1994;Gillaspie etal. ,1999;Akinjogunla etal. ,2008).The objective of the present study was to use a combi-nation of grow-out   ⁄   visual inspection, host range, RT-PCRandsequenceanalysestoassayforseedbornevirusesin cowpea accessions, with a focus on a wide selection,albeitlimitedinnumber,ofseedsoriginatingfromseveralmajorcowpea-producingregionsofdifferentagroclimat-ic conditions in sub-Saharan Africa. The analyses identi-fied the presence of several but not all of the knowncowpea seedborne viruses, confirming their widespreadgeographical distribution. It was also demonstrated thattheidentityofseedbornevirusesfrominfectedgrown-outcowpea plants can be determined by sequencing thecloned RT-PCR products amplified by both virus genus-and family-specific degenerate oligonucleotide primers.Furthermore, using phylogenetic analysis, five new sub-group IB CMV isolates were identified, each srcinatingfromadifferentsub-Saharancountry. Materials and methods Virus isolates Alfalfa mosaic virus  (AMV; genus  Alfamovirus ), CMV-Fny,  Potato virus Y   (PVY; genus  Potyvirus ), PSV-CV1and -West, SBMV and  Tobacco ringspot virus  (TRSV;genus  Nepovirus ) as dried tissues, gifts from D. Mat-thews, University of California, Riverside (UCR), storedover CaCl 2  at 4  C were revived, propagated and subse-quently maintained in  Nicotiana tabacum  cv. Xanthi(AMV and PVY),  N. tabacum  cv. White Burley (PSV-CV1 and -West),  V. unguiculata  (SBMV) and  Cucumissativus  (TRSV) kept in the greenhouse. Plants infectedwith the individual viruses  Angelonia flower break virus (AnFBV; genus  Carmovirus ),  Zucchini yellow mosaicvirus  (ZYMV; genus  Potyvirus ) (D. Matthews, UCR), aswell as  Potato virus M  (PVM; genus  Carlavirus ) and Potato virus S  (PVS; genus  Carlavirus ) (gifts from K.Perry, Cornell University, Ithaca, NY, USA), were main-tainedinthegreenhouse. Grow-out examination of cowpea germplasmaccessions The University of California, Riverside, maintains anappreciable collection of cowpea germplasm (approxi-mately 5500 accessions) obtained from the United StatesDepartment of Agriculture (USDA), the InternationalInstitute of Tropical Agriculture (IITA) in Nigeria, anddifferent countries around the world as part of the TitleXII Bean   ⁄   Cowpea Collaborative Research Support Pro-gram. For this study, seeds of 983 cowpea accessionsfrom 13 countries, including 11 African nations (Fig. 1),ChinaandIndia,wereobtainedfromtheUCRcollection.Five seeds of each accession were planted in clean plasticpotsfilledwithsterilizedsoil,andgrownunderinsect-freegreenhouse conditions. Germinated seedlings wereexamined weekly for symptoms of seedborne viruses onthe primary or first trifoliate leaves. Notes were made onthepresenceorabsenceofvirussymptomsintheseacces-sions. Suspected virus-infected accessions were subjectedto further analyses for seedborne viruses by biologicalassays on indicator plants, and   ⁄   or RT-PCR, cloning andsequenceanalysis(seebelow). Biological assays Leaves with symptoms were harvested from grown-outcowpeaplantsandeithervacuum-driedandstoredat4  Cuntil ready for analysis or freshly inoculated to indicator 774  N. M. Salem  et al. Plant Pathology   (2010)  59 , 773–784  plants.Allbiologicaltestswerecarriedoutbymechanicalinoculation of indicator host species using the sap of homogenized leaves with and in some cases withoutsymptomsasthesourcesofinocula.Indicator plant species used were  Chenopodiumquinoa ,  N. tabacum ,  N  .  benthamiana ,  N  .  clevelandii , N  .  glutinosa ,  V. unguiculata ,  Pisum sativum ,  Glycinemax ,  Solanum lycopersicum  and  Phaseolus vulgaris. Inoculawerepreparedbygrindingleaftissueswithsteril-izedpestlesandmortarsin0 Æ 05  M sodiumphosphatebuf-ferpH7 Æ 0,ataratioof1:2(tissueweight:buffervolume).Plants that served as negative controls were inoculatedwithbuffersolutiononly.Allinoculatedplantswerekeptin the greenhouse and observed for symptoms over aperiod of 15–25 days, during which time localized andsystemicsymptomswererecorded. Extraction of total and virion RNAs, and RT-PCR Suspected virus-infected leaves from grown-out cowpeaplants were harvested and directly frozen at  ) 80  C untilused for total RNA extraction. RT-PCR was performedusing the extracted total RNAs and virus species-specificoligonucleotide primers, as well as virus genus and fam-ily-specific degenerate oligonucleotide primers. VirionRNAs were extracted from purified virions of CPSMVandSBMV(giftsfromG.Bruening,UniversityofCalifor-nia,Davis)andtheCPMVisolatesCPMV-BandCPMV-G (D. Matthews, UCR), essentially according to themethodsof Klaassen  et al.  (1996).For totalRNA extrac-tion, 100 mg leaf tissue were ground in liquid nitrogen,followed by TRIzol  4 (Invitrogen) treatment accordingto the manufacturer’s recommendations. RNA wasresuspended in 30  l L DEPC-treated deionized water.The expression of a host ubiquitin gene was used as aninternal control to ascertain the quality of the total RNAextracts and for determining the RT-PCR amplificationefficiency among test samples. RT-PCR amplification of the ubiquitin mRNA was performed using the primersNtUbiF, 5 ¢ -TGCTTAACACATGCAAGTCGGA-3 ¢  andNtUbiR,5 ¢ -AGCCGTTTCCAGCTGTTGTTC-3 ¢ .Sequences of oligonucleotide primers used for virusdetection were either obtained from published studies orestablishedinthisstudyusingviralsequencesavailableinthe GenBank, and designed using the ‘primer design’functionin VECTORNTIADVANCE TM 10(InforMax)(Table1).Specificity of the primers was tested against virion RNAsof CPMV-B and -G, CPSMV and SBMV, as well as thetotalRNAsextractedfromAMV-,AnFBV-,CMV-Fny-,CPMV-, PSV-CV1-, PSV-West-, PVM-, PVS-, PVY-,TRSV- and ZYMV-infected plants. RT-PCR was carriedout according to previously established protocols forComoviridae (Maliogka  et al. , 2004), Potyviridae (Chen et al. , 2001),  Carlavirus  (Hataya  et al. , 2001),  Carmovi-rus (Morozov etal. ,1995)and Cucumovirus (Choi etal. ,1999) (Table 1). RT-PCR for the detection of AMV,CPMV,CPSMV,SBMVandubiquitinwasperformedbya one-step RT-PCR protocol (Table 1), in which a 25- l Lreaction volume containing 2 Æ 5  l L 10  ·  PCR buffer,1 Æ 1  l L 25 m M  MgCl 2 , 1 Æ 25  l L 0 Æ 1  M  dithiothreitol,0 Æ 5  l L 10 m M  dNTPs, 1 Æ 25  l L each primer (10  l M ),0 Æ 25  l L  Taq  polymerase (1 U  l L ) 1 ) and 0 Æ 1  l L AMVreverse transcriptase (5 U  l L ) 1 , Promega Corp.) wasused. Reactions consisted of one cycle of 52  C for30 min; 35 cycles of 94  C for 30 s, 54  C for 45 s and72  C for 1 min; and a final 72  C extension for 10 min. MSCdBFGBNNiCKBotsB BeninBF Burkina FasoBots BotswanaC CamaroonCd Côte d’lvoireG GhanaK KenyaM MaliN NigeriaNi NigerS Senegal Figure 1  Map indicating the countries in sub-Saharan Africa from which cowpea accessions were analysed in this study. Seedborne cowpea viruses  775 Plant Pathology   (2010)  59 , 773–784  Following amplification, PCR products from each reac-tion mixture were separated by electrophoresis in 1 Æ 0%or 1 Æ 2% agarose gels in TAE buffer, stained with0 Æ 5  l g mL ) 1 ethidiumbromideandvisualizedbyUVillu-mination(Sambrook&Russell,2001). cDNA cloning, nucleotide sequence and phylogeneticanalyses Following agarose gel electrophoresis, DNA fragmentsamplified using Potyviridae- and  Cucumovirus- specificoligonucleotideprimerswerepurifiedusingtheMinEluteGel Extraction kit (Qiagen) according to the manufac-turer’sinstructions.AllDNAfragmentswereligatedintothe pGEM-T Easy vector (Promega Corp.) according tothemanufacturer’sinstructions,followedbytransforma-tion into  Escherichia coli  DH5 a . Recombinant colonieswere selected and grown in Luria-Bertani (LB) brothcontaining ampicillin (100  l g mL ) 1 ; Sigma-Aldrich).Plasmid isolation from randomly selected colonies wasperformed using the FastPlasmid Mini kit (Eppendorf).The purified plasmids were digested with  Eco R I (New Table1  OligonucleotideprimersusedfortheRT-PCRdetectionofcowpeaseedbornevirusesVirus a Oligonucleotide primer information b RT-PCR c Productsize (bp)Sequence Polarity ReferenceComoviridae ComF1: 5 ¢ -ACIWSIGARGGITWYCC-3 ¢ ComR2: 5 ¢ -AVRTTRTCRTCICCRTA-3 ¢ ComNeF3: 5 ¢ -TACGWSGGARGGGTWYCC-3 ¢ ComNeR4: 5 ¢ -ARRTTRTCRTCGCCRTAIAC-3 ¢ ComNeR5: 5 ¢ -AVRTTRTCRTCGCCRTAIGT-3 ¢ + ) + )) 1 Two stepNested737–789Potyviridae Sprimer: 5 ¢ -GGNAAYAAYAGYGGNCARCC-3 ¢ M4: 5 ¢ -GTTTTCCCAGTCACGAC-3 ¢ M4T: 5 ¢ -GTTTTCCCAGTCACGAC (T) 15 -3 ¢ + )) 2 Two step 1700 Cucumovirus   CPTALL-3: 5 ¢ -GACTGACCATTTTAGCCG-3 ¢ CPTALL-5: 5 ¢ -YASYTTTDRGGTTCAATTCC-3 ¢ + ) 3 Two step 938–966 Carlavirus   CARORF3P: 5 ¢ -CCNRATGCCACTTACACCNCCDCCT-3 ¢ Oligo(dT)-AP2: 5 ¢ -CGATGGTACCTGCAGGCGCGCC(T) 18  -3 ¢ 3NTR-AP2: 5 ¢ -CGATGGTACCTGCAGGCGCGCC-3 ¢ + )) 4 Two step 120 Carmovirus   Carmo-II: 5 ¢ -ARGTCGACCCGWNCCNMGNGTNATHCAACC-3 ¢ Carom-VI: 5 ¢ -GMMCTGCAGNACRCARTCRTCNCCRTTRTT-3 ¢ + ) 5 Two step 51AMV CPAMV1: 5 ¢ -TTCCATCATGAGTTCTTCAC-3 ¢ CPAMV2: 5 ¢ -AGGACTTCATACCTTGACC-3 ¢ + ) 6 One step 751CPMV CPMV For: 5 ¢ -GACACGTAGTGCGGCGCCATTA-3 ¢ CPMV Rev: 5 ¢ -AATCCCATCCCAGCAGCTGC-3 ¢ + ) 7 One step 522CPSMV CPSMV For: 5 ¢ -GTCACATGCGTGACAGGATG-3 ¢ CPSMV Rev: 5 ¢ -GTATCGGGCCTAACCTGTGT-3 ¢ + ) 7 One step 500SBMV SBMV For: 5 ¢ -TGGTCCTTCGACGCAATCT-3 ¢ SBMV Rev: 5 ¢ -GTCTGCTTCAGCTGCAGGACA-3 ¢ + ) 7 One step 501 a Virus families (Comoviridae and Potyviridae) and genera ( Cucumovirus  ,  Carlavirus   and  Carmovirus  ) with known cowpea seedbornemembers, as well as the specific cowpea seedborne virus species AMV ( Alfalfa mosaic virus  ), CPMV ( Cowpea mosaic virus  ), CPSMV( Cowpea severe mosaic virus  ) and SBMV ( Southern bean mosaic virus  ). b Virus family-, genus- and species-specific oligonucleotide primers for the detection of known cowpea seedborne viruses. Oligonucleotideprimers for the detection of members of the Comoviridae were designed based on two conserved amino acid motifs: (T   ⁄   V)YGDDN(V   ⁄   L)(primers ComF1 and ComNeF3) and TSEG(Y   ⁄   F)P (primers ComR2, ComNeR4 and ComNeR5), within the RNA-dependent RNA polymerase(RdRp) region of the virus genome (ref. 1: Maliogka  et al. , 2004). The oligonucleotide primer (Sprimer) for the detection of members of thePotyviridae was designed based on the conserved amino acid motif GNNSGQP in the NIb region of the virus genome (ref. 2: Chen  et al. ,2001). Genus-specific oligonucleotide primers correspond to regions flanking the overlapping region between open reading frames (ORFs) 2and 3 in the carlavirus genome (ref. 4: Hataya  et al. , 2001), the RdRp gene in the carmovirus genome (ref.5: Morozov  et al. , 1995), the coatprotein gene (CP) of the cucumovirus genome (ref. 3: Choi  et al. , 1999). Species-specific primers for the amplification of the complete CPgene of AMV were designed according to published information (ref. 6: Parrella  et al. , 2000). Species-specific primers for CPMV, CPSMV andSBMV were designed using genome sequence information obtained from GenBank (Accession Nos NC 003550, M 83309 and M 23021,respectively). Specifically, oligonucleotide primers complementary to nucleotides 704–723 (primer CPMV Rev) and 202–203 (primer CPMVFor) of the CPMV RNA 2 genome, and primers complementary to nucleotides 2481–2500 (primer CPSMV Rev) and 2001–2020 (primerCPSMV For) of the CPSMV RNA 2 genome, were designed to amplify part of the CP gene of the respective viruses (ref. 7: this study).Oligonucleotide primers for the detection of SBMV are complementary to nucleotides 2670–2690 (primer SBMV Rev) and 2191–2208 (primerSBMV For) in the RdRp gene of SBMV (ref. 7: this study). c A one-step, two-step, or two-step enriched-nested RT-PCR assay was used for these primers. 776  N. M. Salem  et al. Plant Pathology   (2010)  59 , 773–784  England Biolabs) to determine the size of inserts by aga-rose gel electrophoresis. Sequences of selected clones(morethantwoforeachsample)weredeterminedinbothdirectionsby means of an ABI3730xlautomated sequen-cer. Sequences were edited in  VECTOR NTI ADVANCE TM 10andsubjectedto BLAST searches(Altschul etal. ,1997).The phylogenetic relationships of cowpea-infectingCMVisolateswerededucedbycomparingtheirpredictedCPaminoacidsequencestothatofisolatesretrievedfromthe GenBank (accession numbers are shown in Fig. 3).Multiple alignments and phylogenetic relationshipswere determined using the  MUSCLE  (Edgar, 2004) and DISTANCES  (ProtDist   ⁄   FastDist + Neighbour with a 1000-replicate bootstrap search) functions, respectively, fromthe website (Dereeper  et al. ,2008).Phylogenetic trees were visualized using  TREEDYN (Chevenet etal. ,2006),alsofromthesamewebsite. Enzyme-linked immunosorbent assay Antigen-coated plate (ACP)-ELISA was performed by aprocedure modified from that described by Mowat &Dawson (1987). Vacuum-dried cowpea tissues (approx.0 Æ 03 g) were homogenized in 2 mL carbonate coatingbuffer (0 Æ 015  M  sodium carbonate and 0 Æ 035  M  sodiumbicarbonate, pH 9 Æ 6). Then, 100  l L of the homogenizedleaf extract of each sample were added to a well of aPolysorp TM microtitre plate (Nunc) and incubated in ahumid chamber at 4  C overnight. After rinsing threetimes in PBS-Tween [phosphate-buffered saline (0 Æ 02  M phosphate, 0 Æ 15  M  NaCl, pH 7 Æ 4], containing 0 Æ 05%Tween 20], wells in the plate were blocked with 200  l L10% (w   ⁄   v) non-fat dried milk in PBS-Tween for 2 h at37  C. Following rinsing, primary antibody diluted inPBS-TPO buffer [PBS-Tween, 2% (w   ⁄   v) polyvinylpy-rollidone, 0 Æ 2% ovalbumin (w   ⁄   v), 0 Æ 02% sodium azide]was added to the wells. The following primary antibod-ies and fold dilutions were used: anti-CMV-Fny serum(1   ⁄   1000), anti-CABMV serum (1   ⁄   1000; a gift from J.A. M. Rezende, Universidade de Sa ˜ o Paulo, Brazil) andanti-SBMV IgG alkaline phosphatase conjugate (1   ⁄   100;Agdia Inc). Following plate incubation (2–3 h at 37  C)and rinsing, a goat-anti-rabbit alkaline phosphataseconjugate diluted 1   ⁄   3000 fold in PBS-TPO buffer wasadded to the anti-CMV-Fny and anti-CABMV sera-coated wells. Following incubation, the plate was rinsedand 100  l L 1-Step TM  p -nitrophenyl phosphate sub-strate (Thermo Scientific) added at room temperatureand left for 15 min for colour development. The absor-bance at 405 nm was then measured in a Wallac VictorII Multilabel counter (Perkin Elmer). Results Evaluation of oligonucleotide primers and RT-PCRconditions Initially,the genus-and family- specificdegenerateprim-ers as well as species-specific primers were evaluated bytesting them in RT-PCR using the total RNA extracts of tissues infected with known cowpea-infecting virusesand,insomecases,virionRNAsofsomeoftheseviruses.The results indicated that they were effective under theamplification conditions established in this and previousreports (Table 1). These RT-PCR amplifications yieldedproducts with the expected sizes for all primers tested.SelectedresultsarepresentedinFig.2. Assessment of cowpea accessions for seedborneviruses A total of 983 randomly selected cowpea accessionsfrom 11 sub-Saharan countries (Fig. 1) plus China andIndia were grown-out and assayed visually for virussymptoms. Plants displayed no symptoms to a variety of systemic symptoms, ranging from mild vein clearing,interveinal chlorosis, yellowing, mosaic, mottling,stunting, leaf deformation and reduction in leaf size.Reddening and necrosis of cotyledons and growing tipswere also observed in a few cases. In total, plants from69 accessions, a number of which were symptomless, M 1 2 3 4 5 6 7 8 9 10 11 M 12 13 14 15 16 17 18 19 20 21 10050010001650CPSbp Figure 2  RT-PCR analysis of cowpea accession lines infected with seedborne viruses. Total RNAs extracted from cowpea plants weresubjected to RT-PCR and the resulting amplified products were subjected to agarose (1 Æ 5%) gel electrophoresis. Representative samplesUCR 815 B043, UCR 924, Simbiri 94, Beng G Bere 94, UCR 521, UCR 2788, UCR 2632 and TVu 5609 (lanes 1–8, respectively) were testedusing  Cucumovirus  -specific degenerate oligonucleotide primers (Table 1). Representative samples TVu 5609, TVu 10362, TVu 3745 and TVu4943 (lanes 12–15, respectively) were tested with Potyviridae-specific degenerate oligonucleotide primers (Table 1). A representative sample,UCR 5269 (lane 18), was tested with SBMV-specific oligonucleotide primers. Positive controls were CMV-Fny ( Cucumovirus  ; lane 9), PSV-CV1( Cucumovirus  ; lane 10), PVY ( Potyviridae  ; lane 16) and SBMV (lane 19). Negative controls were total RNAs of uninfected cowpea plantsamplified with the  Cucumovirus  -specific (lane 11) and Potyviridae-specific (lane 17) degenerate oligonucleotide primers, and the SBMV-specific oligonucleotide primers (lane 20). Lanes 21 and M contained RT-PCR amplified water control and a 1-kb DNA ladder, respectively.The positions of several DNA size (bp) markers are indicated to the left. The expected sizes of the amplified products are indicated by thearrows: C ( Cucumovirus  ; 938–966 bp), P (Potyviridae; 1700 bp) and S (SBMV; 501 bp). Seedborne cowpea viruses  777 Plant Pathology   (2010)  59 , 773–784

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