Genomic variability of the coat protein gene of Citrus tristeza virus isolates obtained from old Meyer lemon introductions in Morocco and more recent budwood introductions from Spain were studied. The coat protein gene of the virus was amplified directly from infected tissue by immunocapture RT-PCR and analysed by single stranded conformation polymorphism (SSCP) and sequencing. Each isolate consisted of several related genomic variants, typical of a quasi-species. Although SSCP analysis has only limited typing ability it could be used in an initial screening to discriminate between isolates of different origin and to analyse the genomic structure of each isolate. Sequence analysis showed that the isolates of Spanish origin were closely related to mild isolates characterised in Florida and in Portugal. The Meyer lemon isolate on the other hand was related to severe strains of Meyer lemon characterised in Florida some years ago and to other severe strains from Brasil. A knowledge of the coat protein gene sequence is useful to trace the origin of the isolates

Bennani, A.

Fonseca, Filomena

Lbida, B.

Nolasco, Gustavo

Santos, C.

Zemzami, M.

Sapientia

1Phytopathol. Mediterr. (2004) 43, 00–00IntroductionCitrus tristeza virus (genus Closterovirus, fam-ily Closteroviridae, CTV) is the causal agent of themost serious disease of Citrus spp. (Rocha-Pena etal., 1995). The virus causes various disease types,the most important of which are the quick declineof trees grafted on sour orange rootstock; reducedvigour; reduced tree quality and stem pitting ofGenomic variability of Citrus tristeza virus (CTV) isolatesintroduced into Morocco.BOUABID LBIDA1, FILOMENA FONSECA2, CARLA SANTOS2, MUSTAPHA ZEMZAMI3, AMINA BENNANI1AND  GUSTAVO NOLASCO21Université My Ismail, Faculté des Sciences Meknès, PLEASE COMPLETE FULL MAIL ADDRESS,Morocco2Centro de Desenvolvimento de Ciencias e Técnicas de Produção vegetal, Universidade do Algarve – FERN,Campus de Gambelas, 8005-139 Faro, Portugal3Unité de controle des plantes, Direction des Domaines Agricoles, Km 11, Hussain, Salé 10.000, MoroccoSummary. Genomic variability of the coat protein gene of Citrus tristeza virus isolates obtained from old Meyerlemon introductions in Morocco and more recent budwood introductions from Spain were studied. The coat proteingene of the virus was amplified directly from infected tissue by immunocapture RT-PCR and analysed by singlestranded conformation polymorphism (SSCP) and sequencing. Each isolate consisted of several related genomicvariants, typical of a quasi-species. Although SSCP analysis has only limited typing ability it could be used in aninitial screening to discriminate between isolates of different origin and to analyse the genomic structure of eachisolate. Sequence analysis showed that the isolates of Spanish origin were closely related to mild isolates character-ised in Florida and in Portugal. The Meyer lemon isolate on the other hand was related to severe strains of Meyerlemon characterised in Florida some years ago and to other severe strains from Brasil. A knowledge of the coatprotein gene sequence is useful to trace the origin of the isolates.Key words: Closterovirus, KEY WORDS DIFFERENT FROM TITLE, coat protein gene.Corresponding author: G. NolascoFax: XXXXXXXXXXXXE-mail: gnolasco@ualg.ptbranches of trees grafted on tolerant rootstocks;leaf yellowing and stop of growth of sour orangeand lemon seedlings. In each region affected theparticular disease syndrome depends on the kindof CTV strains present. Strains may occur singlyor mixed (Grand and Higgins, 1957). A knowledgeof which strains are present in an area is funda-mental to define a strategy to control the disease.The virus is naturally transmitted in a semi-per-sistent manner by several species of aphids, ofwhich Toxoptera citricida is the most effective; thisaphid does not yet occur in the Mediterranean ba-sin. Budwood movement is responsible for the longdistance virus dissemination.2 Phytopathologia MediterraneaBouabid Lbida et al.The CTV genome consists of a single-stranded,positive sense RNA molecule of 19,296 nucleotides,encapsulated in flexuous filamentous particlesabout 11 nm wide  2,000 nm long (Bar-joseph etal., 1989). The virions contain two capsid proteins,a major 25-kDa coat protein (CP) covering about95% of the particle length and a minor 27-kDa cap-sid protein (Febres et al., 1996), which covers onlyone end of the particle.In Morocco CTV has been detected and infectedtrees have been eradicated on several occasions.Some of the isolates were biologically and serolog-ically characterised (Zemzami et al., 1999). A com-parison between the biological data and hybridi-sation patterns obtained with a panel of strain-dif-ferentiating probes has been reported for some ofthese isolates (Zemzami et al., 2002). In this workwe report on the further characterisation of CTVby SSCP analysis and on the sequencing of themajor coat protein gene of additional isolates in-troduced to Morocco a long time ago, and of otherscollected recently at the Moroccan border.Materials and methodsVirus isolatesThe CTV isolates P2 and R1 were interceptedby the Moroccan quarantine system as germplasmcoming from Spain, in 1998 and 2000 respectively.Virus isolate P1 was detected in Lemon Meyer in afield near Marrakesh in 1983. Isolates were prop-agated by graft transmission to Mexican Lime (Cit-rus aurantifolia) and maintained in an insect proofgreenhouse at 21–33°C. Additional biological char-acterisation of these isolates was done on grape-fruit, sweet orange, and mandarin grafted on sourorange. Isolate MZ3 was obtained from Troyer cit-range and corresponded to isolate number 3 previ-ously characterised (Zemzami et al., 1999, 2002)PCR amplification of the coat protein geneFresh bark and leaf tissue (0.2 g) were used asthe source of the virus. Amplification of the coatprotein gene was done by immunocapture reversetranscriptional polymerase chain reaction (IC/RT-PCR), using a one-step combined reverse transcrip-tion and amplification based on methods previouslydescribed (Nolasco et al., 1993). Polyclonal anti-bodies against an E. coli expressed CP gene (Se-queira and Nolasco, 2002) were used to trap thevirions. The whole CP gene and the stop codon wereamplified with primers CTV1 (5'-ATGGACGAC-GAAACAAAGAA-3') and CTV10 (5'-ATCAACGT-GTGTTGAATTTCC-3'). The PCR-products wereseparated by migration on 1% agarose gel electro-phoresis, and visualized under UV light after stain-ing with ethidium bromide.SSCP analysis of IC/RT-PCR or PCR productSSCP analysis was performed directly on theIC/RT-PCR products (from plant tissue) or on thePCR products (from E. coli colonies), following con-firmation of the specificity of the amplified prod-uct. The amplified product (1 µl) was added to 9 µlof denaturing buffer (950 µl of 95% formamide, 40µl of 500 mM EDTA, [pH 8], and a minimal amountof bromophenol blue), the sample was then heatedto 90°C for 5 min and quickly transferred to ice.Denatured products were electrophoresed in therefrigerator (4ºC) for 3 h at 200 V in a native 8%polyacrylamide gel (78 cm, 0.75 mm, Bio-RadMini-Protean II), using TBE as buffer. The gelswere stained with silver nitrate using the proce-dure of Beidler et al. (1982) and dried.Cloning and sequencingThe amplified RT-PCR product was TA-ligatedinto pGEMT-Easy vector (Promega, Madison, WI,USA), according to manufacturer’s instructions,transformed into competent E. coli INVaF’ cells(Invitrogen, Carlsbad, CA, USA) and plated accord-ing to standard procedures (Sambrook et al., 1989).Transformed colonies harbouring the recombinantplasmid were selected by α-complementation on theplates supplied with X-gal and ampicilin (Sam-brook et al., 1989). The occurrence of the coat pro-tein gene insert was confirmed by direct PCR of aportion of selected white colonies, using the sameset of primers as above. This PCR product was insome instances analyzed by SSCP.Selected colonies harbouring the CP gene insertwere transferred to liquid medium and the respec-tive plasmids purified using the WizardTM plusminiprep DNA Purification System (Promega) ac-cording to manufacturer’s instructions. An addi-tional confirmation of the insert was done by re-striction digestion of an aliquot of the plasmid withEco RI. Sequencing reactions were done using theforward and reverse universal primers by Macro-gen (Seoul, Korea).3Vol. 43, No. 2, August 2004Genomic  variability of CTV introduced in MoroccoSequence analysisSequence analysis, alignment and clusteringwere done using the software packages Bioedit(Hall, 1999) and Mega 2 (Kumar et al., 2001). Inaddition to the sequences obtained in this work,the following sequences of the CP gene were re-trieved from the GenBank and used in the com-parisons: AF184118 (haplotype 28C), AF184115(haplotype 25-120), AF184114 (haplotype 19-121),AF184113 (haplotype 13C), M76485 (haplotypeT36). The CP gene sequences from Florida isolatesT30, T3 and Meyer Lemon FL-7 were kindly pro-vided by C.L. Niblett. The sequence of haplotypeCB3-104 from the Capão Bonito isolate from Bras-il was reported in Targon et al. (2000).Results and discussionFragments of approximately 672 bp correspond-ing to the expected size of the CP gene were ob-tained by IC/RT-PCR (results not shown). A pre-liminary assessment of the variability of the CPgene was done by SSCP analysis of the amplifiedfragments (Fig. 1). SSCP patterns consisted of twoor three conspicuous bands in addition to a fastermigrating band corresponding to the non-dena-tured or partially renatured PCR product. Some ofthe single-stranded bands were doublets that couldbe better observed in an amplified picture. Sam-ples of Spanish origin depicted a very similar pat-tern, which differed from the Meyer lemon isolateP1. This indicated that SSCP of the whole CP genemight be useful in an initial discrimination of CTVisolates without the need for smaller fragments assuggested by Rubio et al. (1996), which would havebrought lengths closer to a putative optimum of200 bp (Sheffield et al., 1993).The IC/RT-PCR products were cloned and anumber of the transformed E. coli colonies wereanalyzed by PCR using the same pair of primers.Most of these PCR products were of the expectedsize for the specific amplicon (672 bp) and wereanalysed by SSCP (Fig. 2). Only two clones wereobtained from isolate MZ3; these were not analysedFig. 1. Single-stranded conformation patterns of IC/RT-PCR fragments containing the coat protein gene. Bot-tom bands on the left side panel correspond to ds-DNA.Upper bands correspond to ss-DNA. The picture on theright is a magnification of the ss-DNA region.Fig. 2. Single-stranded conformation patterns of thecloned coat protein gene from isolates P1, P2 and R1.The lane number indicates the haplotype.4 Phytopathologia MediterraneaBouabid Lbida et al.by SSCP. Six clones were analysed from isolate P1and eight each from isolates P2 and R1. Two, 2 and3 SSCP patterns were obtained from P1, P2 andR1 respectively; this polymorphism is typical for aviral quasi-species. As in Kong et al. (2000), wefound a predominant pattern which occurred in83% of clones from P1 and 75% of clones with aSpanish origin. If each SSCP pattern is assumedto correspond to a genomic variant (haplotype), thedistribution of the variants is characterised byheterozygocity (Nei and Kumar, 2000). Heterozy-gocity may take values ranging from 0, if there isonly one haplotype, to 1, if all the haplotypes aredifferent. The heterozygocity of P1 was 0.33, of R1,0.43, and of P2, 0.46. The Spanish isolates appearrelated in their composition of variants since theirmajor SSCP pattern was very similar.The cloned IC/RT-PCR products giving the mostrepresentative SSCP patterns of each isolate weresequenced and the portions corresponding to theprimers were excised to eliminate any bias intro-duced by primer sequence. The sequences werealigned and compared with other available acces-sions of diverse origin (Fig. 3), representative ofthe seven group types reported by Zemzami et al.(2002). The overall genetic diversity of these se-quences (mean nucleotide distance between tworandom selected variants) was 0.072 (SD 0.006).This value increased to 0.081 (SD 0.007) when thevariants sequenced in this work were excluded. Asthese new sequences did not contribute to increaseoverall diversity, it was concluded that they repre-sented true-to-type CTV CP gene variants.The CP gene variants clustered very clearly, asinferred from the high bootstrap values, in fourdistinct groups. The haplotypes of Spanish origin MZ3-8 MZ3-5 13C 28C T3 P1-5 19-121 T36 25-120 T30 R1-4 P2-5 R1-2 P2-8 P2-4 Fl7 P1-3 CB3-10410086100999910099830.01Gr 3aGr 4Gr 2Gr 1Gr MGr 3bGr 5Fig. 3. Dendrogram showing the clustering pattern of the coat protein gene sequences as obtained by UPGMA.Groups mentioned in Zemzami et al. (2002) are shown on the right.5Vol. 43, No. 2, August 2004Genomic  variability of CTV introduced in Moroccodiffered by less than 1% at nucleotide level fromthe well characterised isolate T30 (Garnsey et al.,1987) originating in Florida, or from isolate 25-120which, although characterised in Portugal (Bona-calza, 1998), was detected in certified budwoodimported from Spain in 1996. These isolates wererepresentative of Group M. Isolates T30 and 25-120 both produced mild vein clearing and mild orno stem-pitting when indexed in Mexican lime; theydid not cause stem-pitting on sweet orange orgrapefruit, nor induce decline on sweet orangegrafted on sour orange. This behaviour coincideswith the biological data obtained in this work onisolates P2 and R1.On the other hand, the predominant genomicvariant of isolate P1 (clone P1-3) clustered at lessthan 4% nucleotide distance to sequences obtainedfrom the severe isolates Capão Bonito from Brasiland Fl-7 (Group 5). It is very interesting that iso-late Fl7 was found on Meyer lemon-infected plantsfrom Florida (Cevik et al., 1996). The minor vari-ant P1-5 clustered close to the severe isolate T3(Group 4), also from Florida. The Meyer lemon iso-lates from Florida produced severe vein clearingand stem pitting on Mexican lime, decline of sweetorange grafted on sour orange, and stem-pittingon grapefruit; all of which are symptoms consist-ent with the biological properties so far determinedfor isolate P1. The introduction of the Meyer lem-on isolate in Morocco can be traced back to the firsthalf of the 20th century and confirmation that itcaused CTV infection came in 1963 (Cassin, 1963).However, twenty years later, infected Meyer lem-on trees could still be found in the field, as it wasin this study. Isolate P1 was very similar to theisolates from Florida and California, which have alow transmission rate to other plants (Grant andHiggins, 1957), and this may explain why the se-vere isolate P1 remained hidden for so long.The two clones from isolate MZ3 clustered ingroup 3a, with a severe sweet orange stem pittingisolate from Madeira island (Group 3a).In a previous work (Zemzami et al., 2002) mo-lecular data on the CP gene showed that severestrains occurred in material from old budwood col-lections in Morocco. These findings strongly sug-gest the need for continuous monitoring of severestrains of CTV before symptoms appear in the field.This work also illustrates the importance of thetransport of infected budwood as causing a contin-uous flow of exotic CTV strains to new areas. For acertain period, the newly introduced strains ap-peared to maintain their original biological andmolecular properties, while they remained hidden.During this period the CP gene could be charac-terised to trace their origins. Changes in culturalpractices or in the vector population may inducechanges in the spread and mixture of existingstrains, leading to disease outbreaks.Literature citedBar-joseph M. and F.R. Lee, 1989. Citrus tristeza virus.CMI/AAB Descriptions of Plant Viruses 353.Beidler L., P.R. Hilliard and R.L. Rill, 1982. Ultrasensitivestaining of nucleic acids with silver. Analytical Biochem-istry 126, 374–380.Bonacalza B., 1998 (ed.). Tipificação molecular de isolamen-tos do Virus da tristeza dos citrinos em Portugal. MScthesis, Instituto Superior de Agronomia, UniversidadeTécnica de Lisboa, Lisboa, Portugal, 112 pp.Cassin J., 1963. Découverte de 8 cas de tristeza parmi unlot de plants âgés de citrus introduit au Maroc. Al-Awa-mia 9, 53–77.Cevik B., 1996. Molecular cloning and sequencing of coatprotein genes of citrus tristeza virus isolated from MeyerLemon and Homely Tangor Trees in Florida. Proceed-ings of the 13th Conference of the International Organi-zation of Citrus Virologists (IOCV). IOCV, Riverside, CA,USA, 47–53.Febres V.J., L. Ashoulin, M. Mawassi, A. Frank, M. Bar-joseph, K.L. Manjunath, R.F. Lee and C.L. Niblett, 1996.The P 27 protein is present at one end of citrus tristezavirus particles. Phytopathology 86, 1331–1335.Garnsey S.M., D.J. Gumpf, C.N Roistacher, E.L. Civerolo,R.F Lee, R.K. Yokomi and M. Bar-Joseph, 1987. To-ward a standardized evaluation of the biological prop-erties of citrus tristeza virus. Phytophylactica 19,151–157.Grant T.J. and R.P. Higgins, 1957. Occurrence of mixturesof tristeza virus strains in citrus. Phytopathology 47,272–276.Hall T.A., 1999. BioEdit: a user-friendly biological sequencealignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposia, Ser. 41, 95–98. Kong P., Rubio, L., Polek, M. and B.W. Falk, 2000. Popula-tion structure and genetic diversity within CaliforniaCitrus tristeza virus (CTV) isolates. Virus Genes 21,139–145.Kumar S, K. Tamura, I.B. Jacobsen and M. Nei, 2001. Mega.Molecular Evolutionary Genetic Analysis Version 2.1.http://www.megasoftware.netNei M. and S. Kumar, 2000. Molecular Evolution and Phy-logenetics. Oxford University Press, New York, NY, USA,XX pp.Nolasco G., C. de Blas, V. Torres and F. Ponz, 1993. A meth-od combining immunocapture and PCR amplification6 Phytopathologia MediterraneaBouabid Lbida et al.in a microtiter plate for the routine diagnosis of plantviruses and subviral pathogens. Journal of VirologicalMethods 45,201–218.Rocha-Pena M.A., R.F. Lee, R. Lastra, C.L. Niblett, F.M.Ochoa-Corona, S.M. Garnsey and R.K. Yokomi, 1995.Citrus tristeza virus and its aphid vector Toxoptera cit-ricida: threats to citrus production in the Caribbean andCentral and North America. Plant Disease 79, 437–445Rubio L., M.A. Ayllon, J. Guerri, H. Pappu, C.L. Niblettand P. Moreno, 1996. Differentiation of citrus tristezaclosterovirus (CTV) isolates by single stranded confor-mation polymorphism analysis of the coat protein gene.Annals of Applied Biology 129, 479–489.Sambrook J., E.F Fritsh, and T. Maniatis, 1989. Molecularcloning. A Laboratory Manual. Cold Spring HarborLaboratory, Cold Spring Harbor, NY, USA, XX pp.Sequeira Z. and G. Nolasco, 2002. Bacterial expressed coatprotein: development of a single antiserum for routinedetection of Citrus Tristeza Virus. PhytopathologiaMediterranea 41, 55–-62.Accepted for publication: June 29, 2004Sheffield V.C., J.S. Beck, A.E. Kwitek, D.W. Sanstrom andE.M. Stone, 1993. The Sensitivity of single-strand con-formation polymorphism analysis for the detection ofsingle base substitutions. Genomics 16, 325–332.Targon M.L.P.N., M.A. Machado, G.W. Muller, H.D. Colet-ta Filho, K.L. Manjunath and R.F. Lee, 2000. Sequenceof coat protein gene of the severe Citrus tristeza viruscomplex Capão Bonito. Proceedings of the 14th Confer-ence of the International Organization of Citrus Virolo-gists (IOCV). IOCV, Riverside, CA, USA, 121–126.Zemzami M., S.M. Garnsey, E.B. Nadori and J.H. Hill, 1999.Biological and serological characterization of citrus tris-teza virus (CTV) isolates from Morocco. Phytopatholo-gia Mediterranea 35, 95–100.Zemzami M., C.M. Soares, A.M. Bailey, C.L. Niblett and G.Nolasco, 2002. Molecular characterization and classifi-cation of Morrocan isolates of Citrus tristeza clostero-virus. Proceedings of the 15th Conference of the Interna-tional Organization of Citrus Virologists (IOCV). IOCV,Riverside, CA, USA, 8–12.

Genomic variability of Citrus tristeza virus (CTV) isolates introduced into Morocco

Universidade do Algarve

1Phytopathol. Mediterr. (2004) 43, 00–00
Introduction
Citrus tristeza virus (genus Closterovirus, fam-
ily Closteroviridae, CTV) is the causal agent of the
most serious disease of Citrus spp. (Rocha-Pena et
al., 1995). The virus causes various disease types,
the most important of which are the quick decline
of trees grafted on sour orange rootstock; reduced
vigour; reduced tree quality and stem pitting of
Genomic variability of Citrus tristeza virus (CTV) isolates
introduced into Morocco.
BOUABID LBIDA1, FILOMENA FONSECA2, CARLA SANTOS2, MUSTAPHA ZEMZAMI3, AMINA BENNANI1
AND  GUSTAVO NOLASCO2
1Université My Ismail, Faculté des Sciences Meknès, PLEASE COMPLETE FULL MAIL ADDRESS,
Morocco
2Centro de Desenvolvimento de Ciencias e Técnicas de Produção vegetal, Universidade do Algarve – FERN,
Campus de Gambelas, 8005-139 Faro, Portugal
3Unité de controle des plantes, Direction des Domaines Agricoles, Km 11, Hussain, Salé 10.000, Morocco
Summary. Genomic variability of the coat protein gene of Citrus tristeza virus isolates obtained from old Meyer
lemon introductions in Morocco and more recent budwood introductions from Spain were studied. The coat protein
gene of the virus was amplified directly from infected tissue by immunocapture RT-PCR and analysed by single
stranded conformation polymorphism (SSCP) and sequencing. Each isolate consisted of several related genomic
variants, typical of a quasi-species. Although SSCP analysis has only limited typing ability it could be used in an
initial screening to discriminate between isolates of different origin and to analyse the genomic structure of each
isolate. Sequence analysis showed that the isolates of Spanish origin were closely related to mild isolates character-
ised in Florida and in Portugal. The Meyer lemon isolate on the other hand was related to severe strains of Meyer
lemon characterised in Florida some years ago and to other severe strains from Brasil. A knowledge of the coat
protein gene sequence is useful to trace the origin of the isolates.
Key words: Closterovirus, KEY WORDS DIFFERENT FROM TITLE, coat protein gene.
Corresponding author: G. Nolasco
Fax: XXXXXXXXXXXX
E-mail: gnolasco@ualg.pt
branches of trees grafted on tolerant rootstocks;
leaf yellowing and stop of growth of sour orange
and lemon seedlings. In each region affected the
particular disease syndrome depends on the kind
of CTV strains present. Strains may occur singly
or mixed (Grand and Higgins, 1957). A knowledge
of which strains are present in an area is funda-
mental to define a strategy to control the disease.
The virus is naturally transmitted in a semi-per-
sistent manner by several species of aphids, of
which Toxoptera citricida is the most effective; this
aphid does not yet occur in the Mediterranean ba-
sin. Budwood movement is responsible for the long
distance virus dissemination.
2 Phytopathologia Mediterranea
Bouabid Lbida et al.
The CTV genome consists of a single-stranded,
positive sense RNA molecule of 19,296 nucleotides,
encapsulated in flexuous filamentous particles
about 11 nm wide  2,000 nm long (Bar-joseph et
al., 1989). The virions contain two capsid proteins,
a major 25-kDa coat protein (CP) covering about
95% of the particle length and a minor 27-kDa cap-
sid protein (Febres et al., 1996), which covers only
one end of the particle.
In Morocco CTV has been detected and infected
trees have been eradicated on several occasions.
Some of the isolates were biologically and serolog-
ically characterised (Zemzami et al., 1999). A com-
parison between the biological data and hybridi-
sation patterns obtained with a panel of strain-dif-
ferentiating probes has been reported for some of
these isolates (Zemzami et al., 2002). In this work
we report on the further characterisation of CTV
by SSCP analysis and on the sequencing of the
major coat protein gene of additional isolates in-
troduced to Morocco a long time ago, and of others
collected recently at the Moroccan border.
Materials and methods
Virus isolates
The CTV isolates P2 and R1 were intercepted
by the Moroccan quarantine system as germplasm
coming from Spain, in 1998 and 2000 respectively.
Virus isolate P1 was detected in Lemon Meyer in a
field near Marrakesh in 1983. Isolates were prop-
agated by graft transmission to Mexican Lime (Cit-
rus aurantifolia) and maintained in an insect proof
greenhouse at 21–33°C. Additional biological char-
acterisation of these isolates was done on grape-
fruit, sweet orange, and mandarin grafted on sour
orange. Isolate MZ3 was obtained from Troyer cit-
range and corresponded to isolate number 3 previ-
ously characterised (Zemzami et al., 1999, 2002)
PCR amplification of the coat protein gene
Fresh bark and leaf tissue (0.2 g) were used as
the source of the virus. Amplification of the coat
protein gene was done by immunocapture reverse
transcriptional polymerase chain reaction (IC/RT-
PCR), using a one-step combined reverse transcrip-
tion and amplification based on methods previously
described (Nolasco et al., 1993). Polyclonal anti-
bodies against an E. coli expressed CP gene (Se-
queira and Nolasco, 2002) were used to trap the
virions. The whole CP gene and the stop codon were
amplified with primers CTV1 (5'-ATGGACGAC-
GAAACAAAGAA-3') and CTV10 (5'-ATCAACGT-
GTGTTGAATTTCC-3'). The PCR-products were
separated by migration on 1% agarose gel electro-
phoresis, and visualized under UV light after stain-
ing with ethidium bromide.
SSCP analysis of IC/RT-PCR or PCR product
SSCP analysis was performed directly on the
IC/RT-PCR products (from plant tissue) or on the
PCR products (from E. coli colonies), following con-
firmation of the specificity of the amplified prod-
uct. The amplified product (1 µl) was added to 9 µl
of denaturing buffer (950 µl of 95% formamide, 40
µl of 500 mM EDTA, [pH 8], and a minimal amount
of bromophenol blue), the sample was then heated
to 90°C for 5 min and quickly transferred to ice.
Denatured products were electrophoresed in the
refrigerator (4ºC) for 3 h at 200 V in a native 8%
polyacrylamide gel (78 cm, 0.75 mm, Bio-Rad
Mini-Protean II), using TBE as buffer. The gels
were stained with silver nitrate using the proce-
dure of Beidler et al. (1982) and dried.
Cloning and sequencing
The amplified RT-PCR product was TA-ligated
into pGEMT-Easy vector (Promega, Madison, WI,
USA), according to manufacturer’s instructions,
transformed into competent E. coli INVaF’ cells
(Invitrogen, Carlsbad, CA, USA) and plated accord-
ing to standard procedures (Sambrook et al., 1989).
Transformed colonies harbouring the recombinant
plasmid were selected by α-complementation on the
plates supplied with X-gal and ampicilin (Sam-
brook et al., 1989). The occurrence of the coat pro-
tein gene insert was confirmed by direct PCR of a
portion of selected white colonies, using the same
set of primers as above. This PCR product was in
some instances analyzed by SSCP.
Selected colonies harbouring the CP gene insert
were transferred to liquid medium and the respec-
tive plasmids purified using the WizardTM plus
miniprep DNA Purification System (Promega) ac-
cording to manufacturer’s instructions. An addi-
tional confirmation of the insert was done by re-
striction digestion of an aliquot of the plasmid with
Eco RI. Sequencing reactions were done using the
forward and reverse universal primers by Macro-
gen (Seoul, Korea).
3Vol. 43, No. 2, August 2004
Genomic  variability of CTV introduced in Morocco
Sequence analysis
Sequence analysis, alignment and clustering
were done using the software packages Bioedit
(Hall, 1999) and Mega 2 (Kumar et al., 2001). In
addition to the sequences obtained in this work,
the following sequences of the CP gene were re-
trieved from the GenBank and used in the com-
parisons: AF184118 (haplotype 28C), AF184115
(haplotype 25-120), AF184114 (haplotype 19-121),
AF184113 (haplotype 13C), M76485 (haplotype
T36). The CP gene sequences from Florida isolates
T30, T3 and Meyer Lemon FL-7 were kindly pro-
vided by C.L. Niblett. The sequence of haplotype
CB3-104 from the Capão Bonito isolate from Bras-
il was reported in Targon et al. (2000).
Results and discussion
Fragments of approximately 672 bp correspond-
ing to the expected size of the CP gene were ob-
tained by IC/RT-PCR (results not shown). A pre-
liminary assessment of the variability of the CP
gene was done by SSCP analysis of the amplified
fragments (Fig. 1). SSCP patterns consisted of two
or three conspicuous bands in addition to a faster
migrating band corresponding to the non-dena-
tured or partially renatured PCR product. Some of
the single-stranded bands were doublets that could
be better observed in an amplified picture. Sam-
ples of Spanish origin depicted a very similar pat-
tern, which differed from the Meyer lemon isolate
P1. This indicated that SSCP of the whole CP gene
might be useful in an initial discrimination of CTV
isolates without the need for smaller fragments as
suggested by Rubio et al. (1996), which would have
brought lengths closer to a putative optimum of
200 bp (Sheffield et al., 1993).
The IC/RT-PCR products were cloned and a
number of the transformed E. coli colonies were
analyzed by PCR using the same pair of primers.
Most of these PCR products were of the expected
size for the specific amplicon (672 bp) and were
analysed by SSCP (Fig. 2). Only two clones were
obtained from isolate MZ3; these were not analysed
Fig. 1. Single-stranded conformation patterns of IC/RT-
PCR fragments containing the coat protein gene. Bot-
tom bands on the left side panel correspond to ds-DNA.
Upper bands correspond to ss-DNA. The picture on the
right is a magnification of the ss-DNA region.
Fig. 2. Single-stranded conformation patterns of the
cloned coat protein gene from isolates P1, P2 and R1.
The lane number indicates the haplotype.
4 Phytopathologia Mediterranea
Bouabid Lbida et al.
by SSCP. Six clones were analysed from isolate P1
and eight each from isolates P2 and R1. Two, 2 and
3 SSCP patterns were obtained from P1, P2 and
R1 respectively; this polymorphism is typical for a
viral quasi-species. As in Kong et al. (2000), we
found a predominant pattern which occurred in
83% of clones from P1 and 75% of clones with a
Spanish origin. If each SSCP pattern is assumed
to correspond to a genomic variant (haplotype), the
distribution of the variants is characterised by
heterozygocity (Nei and Kumar, 2000). Heterozy-
gocity may take values ranging from 0, if there is
only one haplotype, to 1, if all the haplotypes are
different. The heterozygocity of P1 was 0.33, of R1,
0.43, and of P2, 0.46. The Spanish isolates appear
related in their composition of variants since their
major SSCP pattern was very similar.
The cloned IC/RT-PCR products giving the most
representative SSCP patterns of each isolate were
sequenced and the portions corresponding to the
primers were excised to eliminate any bias intro-
duced by primer sequence. The sequences were
aligned and compared with other available acces-
sions of diverse origin (Fig. 3), representative of
the seven group types reported by Zemzami et al.
(2002). The overall genetic diversity of these se-
quences (mean nucleotide distance between two
random selected variants) was 0.072 (SD 0.006).
This value increased to 0.081 (SD 0.007) when the
variants sequenced in this work were excluded. As
these new sequences did not contribute to increase
overall diversity, it was concluded that they repre-
sented true-to-type CTV CP gene variants.
The CP gene variants clustered very clearly, as
inferred from the high bootstrap values, in four
distinct groups. The haplotypes of Spanish origin
 MZ3-8
 MZ3-5
 13C
 28C
 T3
 P1-5
 19-121
 T36
 25-120
 T30
 R1-4
 P2-5
 R1-2
 P2-8
 P2-4
 Fl7
 P1-3
 CB3-104
100
86
100
99
99
100
99
83
0.01
Gr 3a
Gr 4
Gr 2
Gr 1
Gr M
Gr 3b
Gr 5
Fig. 3. Dendrogram showing the clustering pattern of the coat protein gene sequences as obtained by UPGMA.
Groups mentioned in Zemzami et al. (2002) are shown on the right.
5Vol. 43, No. 2, August 2004
Genomic  variability of CTV introduced in Morocco
differed by less than 1% at nucleotide level from
the well characterised isolate T30 (Garnsey et al.,
1987) originating in Florida, or from isolate 25-120
which, although characterised in Portugal (Bona-
calza, 1998), was detected in certified budwood
imported from Spain in 1996. These isolates were
representative of Group M. Isolates T30 and 25-
120 both produced mild vein clearing and mild or
no stem-pitting when indexed in Mexican lime; they
did not cause stem-pitting on sweet orange or
grapefruit, nor induce decline on sweet orange
grafted on sour orange. This behaviour coincides
with the biological data obtained in this work on
isolates P2 and R1.
On the other hand, the predominant genomic
variant of isolate P1 (clone P1-3) clustered at less
than 4% nucleotide distance to sequences obtained
from the severe isolates Capão Bonito from Brasil
and Fl-7 (Group 5). It is very interesting that iso-
late Fl7 was found on Meyer lemon-infected plants
from Florida (Cevik et al., 1996). The minor vari-
ant P1-5 clustered close to the severe isolate T3
(Group 4), also from Florida. The Meyer lemon iso-
lates from Florida produced severe vein clearing
and stem pitting on Mexican lime, decline of sweet
orange grafted on sour orange, and stem-pitting
on grapefruit; all of which are symptoms consist-
ent with the biological properties so far determined
for isolate P1. The introduction of the Meyer lem-
on isolate in Morocco can be traced back to the first
half of the 20th century and confirmation that it
caused CTV infection came in 1963 (Cassin, 1963).
However, twenty years later, infected Meyer lem-
on trees could still be found in the field, as it was
in this study. Isolate P1 was very similar to the
isolates from Florida and California, which have a
low transmission rate to other plants (Grant and
Higgins, 1957), and this may explain why the se-
vere isolate P1 remained hidden for so long.
The two clones from isolate MZ3 clustered in
group 3a, with a severe sweet orange stem pitting
isolate from Madeira island (Group 3a).
In a previous work (Zemzami et al., 2002) mo-
lecular data on the CP gene showed that severe
strains occurred in material from old budwood col-
lections in Morocco. These findings strongly sug-
gest the need for continuous monitoring of severe
strains of CTV before symptoms appear in the field.
This work also illustrates the importance of the
transport of infected budwood as causing a contin-
uous flow of exotic CTV strains to new areas. For a
certain period, the newly introduced strains ap-
peared to maintain their original biological and
molecular properties, while they remained hidden.
During this period the CP gene could be charac-
terised to trace their origins. Changes in cultural
practices or in the vector population may induce
changes in the spread and mixture of existing
strains, leading to disease outbreaks.
Literature cited
Bar-joseph M. and F.R. Lee, 1989. Citrus tristeza virus.
CMI/AAB Descriptions of Plant Viruses 353.
Beidler L., P.R. Hilliard and R.L. Rill, 1982. Ultrasensitive
staining of nucleic acids with silver. Analytical Biochem-
istry 126, 374–380.
Bonacalza B., 1998 (ed.). Tipificação molecular de isolamen-
tos do Virus da tristeza dos citrinos em Portugal. MSc
thesis, Instituto Superior de Agronomia, Universidade
Técnica de Lisboa, Lisboa, Portugal, 112 pp.
Cassin J., 1963. Découverte de 8 cas de tristeza parmi un
lot de plants âgés de citrus introduit au Maroc. Al-Awa-
mia 9, 53–77.
Cevik B., 1996. Molecular cloning and sequencing of coat
protein genes of citrus tristeza virus isolated from Meyer
Lemon and Homely Tangor Trees in Florida. Proceed-
ings of the 13th Conference of the International Organi-
zation of Citrus Virologists (IOCV). IOCV, Riverside, CA,
USA, 47–53.
Febres V.J., L. Ashoulin, M. Mawassi, A. Frank, M. Bar-
joseph, K.L. Manjunath, R.F. Lee and C.L. Niblett, 1996.
The P 27 protein is present at one end of citrus tristeza
virus particles. Phytopathology 86, 1331–1335.
Garnsey S.M., D.J. Gumpf, C.N Roistacher, E.L. Civerolo,
R.F Lee, R.K. Yokomi and M. Bar-Joseph, 1987. To-
ward a standardized evaluation of the biological prop-
erties of citrus tristeza virus. Phytophylactica 19,151–
157.
Grant T.J. and R.P. Higgins, 1957. Occurrence of mixtures
of tristeza virus strains in citrus. Phytopathology 47,
272–276.
Hall T.A., 1999. BioEdit: a user-friendly biological sequence
alignment editor and analysis program for Windows 95/
98/NT. Nucleic Acids Symposia, Ser. 41, 95–98.
 Kong P., Rubio, L., Polek, M. and B.W. Falk, 2000. Popula-
tion structure and genetic diversity within California
Citrus tristeza virus (CTV) isolates. Virus Genes 21,139–
145.
Kumar S, K. Tamura, I.B. Jacobsen and M. Nei, 2001. Mega.
Molecular Evolutionary Genetic Analysis Version 2.1.
http://www.megasoftware.net
Nei M. and S. Kumar, 2000. Molecular Evolution and Phy-
logenetics. Oxford University Press, New York, NY, USA,
XX pp.
Nolasco G., C. de Blas, V. Torres and F. Ponz, 1993. A meth-
od combining immunocapture and PCR amplification
6 Phytopathologia Mediterranea
Bouabid Lbida et al.
in a microtiter plate for the routine diagnosis of plant
viruses and subviral pathogens. Journal of Virological
Methods 45,201–218.
Rocha-Pena M.A., R.F. Lee, R. Lastra, C.L. Niblett, F.M.
Ochoa-Corona, S.M. Garnsey and R.K. Yokomi, 1995.
Citrus tristeza virus and its aphid vector Toxoptera cit-
ricida: threats to citrus production in the Caribbean and
Central and North America. Plant Disease 79, 437–445
Rubio L., M.A. Ayllon, J. Guerri, H. Pappu, C.L. Niblett
and P. Moreno, 1996. Differentiation of citrus tristeza
closterovirus (CTV) isolates by single stranded confor-
mation polymorphism analysis of the coat protein gene.
Annals of Applied Biology 129, 479–489.
Sambrook J., E.F Fritsh, and T. Maniatis, 1989. Molecular
cloning. A Laboratory Manual. Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY, USA, XX pp.
Sequeira Z. and G. Nolasco, 2002. Bacterial expressed coat
protein: development of a single antiserum for routine
detection of Citrus Tristeza Virus. Phytopathologia
Mediterranea 41, 55–-62.
Accepted for publication: June 29, 2004
Sheffield V.C., J.S. Beck, A.E. Kwitek, D.W. Sanstrom and
E.M. Stone, 1993. The Sensitivity of single-strand con-
formation polymorphism analysis for the detection of
single base substitutions. Genomics 16, 325–332.
Targon M.L.P.N., M.A. Machado, G.W. Muller, H.D. Colet-
ta Filho, K.L. Manjunath and R.F. Lee, 2000. Sequence
of coat protein gene of the severe Citrus tristeza virus
complex Capão Bonito. Proceedings of the 14th Confer-
ence of the International Organization of Citrus Virolo-
gists (IOCV). IOCV, Riverside, CA, USA, 121–126.
Zemzami M., S.M. Garnsey, E.B. Nadori and J.H. Hill, 1999.
Biological and serological characterization of citrus tris-
teza virus (CTV) isolates from Morocco. Phytopatholo-
gia Mediterranea 35, 95–100.
Zemzami M., C.M. Soares, A.M. Bailey, C.L. Niblett and G.
Nolasco, 2002. Molecular characterization and classifi-
cation of Morrocan isolates of Citrus tristeza clostero-
virus. Proceedings of the 15th Conference of the Interna-
tional Organization of Citrus Virologists (IOCV). IOCV,
Riverside, CA, USA, 8–12.


Genomic variability of the coat protein gene of Citrus tristeza virus isolates obtained from old Meyer
lemon introductions in Morocco and more recent budwood introductions from Spain were studied. The coat protein
gene of the virus was amplified directly from infected tissue by immunocapture RT-PCR and analysed by single
stranded conformation polymorphism (SSCP) and sequencing. Each isolate consisted of several related genomic
variants, typical of a quasi-species. Although SSCP analysis has only limited typing ability it could be used in an
initial screening to discriminate between isolates of different origin and to analyse the genomic structure of each
isolate. Sequence analysis showed that the isolates of Spanish origin were closely related to mild isolates characterised
in Florida and in Portugal. The Meyer lemon isolate on the other hand was related to severe strains of Meyer
lemon characterised in Florida some years ago and to other severe strains from Brasil. A knowledge of the coat
protein gene sequence is useful to trace the origin of the isolates

Lbida, Bouabid

Fonseca, F.

Nolasco, G.

Firenze University Press: E-Journals

205
Phytopathol. Mediterr. (2004) 43, 205–210
Introduction
Citrus tristeza virus (genus Closterovirus, fam-
ily Closteroviridae, CTV) is the causal agent of the
most serious disease of Citrus spp. (Rocha-Pena et
al., 1995). The virus causes various disease types,
the most important of which are the quick decline
of trees grafted on sour orange rootstock: reduced
vigour, reduced tree quality and stem pitting of
Genomic variability of Citrus tristeza virus (CTV) isolates
introduced into Morocco
BOUABID LBIDA1, FILOMENA FONSECA2, CARLA SANTOS2, MUSTAPHA ZEMZAMI3, AMINA BENNANI1
and  GUSTAVO NOLASCO2
1Université My Ismail, Faculté des Sciences, BP 4010 Beni Mhamed, Meknès, Morocco
2Centro de Desenvolvimento de Ciencias e Técnicas de Produção vegetal, Universidade do Algarve – FERN,
Campus de Gambelas, 8005-139 Faro, Portugal
3Unité de Controle des Plantes, Direction des Domaines Agricoles, Km 11, Hussain, Salé 10.000, Morocco
Summary. Genomic variability of the coat protein gene of Citrus tristeza virus isolates obtained from old Meyer
lemon introductions in Morocco and more recent budwood introductions from Spain were studied. The coat protein
gene of the virus was amplified directly from infected tissue by immunocapture RT-PCR and analysed by single
stranded conformation polymorphism (SSCP) and sequencing. Each isolate consisted of several related genomic
variants, typical of a quasi-species. Although SSCP analysis has only limited typing ability it could be used in an
initial screening to discriminate between isolates of different origin and to analyse the genomic structure of each
isolate. Sequence analysis showed that the isolates of Spanish origin were closely related to mild isolates character-
ised in Florida and in Portugal. The Meyer lemon isolate on the other hand was related to severe strains of Meyer
lemon characterised in Florida some years ago and to other severe strains from Brasil. A knowledge of the coat
protein gene sequence is useful to trace the origin of the isolates.
Key words: Closterovirus, heterozigocity, coat protein gene.
Corresponding author: G. Nolasco
Fax: +35 128 9818419
E-mail: gnolasco@ualg.pt
branches of trees grafted on tolerant rootstocks,
leaf yellowing and stop of growth of sour orange
and lemon seedlings. In each region affected the
particular disease syndrome depends on the kind
of CTV strains present. Strains may occur singly
or mixed (Grand and Higgins, 1957). A knowledge
of which strains are present in an area is funda-
mental to define a strategy to control the disease.
The virus is naturally transmitted in a semi-per-
sistent manner by several species of aphids, of
which Toxoptera citricida is the most effective; this
aphid does not yet occur in the Mediterranean ba-
sin. Budwood movement is responsible for the long
distance virus dissemination.
206 Phytopathologia Mediterranea
B. Lbida et al.
The CTV genome consists of a single-stranded,
positive sense RNA molecule of 19,296 nucleotides,
encapsulated in flexuous filamentous particles
about 11 nm wide  2,000 nm long (Bar-Joseph et
al., 1989). The virions contain two capsid proteins,
a major 25-kDa coat protein (CP) covering about
95% of the particle length and a minor 27-kDa cap-
sid protein (Febres et al., 1996), which covers only
one end of the particle.
In Morocco CTV has been detected and infected
trees have been eradicated on several occasions.
Some of the isolates were biologically and serolog-
ically characterised (Zemzami et al., 1999). A com-
parison between the biological data and hybridi-
sation patterns obtained with a panel of strain-dif-
ferentiating probes has been reported for some of
these isolates (Zemzami et al., 2002). In this work
we report on the further characterisation of CTV
by SSCP analysis and on the sequencing of the
major coat protein gene of additional isolates in-
troduced to Morocco a long time ago, and of others
collected recently at the Moroccan border.
Materials and methods
Virus isolates
The CTV isolates P2 and R1 were intercepted
by the Moroccan quarantine system as germplasm
coming from Spain, in 1998 and 2000 respectively.
Virus isolate P1 was detected in Meyer lemon in a
field near Marrakesh in 1983. Isolates were prop-
agated by graft transmission to Mexican Lime (Ci-
trus aurantifolia) and maintained in an insect proof
greenhouse at 21–33°C. Additional biological char-
acterisation of these isolates was done on grape-
fruit, sweet orange, and mandarin grafted on sour
orange. Isolate MZ3 was obtained from Troyer cit-
range and corresponded to isolate number 3 previ-
ously characterised (Zemzami et al., 1999, 2002)
PCR amplification of the coat protein gene
Fresh bark and leaf tissue (0.2 g) were used as
the source of the virus. Amplification of the coat
protein gene was done by immunocapture reverse
transcriptional polymerase chain reaction (IC/RT-
PCR), using a one-step combined reverse transcrip-
tion and amplification based on methods previously
described (Nolasco et al., 1993). Polyclonal anti-
bodies against an E. coli expressed CP gene (Se-
queira and Nolasco, 2002) were used to trap the
virions. The whole CP gene and the stop codon were
amplified with primers CTV1 (5'-ATGGACGAC-
GAAACAAAGAA-3') and CTV10 (5'-ATCAACGT-
GTGTTGAATTTCC-3'). The PCR-products were
separated by migration on 1% agarose gel electro-
phoresis, and visualized under UV light after stain-
ing with ethidium bromide.
SSCP analysis of IC/RT-PCR or PCR product
SSCP analysis was performed directly on the IC/
RT-PCR products (from plant tissue) or on the PCR
products (from E. coli colonies), following confirma-
tion of the specificity of the amplified product. The
amplified product (1 µl) was added to 9 µl of denatur-
ing buffer (950 µl of 95% formamide, 40 µl of 500 mM
EDTA, [pH 8], and a minimal amount of bromophe-
nol blue), the sample was then heated to 90°C for 5
min and quickly transferred to ice. Denatured prod-
ucts were electrophoresed in the refrigerator (4ºC)
for 3 h at 200 V in a native 8% polyacrylamide gel
(78 cm, 0.75 mm, Bio-Rad Mini-Protean II, Bio Rad
Laboratories, Hercules, CA, USA), using TBE as buff-
er. The gels were stained with silver nitrate using
the procedure of Beidler et al. (1982) and dried.
Cloning and sequencing
The amplified RT-PCR product was TA-ligated
into pGEMT-Easy vector (Promega, Madison, WI,
USA), according to manufacturer’s instructions,
transformed into competent E. coli INVaF’ cells
(Invitrogen, Carlsbad, CA, USA) and plated accord-
ing to standard procedures (Sambrook et al., 1989).
Transformed colonies harbouring the recombinant
plasmid were selected by α-complementation on the
plates supplied with X-gal and ampicilin (Sam-
brook et al., 1989). The occurrence of the coat pro-
tein gene insert was confirmed by direct PCR of a
portion of selected white colonies, using the same
set of primers as above. This PCR product was in
some instances analyzed by SSCP.
Selected colonies harbouring the CP gene insert
were transferred to liquid medium and the respec-
tive plasmids purified using the WizardTM plus min-
iprep DNA Purification System (Promega) accord-
ing to manufacturer’s instructions. An additional
confirmation of the insert was done by restriction
digestion of an aliquot of the plasmid with Eco RI.
Sequencing reactions were done using the forward
and reverse universal primers by Macrogen (Seoul,
Korea).
207Vol. 43, No. 2, August 2004
Genomic  variability of CTV introduced in Morocco
Sequence analysis
Sequence analysis, alignment and clustering
were done using the software packages Bioedit
(Hall, 1999) and Mega 2 (Kumar et al., 2001). In
addition to the sequences obtained in this work,
the following sequences of the CP gene were re-
trieved from the GenBank and used in the com-
parisons: AF184118 (haplotype 28C), AF184115
(haplotype 25-120), AF184114 (haplotype 19-121),
AF184113 (haplotype 13C), M76485 (haplotype
T36). The CP gene sequences from Florida isolates
T30, T3 and Meyer Lemon FL-7 were kindly pro-
vided by C.L. Niblett. The sequence of haplotype
CB3-104 from the Capão Bonito isolate from Bras-
il was reported in Targon et al. (2000).
Results and discussion
Fragments of approximately 672 bp correspond-
ing to the expected size of the CP gene were ob-
tained by IC/RT-PCR (results not shown). A pre-
liminary assessment of the variability of the CP
gene was done by SSCP analysis of the amplified
fragments (Fig. 1). SSCP patterns consisted of two
or three conspicuous bands in addition to a faster
migrating band corresponding to the non-dena-
tured or partially renatured PCR product. Some of
the single-stranded bands were doublets that could
be better observed in an amplified picture. Sam-
ples of Spanish origin depicted a very similar pat-
tern, which differed from the Meyer lemon isolate
P1. This indicated that SSCP of the whole CP gene
might be useful in an initial discrimination of CTV
isolates without the need for smaller fragments as
suggested by Rubio et al. (1996), which would have
brought lengths closer to a putative optimum of
200 bp (Sheffield et al., 1993).
The IC/RT-PCR products were cloned and a
number of the transformed E. coli colonies were
analyzed by PCR using the same pair of primers.
Most of these PCR products were of the expected
size for the specific amplicon (672 bp) and were
analysed by SSCP (Fig. 2). Only two clones were
obtained from isolate MZ3; these were not analysed
Fig. 1. Single-stranded conformation patterns of IC/RT-
PCR fragments containing the coat protein gene. Bot-
tom bands on the left side panel correspond to ds-DNA.
Upper bands correspond to ss-DNA. The picture on the
right is a magnification of the ss-DNA region.
Fig. 2. Single-stranded conformation patterns of the
cloned coat protein gene from isolates P1, P2 and R1.
The lane number indicates the haplotype.
208 Phytopathologia Mediterranea
B. Lbida et al.
by SSCP. Six clones were analysed from isolate P1
and eight each from isolates P2 and R1. Two, 2 and
3 SSCP patterns were obtained from P1, P2 and
R1 respectively; this polymorphism is typical for a
viral quasi-species. As in Kong et al. (2000), we
found a predominant pattern which occurred in
83% of clones from P1 and 75% of clones with a
Spanish origin. If each SSCP pattern is assumed
to correspond to a genomic variant (haplotype), the
distribution of the variants is characterised by
heterozygocity (Nei and Kumar, 2000). Heterozy-
gocity may take values ranging from 0, if there is
only one haplotype, to 1, if all the haplotypes are
different. The heterozygocity of P1 was 0.33, of R1,
0.43, and of P2, 0.46. The Spanish isolates appear
related in their composition of variants since their
major SSCP pattern was very similar.
The cloned IC/RT-PCR products giving the most
representative SSCP patterns of each isolate were
sequenced and the portions corresponding to the
primers were excised to eliminate any bias intro-
duced by primer sequence. The sequences were
aligned and compared with other available acces-
sions of diverse origin (Fig. 3), representative of
the seven group types reported by Zemzami et al.
(2002). The overall genetic diversity of these se-
quences (mean nucleotide distance between two
random selected variants) was 0.072 (SD 0.006).
This value increased to 0.081 (SD 0.007) when the
variants sequenced in this work were excluded. As
these new sequences did not contribute to increase
overall diversity, it was concluded that they repre-
sented true-to-type CTV CP gene variants.
The CP gene variants clustered very clearly, as
inferred from the high bootstrap values, in four
distinct groups. The haplotypes of Spanish origin
 MZ3-8
 MZ3-5
 13C
 28C
 T3
 P1-5
 19-121
 T36
 25-120
 T30
 R1-4
 P2-5
 R1-2
 P2-8
 P2-4
 Fl7
 P1-3
 CB3-104
100
86
100
99
99
100
99
83
0.01
Gr 3a
Gr 4
Gr 2
Gr 1
Gr M
Gr 3b
Gr 5
Fig. 3. Dendrogram showing the clustering pattern of the coat protein gene sequences as obtained by UPGMA.
Groups mentioned in Zemzami et al. (2002) are shown on the right.
r 3a
r 
209Vol. 43, No. 2, August 2004
Genomic  variability of CTV introduced in Morocco
differed by less than 1% at nucleotide level from
the well characterised isolate T30 (Garnsey et al.,
1987) originating in Florida, or from isolate 25-120
which, although characterised in Portugal (Bona-
calza, 1998), was detected in certified budwood
imported from Spain in 1996. These isolates were
representative of Group M. Isolates T30 and 25-
120 both produced mild vein clearing and mild or
no stem-pitting when indexed in Mexican lime; they
did not cause stem-pitting on sweet orange or
grapefruit, nor induce decline on sweet orange
grafted on sour orange. This behaviour coincides
with the biological data obtained in this work on
isolates P2 and R1.
On the other hand, the predominant genomic
variant of isolate P1 (clone P1-3) clustered at less
than 4% nucleotide distance to sequences obtained
from the severe isolates Capão Bonito from Brasil
and Fl-7 (Group 5). It is very interesting that iso-
late Fl7 was found on Meyer lemon-infected plants
from Florida (Cevik et al., 1996). The minor vari-
ant P1-5 clustered close to the severe isolate T3
(Group 4), also from Florida. The Meyer lemon iso-
lates from Florida produced severe vein clearing
and stem pitting on Mexican lime, decline of sweet
orange grafted on sour orange, and stem-pitting
on grapefruit; all of which are symptoms consist-
ent with the biological properties so far determined
for isolate P1. The introduction of the Meyer lem-
on isolate in Morocco can be traced back to the first
half of the 20th century and confirmation that it
caused CTV infection came in 1963 (Cassin, 1963).
However, twenty years later, infected Meyer lem-
on trees could still be found in the field, as it was
in this study. Isolate P1 was very similar to the
isolates from Florida and California, which have a
low transmission rate to other plants (Grant and
Higgins, 1957), and this may explain why the se-
vere isolate P1 remained hidden for so long.
The two clones from isolate MZ3 clustered in
group 3a, with a severe sweet orange stem pitting
isolate from Madeira island (Group 3a).
In a previous work (Zemzami et al., 2002) mo-
lecular data on the CP gene showed that severe
strains occurred in material from old budwood col-
lections in Morocco. These findings strongly sug-
gest the need for continuous monitoring of severe
strains of CTV before symptoms appear in the field.
This work also illustrates the importance of the
transport of infected budwood as causing a contin-
uous flow of exotic CTV strains to new areas. For a
certain period, the newly introduced strains ap-
peared to maintain their original biological and
molecular properties, while they remained hidden.
During this period the CP gene could be charac-
terised to trace their origins. Changes in cultural
practices or in the vector population may induce
changes in the spread and mixture of existing
strains, leading to disease outbreaks.
Literature cited
Bar-Joseph M. and F.R. Lee, 1989. Citrus tristeza virus.
CMI/AAB Descriptions of Plant Viruses 353.
Beidler L., P.R. Hilliard and R.L. Rill, 1982. Ultrasensitive
staining of nucleic acids with silver. Analytical Biochem-
istry 126, 374–380.
Bonacalza B., 1998 (ed.). Tipificação molecular de isolamen-
tos do Virus da tristeza dos citrinos em Portugal. MSc
thesis, Instituto Superior de Agronomia, Universidade
Técnica de Lisboa, Lisboa, Portugal, 112 pp.
Cassin J., 1963. Découverte de 8 cas de tristeza parmi un
lot de plants âgés de citrus introduit au Maroc. Al-Awa-
mia 9, 53–77.
Cevik B., S.S. Pappu, H.R. Pappu, D. Tight, D. Benscher,
S.H. Futch, P. Rucks, R.F. Lee and C.L. Niblett, 1996.
Molecular cloning and sequencing of coat protein genes
of citrus tristeza virus isolated from Meyer Lemon and
Homely Tangor Trees in Florida. Proceedings of the 13th
Conference of the International Organization of Citrus
Virologists (IOCV). IOCV, Riverside, CA, USA, 47–53.
Febres V.J., L. Ashoulin, M. Mawassi, A. Frank, M. Bar-
Joseph, K.L. Manjunath, R.F. Lee and C.L. Niblett,
1996. The P 27 protein is present at one end of citrus
tristeza virus particles. Phytopathology 86, 1331–1335.
Garnsey S.M., D.J. Gumpf, C.N. Roistacher, E.L. Civero-
lo, R.F. Lee, R.K. Yokomi and M. Bar-Joseph, 1987.
Toward a standardized evaluation of the biological
properties of citrus tristeza virus. Phytophylactica
19,151–157.
Grant T.J. and R.P. Higgins, 1957. Occurrence of mixtures
of tristeza virus strains in citrus. Phytopathology 47,
272–276.
Hall T.A., 1999. BioEdit: a user-friendly biological sequence
alignment editor and analysis program for Windows 95/
98/NT. Nucleic Acids Symposia, Ser. 41, 95–98.
Kong P., Rubio L., Polek M. and B.W. Falk, 2000. Popula-
tion structure and genetic diversity within California
Citrus tristeza virus (CTV) isolates. Virus Genes 21,139–
145.
Kumar S., K. Tamura, I.B. Jacobsen and M. Nei, 2001.
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Genomic Variability of Citrus tristeza virus (CTV) Isolates Introduced into Morocco

Bouabid Lbida

F. Fonseca

C. Santos

M. Zemzami

A. Bennani

G. Nolasco

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Genomic Variability of <em>Citrus tristeza virus</em> (CTV) Isolates Introduced into Morocco

Genomic variability of Citrus tristeza virus (CTV) isolates introduced into Morocco

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