at SciVerse ScienceDirect

Physiological and Molecular Plant Pathology 81 (2013) 45e53

Contents lists available

Physiological and Molecular Plant Pathology

journal homepage: www.elsevier .com/locate/pmpp

Biological characterization of Citrus tristeza virus monophyletic isolates withrespect to p25 gene

Katarina Han�cevi�c a,*, Silvija �Cerni b, Gustavo Nolasco c, Tomislav Radi�c a, Khaled Djelouah d,Dijana �Skori�c b

a Institute for Adriatic Crops and Karst Reclamation, Put Duilova 11, 21000 Split, Croatiab Faculty of Science, Department of Biology, University of Zagreb, Maruli�cev trg 9a, HR-10000 Zagreb, CroatiacCenter for Biodiversity Functional and Integrative Genomics, Universidade do Algarve, Campus de Gambelas, 8005e139 Faro, Portugald Istituto Agronomico Mediterraneo, Via Ceglie 09, I-70010 Valenzano (Ba), Italy

a r t i c l e i n f o

Article history:Accepted 27 October 2012

Keywords:Citrus tristeza virusIndicator plantsPathogenicitySymptoms

* Corresponding author. Fax: þ385 21 316584.E-mail address: katarina@krs.hr (K. Han�cevi�c).

0885-5765/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.pmpp.2012.10.005

a b s t r a c t

Citrus tristeza virus (CTV) is the most destructive viral pathogen of citrus and displays a high level ofgenetic and phenotypic diversity. In this study the biological characterization of monophyleticCTV-isolates based on p25 gene (Gp 1, Gp 2, Gp 3a, Gp 4, Gp 5, Gp M) was analyzed for the first time onthe set of standard indicator plants and unde r the same environmental conditions, in order to comparethe phenotypic characteristics of p25 genomic variants. The results showed that tested CTV-isolatesvaried in their ability to induced symptoms as well as in severity of symptoms e.g. pathogenicity.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Citrus tristeza virus (CTV, Closteroviridae) is the causal agent oftristeza, the most devastating citrus viral disease [1]. CTV mayinduce the development of three distinctive syndromes: quickdecline (QD), stem pitting (SP) and seedling yellows (SY). QD, ortristeza sensu stricto, is a decline disease of most citrus speciesgrafted on sour orange rootstock (Citrus aurantium L.) and in themost severe cases a tree may collapse in just a few weeks [1].Stem-pitting, stunting, reduced yield and poor fruit quality of sweetorange, grapefruit andmandarin, regardless of the rootstock are themain features of SP, whilst the leaf yellowing and stunting of sourorange, grapefruit (Citrus paradisi Macf.) or lemon (Citrus limon (L.)Burn. f.) characterize the SY syndrome [1].

CTV phenotype characterization by biological assay is usuallyperformed in plants from the family Rutaceae. The types of symp-toms expressed by individual indicator species are well known anddocumented [2]. A general indicator able to express all symptomtypes of tristeza disease, as well as to detect mild field isolates, isMexican lime (Citrus aurantifolia Swing.). Sweet orange (Citrussinensis (L.) Osbeck), sour orange, grapefruit, and sweet orange onsour orange rootstock are used for more specific CTV biologicalcharacterization. Symptoms developed on such indicators may vary

All rights reserved.

from mild to very distinct or severe ones [3,4]. Although thesymptom expression may be affected by environmental conditions,besides rootstock/scion combination, the major factor influencingthe disease outcome is the viral genotype [1].

Considerable effort has been made toward determining molec-ular markers that could be used to quickly identify CTV strains incorrelationwith their particular pathogenicity profile or phenotype[5e8]. Although there are more gene sequences used for CTVgenotype differentiation, the systematic investigation of capsidprotein (CP) gene sequence in relation to biological properties hasnot been reported. The three-kb-terminal region, which includesCP gene sequence also comprises other genes whose products havebeen proven tomodulate the host response to the infection throughpost-transcriptional gene silencing [9] and that are also the path-ogenicity determinants, e.g. p23 [10]. Phylogenetic analysis of p23sequence variants from a set of CTV isolates separated mild fromthe most severe CTV isolates [11].

As reported for other viruses, naturally occurring CTV isolatesexist mostly as populations of genomic variants and it is often foundthat a field isolate is a mix of genomic variants having differentphenotype [12e14]. However, in a number of reports the authors didnot fully characterize the array of CTV variants in the isolate [5,15,16].Consequently, contradictory conclusions regarding the relationshipbetween a particular sequence variant obtained from the infectingisolate and the observed symptoms could be easily drawn.

With the aim to investigate the possible relationship betweenphylogenetic and biological results, we based our study on

K. Han�cevi�c et al. / Physiological and Molecular Plant Pathology 81 (2013) 45e5346

biological characterization of individual monophyletic isolates withrespect to p25 gene (isolates whose genomic variants cluster intoone phylogenetic group [17]). The traditional method for differen-tiating CTV strains by biological indexing [2] was performed byinoculating a set of standard bioindicators with CTV isolates,representatives of different phylogenetic groups, previously char-acterized as monophyletic. Additionally, Satsuma mandarin (Citrusunshiu Mak. Marc.) grafted on Poncirus trifoliata L. Raf. rootstock,a combination generally believed to be less sensitive to tristeza, wasincluded in this study. Infected plants were examined for changesthat could be related to CTV-infection, thus making an attempt toassociate different monophyletic CTV isolates with their particularbiological characteristics.

2. Materials and methods

2.1. Virus source material

CTV-isolates used in this study were chosen from the accessionscollected at the Mediterranean Agronomic Institute in Valenzano/Bari (MAIB, Italy) and at the University of Algarve (Faro, Portugal),established by long-term phytosanitary survey and selection oftristeza isolates in the Mediterranean region (Table 1). They wererepresentatives of six phylogenetic groups [17]. As a sourcematerialfor the inoculation, CTV-infected buds of Madam Vinous sweetorange seedlings were maintained in the greenhouse of MAIB.

2.2. Molecular characterization of CTV isolates

2.2.1. RNA extraction and RT-PCR amplificationThe isolation of total RNA from infected young bark was done by

using RNeasy Plant Mini Kit (Qiagen, Germany). Reverse Tran-scription Polymerase Chain Reaction (RT-PCR) was performed usingprimers corresponding to both ends of CTV-CP gene [15]. Onemicroliter of RNA extracts was added to 50 mL of the reactionmixture containing 10 mM Tris, 50 mM KCl, 4 mM MgCl2, 0.2 mMdNTPs, 200 nM primers, 3 U of RNase inhibitor (Applied Bio-systems, USA) and 1 U of Ampli Taq DNA polymerase (AppliedBiosystems, USA). Reverse transcriptionwas performed at 38 �C for45 min and the denaturation step at 94 �C for 2 min. Amplificationof cDNA took place according to the following procedure: incuba-tions at 92 �C for 30 s, 52 �C for 30 s, 72 �C for 30 s (30 cycles), andfinal extension at 72 �C for 20 min. The presence of amplificationproducts (672 bp) was visualized in UV-light after horizontal 1%agarose gel electrophoresis and ethidium-bromide staining [19].The standard for determining the DNA molecular weight wasMarker 8 (Fermentas, Lithuania).

2.2.2. SSCP analysisTo separate different genomic variants from the virus isolate

populations, CP-amplicons were cloned into pTZ57 R/T vector(Fermentas, Lithuania) according to the manufacturer’s instruc-tions. The transformation of competent E. coli INVaF’ cells (Invi-trogen, Germany) was performed according to the standard

Table 1Description of Citrus tristeza virus isolates used for the graft-inoculation of standardCitrus indicators and Satsuma mandarin (C. unshiu Mak. Marc.) plants.

Virus isolate code and origina Q3b 440c 8c Q57b 2c 176c

Phylogenetic group 1 2 3a 4 5 MCountry of origin Albania Croatia Portugal Egypt Portugal Spain

a The accession collection of the grafting material.b Mediterranean Agronomic Institute Valenzano/Bari (MAIB), Italy.c University of Algarve, Faro, Portugal.

procedure [19]. Transformed colonies were selected by a-comple-mentation, and the presence of the insert was confirmed by PCRusing the same primers and PCR reaction conditions as above.

Different CTV variants were identified by the SSCP (Single-StrandConformation Polymorphism) analysis [20] using PCR products from20 clones per sample. All products displaying different SSCPpatterns were considered different genomic variants [21] andplasmids harboring selected variants were purified using Pure-LinkTM Quick Plasmid Miniprep Kit (Invitrogen, Germany).

At least three representatives of each DNA profile weresequenced in both directions (Macrogen Inc, South Korea) using theM13-pUC universal primer pair. Sequences were aligned usingClustalX 1.8 [22] and analyzed by MEGA version 5 [23] using theneighbor-joining method and applying Tamura-Nei evolutionarymodel. The tree topologywas evaluated by bootstrap analysis basedon 1000 repetitions. All different CP-sequences obtained in thiswork were submitted to the GenBank under accession numbers:JQ655276eJQ655293.

2.3. Inoculation of standard indicators and Satsuma mandarinplants

Mexican lime, Madam Vinous sweet orange, Sicilian sour orangeand CRC grapefruit plants were germinated from seeds in plasticbaskets filled with substrate (Brill Citrus fruit, Geo Tec, Italy) andtwenty days after sowing seedlings were transplanted into peatcontainers (Jiffy Products, Netherlands). When the seedlingstrengthened, the plants with “Jiffy” pots were transferred to a 7 Lvolume plastic containers previously filled with sterilized mixtureof 1/3 peat, 1/3 terra rossa and 1/3 agroperlit. Plants were cultivatedin an insect-proof screen house 18 months prior to graft inocula-tion. One group of sweet orange seedlings was used for graftingsour orange in order to obtain sweet on sour orange indicatorplants. The buds of Satsuma mandarin plants (‘Kawano Wase’,‘Miho’ and ‘Kuno’) were grafted on P. trifoliata young seedlings forlater use in biological tests, where these three cultivars wereevaluated as one single Satsuma test. All plants were treated withN:P:K fertilizer (18:18:18; once a week during the autumn andspring, and once in two weeks in the summer) and urea (onceduring the summer and once in two weeks in the spring fertilizer).To prevent insect and mite infestations plants were sprayed asneeded with Confidor SL200 (BayerCropScience) e Phyllocnistiscitrella Vertimec 1.8EC (Syngenta) e Panonychus ulmi Ortus 5SC(Herbos) e Panonychus ulmi.

Inoculations with CTV infected material from source plantswere done in October with 2e3 bark and bud chips per seedling [2].After inoculation, plants were cut back and just one shoot was leftto grow. Each virus isolate was inoculated on six plants of followingindicators: limes, sweet and sour oranges, grapefruits, Satsumamandarins, and sweet orange plants grafted on sour orangerootstock. Six plants of each indicator species were used asnon-inoculated controls. Plants were maintained in shadedgreenhouse with the average temperature range of 20e26 �C(maximum up to 35 �C during the summer months, and 16 �Cduring the winter months).

2.4. DTBIA

All plants were tested twice for the virus presence using DTBIA(Direct Tissue Blott Immunoassay), three and five weeks post inoc-ulation (p. i.).

DTBIA analysis were performed on tissue imprints from youngspring shoots. Selected tissue was blotted on nitrocellulosemembranes (BioRad, USA) and virus detection was carried out byusing polyclonal antibodies following the manufacturers protocol

K. Han�cevi�c et al. / Physiological and Molecular Plant Pathology 81 (2013) 45e53 47

(Agritest, Italy). All imprints were checked for stained areas underthe same stereomicroscope always using the same magnification(20�) indicating the presence of virus particles.

2.5. Evaluation of symptoms

The occurrence and intensity of different symptoms (retardedgrowth, internode shortening, yellowing, vein clearing and leafcupping) were continuously monitored on every indicator plantevery week throughout 7 months. Only one lateral shoot was left togrow and it served for all the measurements, while others wereremoved. Stem-pitting was inspected twice: after seven months onlateral shoot and eighteen months p. i. on main stem. Retardedgrowth was evaluated by measuring the “growth dynamics”, whichdescribes average weekly shoot elongation, while “internodelength dynamics” describes averageweekly changes in shoot lengthdivided by the number of internodes.

For categorizing the symptom intensity, an arbitrary scale from0 to 3, based on the visual assessment of symptom intensity, wasadopted [4]. Zero signifies the absence of symptoms, 1 e thepresence of mild, 2 e moderate, and 3 e severe symptoms (Fig. 1)The intensity of the stem-pitting symptoms was categorized in thesamemanner (scale 0e3), based on the number of pits on the 10 cmlong woody stem (1: 1e10 pits; 1.5: 11e25 pits; 2: 26e50 pits; 2.5:51e100 pits; 3: more than 100 pits). For a detailed monitoring of

Fig. 1. Typical symptoms of Citrus tristeza virus (CTV) infection in different indicators. a) Sb) moderate stem pitting in sweet orange main stem with numerous well defined pits, c) mpitting in lateral branch of Satsuma mandarin, e) severe seedling yellows (SY) symptoms ing) moderate seedlings yellows in sweet orange, h) moderate leaf cupping in Mexican lime, iwith severe vein clearing.

the stem-pitting symptoms, stems were autoclaved and peeled, sothat the changes in woody part of the plant could be clearlydescribed. Representative examples of how the reading were madeare shown in Fig. 1.

In order to associate virus isolate with specific symptoms,diagrams describing separate biological activity for each isolate arepresented. For that purpose, numerical values of shoot growth andinternode length dynamics were classified from 0 to 3 on the basisof differences between the control and inoculated plants only forthe cases that were significantly different. The median of five mostprominent symptoms’ intensities was used to express the maximalobserved pathogenicity for each CTV isolate.

The data obtained from symptom evaluation were comparedusing ANOVA (P � 0.05; Bonferroni post-hoc test), separately foreach virus isolate. Statistical analysis was carried out using Statview(SAS software package, Version 5.0) and Systat (10.2).

3. Results

3.1. Molecular characterization of CTV-isolates

DTBIA results were corroborated by RT-PCR confirming thepresence of CTV in all Madam Vinous inoculum source plants. TheSSCP analysis of separated CP sequence variants (not shown)confirmed the coexistence of different genomic variants in the

evere stem pitting in Mexican lime main stem with numerous pits, often coalesced,ild stem pitting in grapefruit main stem with scattered discrete pits, d) severe stem-grapefruit, f) severe seedlings yellows in sour orange (with reduction in leaf size and),) severe vein clearing and leaf cupping in Mexican lime, j) portion of Mexican lime leaf

K. Han�cevi�c et al. / Physiological and Molecular Plant Pathology 81 (2013) 45e5348

majority of isolates (Fig. 2). All isolates had a population structureconsisting of one predominant SSCP variant (haplotype) whosefrequency ranged from 58 to 100%. The largest number of genomicvariants found was four, within the population structure of isolates440 (Gp2), while isolates 2 (Gp5) and 57 (Gp4) were the mosthomogeneous with a 100% of one genomic variant frequency(Fig. 2). Despite different haplotypes found in the isolates, thephylogenetic relationship analysis of the obtained sequencesproved that all isolates were monophyletic with respect to CP gene,yet showed a clear categorization of CP variants into six welldefined clusters (Fig. 3). The classification scheme of CP sequencesis in accordance with the one published [17]. Main virus groupbranches were supported by high bootstrap values (Fig. 3).

Fig. 2. SSCP-analysis of CP-gene belonging to CTV phylogenetic group (Gp): a) 1, b) 2, c) 3aLetters indicate haplotypes. * Sequenced samples.

3.2. Biological characterization

One month p. i., plants began to produce new shoots. All inocu-lated plants continued to grow in the following months (October,November). In winter time, due to the season conditions (short daysand low temperatures), the plants entered the resting phase inwhichmost symptoms of tristeza disease were not evident. Vegetativegrowth continued in late winter or early spring (February, March).

3.2.1. Retarded growth and internode shorteningStatistically significant differences in shoot growth dynamics

(Table 2) and internode length (Table 3) were observed betweencontrol (uninoculated) plants and those inoculated with CTV

, d) 4, e) 5 and f) M. Transformed bacterial cells are presented with different numbers.

Fig. 3. Neighbor-joining phylogenetic tree obtained by the analysis of CTV coat proteinsequences of isolates Q3, 440, 8, Q57, 2 and 176 belonging to phylogenetic groups Gp 1,Gp 2, Gp 3a, Gp 4, Gp 5, and Gp M, respectively. The bar represents 0.01 nucleotidesubstitutions per site. Reference sequences included in the analysis are in bold. Clus-ters are designated in accordance with the phylogenetic groups of Nolasco et al. (2009).

K. Han�cevi�c et al. / Physiological and Molecular Plant Pathology 81 (2013) 45e53 49

isolates, as well as among some indicator plants of the same typeinoculated with different CTV isolates.

Sour orange and grapefruit displayed reactions of retardedgrowth for the greatest number of tested isolates, calculated fromthe number of statistically different values in relation to the controlones (Table 2). The isolates which affected the growth dynamics ofthe most citrus species tested were those from groups Gp 2 and GpM (Table 2). Average internodes length in almost all plantsdecreased during March and/or April, which coincides with thebeginning of vegetative plant growth. After this period, internodes

elongation began (not shown). The most intense reaction of inter-nodes shortening was observed in sweet orange and Satsumamandarin plants (Fig. 5). Gp 1, Gp 2 and Gp M isolates hada significant impact on the average internodes length of the mostcitrus species tested (Table 3, Fig. 5).

3.2.2. Leaf cupping, vein clearing and yellowing symptomsDuring the experiment, the symptom of leaf cupping in Mexican

lime was recorded in two periods, in winter on old leaves, and inspring on the young ones. Symptoms proved to be very variableregarding the intensity of reaction, depending on the isolates(Fig. 4). Besides Mexican lime plants, this symptom was alsoobserved in some plants of Satsuma mandarin.

The symptoms of vein clearing in Mexican lime began todevelop five months p. i. with the highest intensity reached inmid-April, seven months p. i. (not shown). The average intensity ofvein clearing symptom in lime plants is positively correlated to theaverage intensity of leaf cupping symptom with regard to virusisolate (Fig. 4). The greatest average intensity of both symptomswas reached in the plants inoculated with Gp 1 and Gp 2 isolates(Fig. 4). Gp 1 had an effect on appearance of yellowing symptoms insour orange, sweet orange and sweet on sour orange plants, as wellas Gp 2 in Satsuma mandarin. Gp 3a induced yellowing in grape-fruit plants. Some plants of sweet orange also developed veinclearing symptom. Foliar symptoms on indicators other than limeplants appeared sporadically, so only leaf cupping and vein clearingsymptoms in Mexican lime plants are here presented. Followingthe temperature rise in the greenhouse, the intensity of these twosymptoms in early May began to diminish.

3.2.3. Stem-pittingThe first SP recording was performed seven months p. i. on one

piece of stem (10 cm long), and the second one 18 months p. i. on3e5 stems per each plant. Mexican lime and grapefruit plantsreacted with most of the isolates tested (Table 4). Gp 4 isolatedeveloped the most prominent SP-symptom in most indicatorstested, followed by Gp 3a and Gp 2, respectively. Gp 5 and Gp Misolates were not found to cause pits (Table 4, Fig. 5) in any of theindicator species. No development of SP symptoms was observedon uninoculated indicator control plants, as expected.

Statistically significant differences in symptoms observedbetween control and indicator plants inoculated with differentmonophyletic isolate are summarized in Fig. 5.

4. Discussion

Molecular characterization of CTV isolates revealed the genomicdiversity within and between virus isolates to which certainpathogenicity (phenotype, biological characteristics) may beascribed [5,10,15,16,24,25]. These differences in CTV phenotypemay be manifested as rapid plant decline, stem-pitting and seed-ling yellows. However, the association of particular CTV genotypeand its pathogenicity was not always unequivocal. The pathoge-nicity of the virus depends on the population structure of viralRNAs present in an isolate and it is common for CTV hosts to beinfected with different CTV-genotypes, which may have differentphenotypes [12e15,18,21].

The representatives of six phylogenetic groups based on p25,were inoculated on a set of standard Citrus indicators (Mexicanlime, Sicilian sour orange, Madam Vinous sweet orange, sourorange on sweet orange, CRC grapefruit) and Satsuma mandaringrowing in the same environmental conditions. The differences insymptoms of retarded growth, internode shortening, stem-pittingand foliar changes were monitored and evaluated for each isolatein order to compare CTV pathogenicity with virus genotype.

Table 2Average shoot length and standard deviation (in cm) of control plants versus indicator and Satsuma mandarin (C. unshiuMak. Marc.) plants infected with monophyletic Citrustristeza virus isolates belonging to six phylogenetic groups.

Phylogenetic group/indicator plant Mexican lime Sour orange Sweet orange Sweet on sour orange Grapefruit Satsuma mandarin

Control 18.2 � 7.4a 20.5 � 7.4a 31.5 � 11.0bc 15.2 � 6.7 ab 28.1 � 16.7a 9.6 � 5.4aGp 1 10.2 � 4.8*b 10.4 � 3.8*bc 23.9 � 9.1c 10.0 � 5.3bc 28.3 � 10.5a 7.8 � 3.7aGp 2 15.1 � 6.9 ab 9.3 � 3.1*c 44.9 � 17.0*a 13.7 � 8.8 ab 14.8 � 3.0*b 3.3 � 0.4*bGp 3a 15.8 � 6.2a 8.8 � 3.0*cd 34.1 � 11.8bc 12.7 � 6.2 ab 14.9 � 5.1*b 9.6 � 5.3 aGp 4 17.4 � 4.2a 5.8 � 1.0*d 34.3 � 12.1b 16.9 � 6.7a 29.1 � 8.6a 8.7 � 1.9aGp 5 14.8 � 4.8 ab 5.5 � 2.1*d 35.4 � 12.3 ab 17.0 � 7.6a 17.8 � 7.3*b 9.1 � 1.9aGp M 16.5 � 6.0 ab 12.8 � 4.5*b 26.2 � 6.2bc 6.4 � 4.5*c 19.3 � 8.8*b 6.6 � 3.1a

*P-value indicates significant difference in shoot growth according to Fisher test (P � 0.05).a, b, c, d e Bonferroni post-hoc test at the significance level P � 0.05. Different letters indicate statistically significant difference in shoot growth between the same indicatorplants infected with six monophyletic isolates (in column).

K. Han�cevi�c et al. / Physiological and Molecular Plant Pathology 81 (2013) 45e5350

Changes in inoculated lime plants were used to initially separatemild from severe CTV isolates. Plants were inspected for veinclearing, leaf cupping, SP and growth changes, which, if observedindicated the presence of severe CTV isolate, especially if SP wasone of the symptom detected. Particular significance was given tofoliar symptoms developed in old leaves, suggesting severephenotype of tested CTV isolate [2,4].

SP potential of isolates was assessed in seedlings of MadamVinous sweet orange [2,4]. Sweet orange plants were additionallyobserved for vein clearing of young leaves indicative of severe SPisolates [2]. For severe SP isolates, woody stems of sour orangewereexamined [2]. SP in seedlings of grapefruit was used to detect SPsyndrome and measure the ability of isolates to cause SP ingrapefruit, but also served as an additional indicator for SY in caseof retarded growth, internode shortening and/or foliar changesappearance [4,26].

Seedlings of sour orange were primarily used to detect the SYsyndrome induced by certain CTV isolates [2,4,27]. Retardedgrowth, internode and foliar changes, namely yellowing, indicatethe presence and severity of SY [2]. SY-pathogenic potential in themost severe form can cause fully retarded plant growth of sourorange, followed by chlorotic changes in leaves and their reducedsurface [28].

Plants composed of sweet orange scions grafted on sourorange seedling rootstocks were used to evaluate the ability ofisolates to cause retarded growth, internode shortening andfoliar changes that reflect a CTV-induced injury to the phloem atthe bud union which is associated with typical tristeza declinesyndrome [29].

4.1. Phylogenetic group 1

Gp 1 had the most significant effect on the overall growthdynamics (Table 2) and internode length (Table 3) of Mexican limeplants. Average symptom intensity of vein clearing and leaf cupping(Fig. 4) and observed pathogenicity (Fig. 5) of these symptomshad maximum values (intensities) when compared to other

Table 3Average internode length (in cm) with standard deviation in control plants versus inmonophyletic Citrus tristeza virus isolates belonging to six phylogenetic groups.

Phylogenetic group/indicator plant Mexican lime Sour orange Swee

Control 1.2 � 0.1 ab 1.5 � 0.1a 2.5 �Gp 1 1.1 � 0.2*c 0.9 � 0.4*b 1.5 �Gp 2 1.1 � 0.2*d 1.2 � 0.5a 2.3 �Gp 3a 1.2 � 0.2b 1.1 � 0.6*b 2.1 �Gp 4 1.2 � 0.3a 1.5 � 0.4a 1.9 �Gp 5 1.3 � 0.3a 1.1 � 0.6*b 1.9 �Gp M 1.1 � 0.2*c 1.6 � 3.1a 1.9 �

*P-value indicates significant difference in internode growth according to Fisher test (Pa, b, c, d e Bonferroni post-hoc test at the significance level P � 0.05. Different letters insame indicator plants infected with six monophyletic isolates (in column).

monophyletic groups. Intensive foliar symptoms not only observedin young but also in old leaves of Mexican lime suggest that Gp 1phylogenetic group clusters with severe CTV isolates. Retardedgrowth (Table 2), internode shortening (Table 3) and leaf yellowingin sour orange plants confirmed the SY pathogenic potential oftested isolate. Gp1 isolates encompass T36-CP-haplotypes [17]characterized by the development of dramatic forms of tristeza[30]. There was a severe deterioration of individual branches ofsweet on sour orange plants and sweet orange, leaf chlorosis andpartial defoliation, which were also accompanied with symptomsof internode shortening, consistent with QD syndrome [29]. In ourexperiment, sweet on sour orange and sweet orange plants werenot completely lost. We suppose that the short period of plantdormancy during the winter time, because of the growing ingreenhouse conditions, favored the regeneration of phloem tissue,so plants eventually recovered. Overall, the pathogenicity profile ofCTV Q3 isolate representing Gp1 in this study is in accordance withQD potential as previously suggested for this lineage of isolates[7,17].

4.2. Phylogenetic group 2

The greatest average intensity of vein clearing and leaf cuppingsymptoms, besides Gp 1, was obtained in Mexican lime plantsinoculated with Gp 2 isolate (Fig. 4). Sour and sweet orange, as wellas grapefruit inoculated with Gp 2 representative developedsymptoms of retarded growth (Table 2) with the maximal intensityof this symptom expressed in sour orange plants. Grapefruit and allother tested indicators except sweet orange plants developed SPafter the first evaluation of this symptom (Table 4) which makesthis Gp 2 representative an SP inducing isolate. The lack of pits insweet orange plants inoculated with monophyletic Gp 2 isolateswas previously reported [25], and that was also the case in ourstudy. It was surprising that the SP intensity decreased after 18months in all indicators that developed SP, although the second SPevaluation was done mostly on main stem which is older thanlateral shoot used for the first SP measurement.

dicator and Satsuma mandarin (C. unshiu Mak. Marc.) plants after infection with

t orange Sweet on sour orange Grapefruit Satsuma mandarin

0.2a 1.5 � 0.2bc 1.7 � 0.2bc 1.7 � 0.2a0.7*e 1.2 � 0.5*d 2.0 � 0.3a 1.4 � 0.4b0.5*b 1.3 � 0.4*d 1.5 � 0.2c 0.7 � 0.4*e0.5*c 1.5 � 0.4c 1.5 � 0.6c 1.3 � 0.8*bc0.6*d 1.5 � 0.2c 2.3 � 0.2a 1.1 � 1.0*cd0.8*d 1.6 � 0.2a 1.5 � 1.7c 1.8 � 0.3a0.4*d 1.1 � 0.4*e 1.8 � 0.5b 1.0 � 0.9*d

� 0.05).dicate statistically significant difference in internode length dynamics between the

0

0,5

1

1,5

2

0 Gp1 Gp2 Gp3a Gp4 Gp5 GpMvirus isolate

av

era

ge

s

ym

pto

m

in

te

ns

ity

(0

-3

)

c

aab

b b

c c

a

0

0,5

1

1,5

2

0 Gp1 Gp2 Gp3a Gp4 Gp5 GpMvirus isolate

av

era

ge

s

ym

pto

m

in

te

ns

ity

(0

-3

)

c

ab

b b

cc

bVein clearing on Mexican lime plants induced by Leaf cupping on Mexican lime plants induced by

different virus isolates different virus isolates

Fig. 4. Average values and standard deviation of vein clearing (a) and leaf cupping symptoms (b) on control Mexican lime plants (0) and Mexican lime plants after infection with sixmonophyletic Citrus tristeza virus isolates from phylogenetic groups Gp 1, Gp 2, Gp 3a, Gp 4, Gp 5 and Gp M. a, b, c e Bonferroni post-hoc test at the significance level P � 0.05.Different letters indicate statisticaly significant difference in observed symptoms between control and infected Mexican lime plants.

K. Han�cevi�c et al. / Physiological and Molecular Plant Pathology 81 (2013) 45e53 51

So far, it has been difficult to correlate CTV variants from thisgroup with characteristic symptoms unequivocally, althoughreports for mild phenotypes [16,17] but also severe SP [5] weregiven for some mix isolates having variants from this group. Ourresults confirm the SP inducing syndrome of GP 2 isolates,assigning this isolate to the severe group of CTV isolates.

4.3. Phylogenetic group 3a

In this study Gp 3a, represented by virus isolate 8 induced SPsymptoms after 18 months on every tested standard indicatorexcept sour orange (Table 4). The appearance of stem pitting insweet orange in our study supports previous classification of Gp 3aisolates [17] among severe SP phenotypes. Other authors also

Fig. 5. Pathogenicity (symptom type and intensity) of: a (Gp 1 isolate), b (Gp 2 isolate), cexpressed on standard Citrus indicator hosts and Satsuma mandarin (C. unshiu Mak. Marc.)orange; SwS e sweet on sour orange; GR e grapefruit; SAT e Satsuma mandarin. Types of shigher values of two measurements are presented); RG e retarded growth; RI e reduced inteprominent symptoms’ measurements.

support the correlation between Gp 3a variants and SP syndrome[25,31,32]. The reduced growth and shortened internodes devel-oped in sour orange, additionally supported by observed leafyellowing and maximal intensity of retarded growth symptomexpressed in grapefruit plants, grouped this isolate as SY isolate aswell (Tables 2 and 3).

4.4. Phylogenetic group 4

The most severe SP symptoms on branches of Mexican lime,sweet orange, sweet on sour orange and grapefruit (Table 4; Fig. 5d)in our biological experiments were caused by Gp 4 monophyleticisolate Q57. The general reaction of Mexican lime to Gp 4 isolatewas very intense (Fig. 5d), as in some previous reports [4]. Leaf

(Gp 3a isolate), d (Gp 4 isolate), e (Gp 5) and f (Gp M) isolate of Citrus tristeza virusplants. Host are abbreviated as: ML e Mexican lime; SwO e sweet orange; SO e sourymptoms abbreviations: LC e leaf cupping; VC e vein clearing; SP e stem-pitting (thernodes. Estimated symptom intensity (0e3) is calculated from the median of five most

Table 4Evaluation of stem-pitting symptoms intensity based on number of pits seven and18 months post inoculation separated by a slash.

Phylogeneticgroup/indicatorplant

Mexicanlime

Sourorange

Sweetorange

Sweet onsour orange

Grapefruit Satsumamandarin

Gp 1 0/1.5 0/0 0/0 0/0 1/0 0/0Gp 2 2.5/1.5 1.25/0 0/0 0.75/0.5 1.5/0 0/0Gp 3a 1.5/2.5 0/0 0/1.5 0.75/1 1/1 0/0.5Gp 4 2.75/3 0/0 1.5/3 1.25/2.5 1.5/1 1.5/1.5Gp 5 0/0 0/0 0/0 0/0 0/0 0/0Gp M 0/0 0/0 0/0 0/0 0/0 0/0

K. Han�cevi�c et al. / Physiological and Molecular Plant Pathology 81 (2013) 45e5352

cupping was particularly discernable symptom (Fig. 4b) developedalso on the old leaves, and appeared earlier than in plants infectedwith other virus isolates.

No other CTV isolate caused SP on sweet orange within onlysevenmonths after inoculation. Also, the shape of pits differed fromthose caused by other investigated CTV isolates. Pits were frequent,deep and protracted therefore; the peeled branches appearedmoredamaged than the ones infected by other SP isolates (Fig. 5). Gp 4variant demonstrates phenotypic characteristics of tristeza T3-likegenotype, associated with SP-symptoms in sweet orange [4,7,16],what was confirmed for monophyletic isolate in our study. In ourexperiments, sweet orange plants developed leaf symptoms of mildvein clearing, while the leaves had a significantly changed shape(not presented), what besides developed SP in all relevant indica-tors confirm its affiliation to severe SP isolate [2].

4.5. Phylogenetic group 5

In the majority of indicators inoculated with virus isolate 2 fromGp 5, the virus has caused the occurrence of mild, barely noticeablesymptoms (Fig. 5e). Plants of sour orange developed symptoms ofretarded growth and internode shortening, while grapefruit seed-ling developed retarded growth (Tables 2 and 3). No reaction inMexican lime was observed and no indication of SP in any of theindicators was observed. Although isolates of these groups areusually associated with severe symptoms of grapefruit and sweetorange SP (e.g. B249 from Venezuela [33]; the association of Gp 5haplotypes with mild symptoms has been reported previously[18,34], which is in agreement with our results.

4.6. Phylogenetic group M

Sour orange, sweet on sour orange and grapefruit plants infectedwith GpM had significantly reduced growth (Table 2). Sweet orangeand sweet on sour orange also developed shorter internodes(Table 3). Stem-pitting, vein clearing, leaf cupping were absent evenin Mexican lime (Fig. 5f). The same results were obtained by [4,6,7]when analyzing the pathogenicity of isolates T30 and T385. Thehypothesis that the T30-genotypes usually develop mild symptomswas confirmed on many samples [6], as well as in our study. Inaddition tomild reactions ofMexican lime, supplementarypatternofT30 biological characteristics including retarded growth and chlo-rosis of sweet on sour orange is proposed [4],whichmainly coincideswith our results. Virus isolate 176 (Gp M) in this study neitherdeveloped symptoms that can be attributed to QD nor to SP virusphenotypes, but someobserved symptomsand their intensity (Fig. 5)suggest the association of Gp M isolate with SY inducing isolates.

4.7. Pathogenicityofmonophyletic CTV isolates in Satsumamandarin

To investigate the impact of monophyletic CTV-isolates onSatsuma mandarin, the plants of different cultivars were subjected

to the same biological characterization assays as the standardindicator species. The infection with isolates Gp 2, Gp 3a and Gp 4caused significant changes in plants. Shortened internodes werethe most common symptom in the above mentioned groups(Table 3). Gp 2 also induced retarded growth (Table 2) andsporadically the leaf yellowing was observed. Plants infected withGp 3a and Gp 4 developed SP-symptoms (Table 4). In the mandarinplants infected with Gp 1, the symptom of leaf cupping was veryevident. If we take into account that the isolate of Gp 1 in our studydemonstrate high pathogenicity, then this symptom appearanceconfirms that the most severe CTV-isolates can cause leaf cuppingin Satsuma mandarins [35].

4.8. Conclusions

This study offers complete biological characterization simulta-neously on six out of seven major groups of CTV monophyleticisolates based on the CP-gene [17], so far missing in the publishedliterature.

The most of the symptoms are found in all isolates with varyingseverity. Severe reactions were associated with Gp 1 reflected indevelopment of symptoms associated with QD and SY syndromes.Gp 2, Gp 3a and Gp 4 isolates developed SP, with Gp 4 as the mostsevere. Besides SP, Gp 3a displayed SY pathogenicity potential.Members of Gp M and Gp 5 phylogenetic group showed lesspathogenicity as evident in the development of mild symptoms,which can be associated with SY syndrome.

These results clearly show that the phenotypic characteristics ofdisease are not associated only with the CP-gene. However, to fullydetermine the pathogenicity of p25 monophyletic groups, a greaternumber of variants should be analyzed. Also, since the same geneticstructure is maintained through the 30-region [36,37]. biologicalcharacterizationof otherpotential pathogenicitydeterminants shouldbe explored in details and the corresponding results compared.

Acknowledgment

This work was supported by Ministry of Science, Education andSports of the Republic of Croatia (Projects 091-0910468-0279 and119-1191192-1222). The authors have no conflict of interest todeclare.

References

[1] Moreno P, Ambros S, Albiach-Marti MR, Guerri J, Peña L. Citrus tristeza virus:a pathogen that changed the course of the citrus industry. Mol Plant Pathol2008;9:251e68.

[2] Roistacher CN. Graft-transmissible diseases of citrus. In: Handbook fordetection and diagnosis. Rome: IOCV/FAO; 1991.

[3] Garnsey SM, Gumpf DJ, Roistacher CN, Civerolo E, Lee RF, Yokomi RK, et al.Toward a standardized evaluation of the biological properties of Citrus tristezavirus. Phytophylactica 1987;19:151e7.

[4] Garnsey SM, Civerolo EL, Gumpf DJ, Paul C, Hilf ME, Lee RF, et al. Biologicalcharacterization of an international collection of Citrus tristeza virus (CTV)isolates. In: Hilf ME, Duran-Vila N, Rocha-Peña MA, editors. Proceedings of the16th conference of the international organization of citrus virologists (IOCV),Mexico; 2005. p. 75e93.

[5] Niblett CL, Genc H, Cevik B, Halbert S, Brown L, Nolasco G, et al. Progress instrain differentiation of Citrus tristeza virus and its application to the epide-miology of Citrus tristeza disease. Virus Res 2000;71:97e106.

[6] Ayllon MA, Lopez C, Navas-Castillo J, Garnsey SM, Guerri J, Flores R, et al.Polymorphism of the 50 terminal region of Citrus tristeza virus (CTV) RNA:incidence of three sequence types in isolates of different origin and patho-genicity. Arch Virol 2001;176:27e40.

[7] Hilf ME, Mavrodieva VA, Garnsey SM. Genetic marker analysis of globalcollection of isolates of Citrus tristeza virus: characterization and distributionof CTV genotypes and association with symptoms. Phytopathology 2005;95:909e17.

[8] Herrera-Isidrón L, Ochoa-Sánchez JC, Rivera-Bustamante R, Martínez-Soriano JP. Sequence diversity on four ORFs of Citrus tristeza virus correlateswith pathogenicity. Virol J 2009;6:116.

K. Han�cevi�c et al. / Physiological and Molecular Plant Pathology 81 (2013) 45e53 53

[9] Lu R, Folimonov A, Shintaku MLWX, Falk BW, Dawson WO, Ding SW. Threedistinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome.Proc Natl Acad Sci USA 2004;101:15742e7.

[10] Albiach-Marti MR, Robertson C, Gowda S, Tatineni S, Belliure B, Garnsey SM,et al. The pathogenicity determinant of Citrus tristeza virus causing theseedling yellows syndrome maps at the 30-terminal region of the viralgenome. Mol Plant Pathol 2010;11:55e67.

[11] Sambade A, Lopez C, Rubio L, Flores R, Guerri J, Moreno P. Polymorphism ofa specific region in gene p23 of Citrus tristeza virus allows discriminationbetween mild and severe isolates. Arch Virol 2003;148:2325e40.

[12] Rubio L, Ayllon MA, Kong P, Fernandez A, Polek ML, Guerri J, et al. Geneticvariation of Citrus tristeza virus isolates from California and Spain: evidencefor mixed infections and recombination. J Virol 2001;75:8054e62.

[13] Velazquez-Monrea JJ, Mathew DM, Dodds JA. Segregation of distinct variantsfrom Citrus tristeza virus isolate SY568 using aphid transmission. Phytopa-thology 2009;99:1168e76.

[14] Harper SJ, Dawson TE, Pearson MN. Isolates of Citrus tristeza virus that over-come Poncirus trifoliata resistance comprise a novel strain. Arch Virol 2010;155:471e80.

[15] GillingsM, Broadbent P, Indsto J, Lee RF. Characterization of isolates and strainsof Citrus tristeza closterovirus using restriction analysis of the coat protein geneamplified by the polymerase chain reaction. J Virol Meth 1993;44:305e17.

[16] Zemzami M, Soares CM, Bailey AM, Niblett CL, Nolasco G. Molecular charac-terization and classification of Morrocan isolates of Citrus tristeza closterovirus.In: da Graça JV, Duran-Vila N, Milne RG, editors. Proceedings of the 15thconference of the international organization of citrus virologists (IOCV)Riverside; 2002. p. 8e12.

[17] Nolasco G, Santos C, Silva G, Fonsceca F. Development of an asymetric PCR-ELISA typing method for Citrus tristeza virus based on the coat protein gene.J Virol Meth 2009;155:97e108.

[18] �Cerni S, Ru�s�ci�c J, Nolasco G, Gatin �Z, Kraja�ci�c M, �Skori�c D. Stem pitting andseedling yellows symptoms of Citrus tristeza virus infections may be deter-mined by minor sequence variants. Virus Genes 2008;36:241e9.

[19] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning e a laboratory manual.Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989.

[20] Rubio L, AyllonM,Guerri J, PappuH,Niblett C,MorenoP.Differentiationof Citrustristeza closterovirus (CTV) isolates by single-strand conformation poly-morphism analysis of the coat protein gene. Ann Appl Biol 1996;129:479e89.

[21] Kong P, Rubio L, Polek M, Falk BW. Population structure and genetic diversitywithin California Citrus tristeza virus isolates. Virus Genes 2000;21:139e45.

[22] Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignmentaided by quality analysis tools. Nucleic Acids Res 1997;25:4876e82.

[23] Tamura K, Peterson D, Peterson N, Steche G, Nei M, Kumar S. MEGA5:molecular evolutionary genetics analysis using maximum likelihood,

evolutionary distance, and maximum parsimony methods. Mol Biol Evol2011;28:2731e9.

[24] Papayiannis LC, Santos C, Kyriakou A, Kapari T, Nolasco G. Molecular char-acterization of Citrus tristeza virus isolates from Cyprus on the basis of the coatprotein gene. J Plant Pathol 2007;89:291e5.

[25] �Cerni S, �Skori�c D, Ru�s�ci�c J, Kraja�ci�c M, Papi�c T, Djelouah K, et al. East adriatic-a reservoir region of severe Citrus tristeza virus strains. Eur J Plant Pathol 2009;124:701e6.

[26] McClean APD. Seedling yellows in South African citrus trees. S Afr J Sci 1960;3:259e79.

[27] Wallace JM. Virus and virus-like diseases. In: Reuther W, Calavan EC,Carman GE, editors. The citrus industry, vol. 1. Berkeley: Univ. of Calif DivAgric Sci; 1978. p. 67e184.

[28] Suastika G, Natsuaki T, Terui H, Kano T, Leki H, Okuda S. Nucleotidesequence of Citrus tristeza virus seedling yellows isolate. J Gen Plant Pathol2001;67:73e7.

[29] Schneider H. The anatomy of tristeza-virus-infected citrus. In: Wallace JM,editor. Citrus virus diseases. Richmond, CA: Univ of Calif Div Agric Sc; 1959. p.73e84.

[30] Karasev AV, Boyko VP, Gowda S, Nikolaeva OV, Hilf ME, Koonin EV, et al.Complete sequence of the Citrus tristeza virus RNA genom. Virology 1995;208:511e20.

[31] Yang ZN, Mathews DM, Dodds JA, Mirkov TE. Molecular characterization of anisolate of Citrus tristeza virus that causes severe symptoms in sweet orange.Virus Genes 1999;19:131e42.

[32] Ruiz-Ruiz S, Moreno P, Guerri J, Ambros S. The complete nucleotide sequenceof severe stem pitting isolate of Citrus tristeza virus from Spain: comparisonwith isolates from different origins. Arch Virol 2006;151:387e98.

[33] Febres VJ, Niblett CL, Lee RF, Moore GA. Characterization of grapefruit plants(Citrus paradisi Macf.) transformed with Citrus tristeza closterovirus genes.Plant Cell Rep 2003;21:421e8.

[34] Iglesias NG, Gago-Zachert SP, Robledo G, Costa N, Inés Plata M, Vera O, et al.Population structure of Citrus tristeza virus from field Argentinean isolates.Virus Genes 2008;36:199e207.

[35] Zhou CY, Zhao XY, Jiang YH. Boat-shaped leaf symptoms of Satsumamandarin associated with Citrus Tristeza Virus (CTV). In: da Graca JV,Moreno P, Yokomi K, editors. Proceedings of the 13th conference of theinternational organization of citrus virologists (IOCV) Fouzhou, China; 1996. p.154e7.

[36] Martín S, Sambade A, Rubio S, Vives MC, Moya P, Guerri J, et al. Contribution ofrecombination and selection to molecular evolution of Citrus tristeza virus.J Gen Virol 2009;90:1527e38.

[37] Ghorbel R, Lopez C, Fagoaga C, Moreno P, Navarro L, Flores R, et al. Transgeniccitrus plants expressing the Citrus tristeza virus p23 protein exhibit viral-likesymptoms. Mol Plant Pathol 2001;2:27e36.