5a-08-molecular characterization and potential sources of tristeza virus in...

Plant Pathology (2011) Doi: 10.1111/j.1365-3059.2011.02553.x

Molecular characterization and potential sources of Citrustristeza virus in Oman

A. M. Al-Sadia*, S. A. Al-Hilalia, R. A. Al-Yahyaia, F. A. Al-Saida, M. L. Deadmana,

I. H. Al-Mahmoolia and G. Nolascob

aDepartment of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, PO Box 34, AlKhoud 123,

Oman; and bCenter for Biodiversity, Functional and Integrative Genomics, Universidade do Algarve, Campus de Gambelas, 8005-

139 Faro, Portugal

A study was conducted to characterize occurrence, molecular variability and potential sources of Citrus tristeza virus (CTV)

in Oman. A survey during 2009 and 2010 showed that CTV occurs in most (91%) of the surveyed districts. Moderate to

high levels of infection with CTV in lime were detected in northern Oman (15–63%) compared to the south (0–12%). This

could be related to the continuous introduction of infected citrus germplasm in the north from abroad, where CTV was

detected in 45% of citrus seedlings imported from Syria, Lebanon, India, Pakistan and Egypt. CTV was detected for the first

time in sweet lime, sweet lemon, citron, mandarin, sweet orange and sour orange and was found to be associated with stem

pitting, stunting, leaf curling and vein clearing symptoms in many of the infected trees and seedlings in Oman. Bi-directional

reverse transcription-PCR analysis of the coat protein (CP) gene of 22 randomly selected CTV-positive samples provided evi-

dence that severe strains of CTV exist in Oman. Cloning and sequencing the CP gene of six isolates showed that they have

91–100% nucleotide identity with each other and 96–99% with representative isolates from other parts of the world. Phylo-

genetic analysis of the CTV isolates showed that four belong to CTV Group 4. However, two isolates formed a separate

clade with 100% bootstrap support for separation from Group 5. Phylogenetic analysis and coefficient of differentiation val-

ues suggest that the two isolates from Oman constitute a new CTV phylogenetic group.

Keywords: CTV phylogeny, lime, Tristeza

Introduction

Citrus is an important genus of fruit crops in the world. InOman, acid limes (Citrus aurantifolia) are the fourthmajor fruit crop in terms of production after date palms,bananas and mangoes (FAO, 2010). There are over350 000 lime trees across the country (MAF, 2005).Other citrus species grown in Oman are: sweet lime(C. limettioides), sweet orange (C. sinensis), citron(C. medica), mandarin (C. reticulata), lemon (C. limon)and sour orange (C. aurantium). Citrus trees are found inall regions in Oman, with production being concentratedin the Al-Batinah region. Most citrus species are grownon their own, with grafting being a very limited practice.However, citrus propagation is commonly practised inOman using seeds and layering. Most lime and sweet limeplants are propagated locally, while many other citrusspecies are imported via nurseries from countries such asIndia, Pakistan, Syria, Lebanon and Egypt.

Most species of citrus are susceptible to several plantpathogenic fungi, prokaryotes, viruses, nematodes and

*E-mail: [email protected]

ª 2011 The Authors

Plant Pathology ª 2011 BSPP

viroids. Citrus tristeza virus (CTV), a member of thegenus Closterovirus of the Closteroviridae family, is themost destructive viral pathogen of citrus in the world.The virus has killed over 100 million citrus trees in Spain,Brazil and Argentina, especially those that are grafted onsour orange rootstocks (Moreno et al., 2008). CTV cancause different symptoms on citrus trees depending onthe strain of the virus, citrus species, environmental con-ditions and propagation method. These include decline ofcitrus trees grafted on sour orange rootstocks, stem pit-ting symptoms on acid lime, pummelo (C. grandis),grapefruit (C. paradise) and sweet orange, and seedlingyellows (McClean, 1960; Garnsey & Lee, 1988; Lee &Bar-Joseph, 2000; Brlansky et al., 2002). CTV can betransmitted by propagation of infected buds, import ofdiseased planting material and several aphid species suchas Toxoptera citricida, Aphis gossypii and A. spiraecola(Roistacher & Bar-Joseph, 1987).

Citrus tristeza virus was first detected in Oman in1986 in seedlings of lemon, mandarin and grapefruitimported from India (Bove, 1995). However, littleattention has been given to the disease. A preliminarysurvey by the Ministry of Agriculture in 2008 in 10 dif-ferent farms in Oman showed that the disease was pres-ent in four of them (MA, 2009). However, because that

1

2 A. M. Al-Sadi et al.

survey was very limited, there is still little known aboutthe distribution of CTV in Oman, or which citrus spe-cies are affected. In addition, there is a lack of knowl-edge about the most common symptoms associatedwith CTV infection and the potential role of commer-cial nurseries in disseminating CTV infected citrus seed-lings. This establishes a barrier towards developmentand implementation of effective management strategiesfor CTV in the country.

Biological indexing has been used in the past for straindifferentiation in CTV. However, because indexing istime consuming (up to 12–15 months) (Mukhopadhyay,2004; Moreno et al., 2008), several serological andmolecular methods have been developed over the pasttwo decades for characterization of CTV strains. Thefinding that severe isolates of CTV (decline or stem pittinginducing isolates) differ from the mild isolates at position371 of the coat protein (CP) gene (Niblett et al., 2000) hashelped develop selective primers, which through bi-direc-tional reverse transcription PCR (RT-PCR) has helped inaccurate, sensitive and fast characterization of CTVstrains (Cevik et al., 1996; Niblett et al., 2000; Huanget al., 2004). Although this test has proved to be efficientin differentiating CTV strains from Florida and someother places into two types, severe and mild (Huang et al.,2004; Jiang et al., 2008), it is not clear whether Omanilime trees developing severe CTV symptoms can be iden-tified using the test.

Restriction analysis as well as phylogenetic analysis ofthe CP gene of CTV isolates has helped group CTV haplo-types from different parts of the world into seven differentgroups (Mawassi et al., 1993; Zemzami et al., 2002;Nolasco et al., 2009). The grouping was found to berelated to biological activities of the haplotypes (Nolascoet al., 2009). A recent study by Nolasco et al. (2009) onmore than 140 CTV haplotypes from 20 different parts ofthe world has shown that all belong to one of the sevenCTV groups. Oman lies on the far eastern part of the Ara-bian Peninsula, a place which has been considered thetransition point through which lime and some other citrusspecies have been moved from India and China to thewest (Davies & Albrigo, 1994). Because no isolates wereincluded in the study by Nolasco et al. (2009) from Omanor other parts of the Arabian Peninsula, it is not known ifisolates from this part of the world belong to any of thepreviously described seven groups. In addition, there is alack of knowledge concerning molecular variability ofCTV from Oman, so that it is not known whether CTV inOman originates from common or multiple sources.

This study was therefore conducted to characterizeoccurrence, phylogeny and sources of CTV in Oman. Spe-cific objectives include: (i) to characterize the distributionof CTV in Oman; (ii) to find out whether commercialnurseries could play a role in spreading CTV infectedseedlings; and (iii) to characterize the phylogenetic rela-tionship of CTV isolates from Oman with CTV fromother parts of the world. Investigations into these aspectsof the disease may help establish a solid backgroundabout distribution and molecular variability of CTV in

Oman, which will be important in delineating a diseasecontrol strategy.

Materials and methods

Distribution of CTV

In order to characterize distribution of CTV in Oman, asurvey was conducted from January 2009 to July 2010.The survey covered 22 districts located in eight differentgeographical regions in the Sultanate of Oman (Fig. 1). Atotal of 137 farms were visited, with at least five randomlysurveyed farms per district, except for four districts whichare limited in the number of citrus farms. Because acidlime is the dominant species of citrus in the country(MAF, 2005), it was the main focus of the survey. Samplecollection was done from a total of 526 lime trees; at leastfive lime trees per farm, except for farms with limitednumbers of lime trees. During the course of the survey,samples were also collected from other citrus trees whichwere present in the surveyed farms. Samples representingthe young developing leaves and twigs were randomlycollected from trees, with each sample consisting of 15–30 leaves. Samples were collected from trees ⁄ seedlingsshowing one or more of the following symptoms: seedlingyellowing, stem pitting, stunting, leaf curling, vein clear-ing or necrosis. In farms where these symptoms were notdetected, the collection of samples was from randomlyselected trees. Samples were placed in sealed plastic bagsand kept in an ice box during transport to the PlantPathology Research Laboratory at Sultan Qaboos Uni-versity, and then stored at )80�C for further analysis.

Double antibody sandwich procedure-enzyme linkedimmunosorbent assay (DAS-ELISA; CTV-IDENTIKIT,Adgen) was used for detection of CTV in all the citrussamples, following the manufacturer’s protocol. CTVpositive and negative samples provided with the kits wereused as controls in every ELISA test. The colour reactionwas monitored by measuring absorbance at 405 nmusing MultiskanEx spectrophotometer (ThermoLabsys-tems). Citrus samples were considered CTV positive if theabsorbance reading of the samples was at least double theabsorbance of the negative control (Garnsey & Cambra,1991).

Occurrence of CTV in nursery seedlings

In order to find out whether citrus seedlings importedfrom abroad could act as potential sources of CTV intoOman, the following experimental approach was fol-lowed. Three separate surveys were carried out in thesummer, autumn and winter of 2010. Each survey cov-ered five different nurseries located in Seeb district,Oman. A total of 119 different citrus samples were col-lected from nurseries, 3–14 samples per nursery. Thesesamples represented different citrus species whichincluded acid lime (21), Tahiti lime (C. latifolia) (5),sweet lime (8), sweet orange (31), sour orange (4), man-darin (24), grapefruit (3), lemon (17) and kumquat (For-

Plant Pathology (2011)

Iran

Dibba

OmanKSA

UAEDibba

Madha

MahadhaMahadha

Samael

YanqulDhank

Rustaq

IbriBahla

Nizwa

Mudhaibi

IbraSamael

Mudhaibi

TaqaMarbatSalalah

Figure 1 A map of Oman showing the main districts where samples were collected.

Molecular characterization of CTV from Oman 3

tunala margarite) (6). Out of the 119 samples, 86 seed-lings were imported from Syria, Egypt, Lebanon, Jordon,Pakistan or India, while 33 seedlings were propagatedlocally. The collection of samples from nurseries wasfrom seedlings which had just arrived from the places oforigin (27) and also from imported seedlings which hadbeen kept in nurseries for 2–12 weeks (92).

RT-PCR for CTV detection

Detection of CTV in the nursery samples was achievedusing RT-PCR. Midribs and petioles of the citrus leavescollected from nurseries were cut and then ground usingliquid nitrogen. RNA was extracted from 0Æ1 g of theground midribs and petioles using RNeasy Mini Kit(QIAGEN) according to the manufacturer’s protocol.RT-PCR was performed using QIAGEN One-StepRT-PCR master mix and primers PIN1 (5¢-GGT TCACGC ATA CGT TAA GCC TCA CTT-3¢) and PIN2 (5¢-TAT CAC TAG ACA ATA ACC GGA TGG GTA-3¢)(Olmos et al., 1999). The master mix for each sample con-sisted of: 10 lL of 5· QIAGEN One-step RT-PCR buffer,2 lL dNTP mix, 10 lL of 5· Q-solution, 0Æ6 lM of each


primer, 2 lL of QIAGEN One-step RT-PCR EnzymeMix, 10 lL of the RNA sample and RNase-free water upto 50 lL. The cycling conditions were as follows: reversetranscription at 50�C for 30 min, then one cycle at 95�Cfor 15 min, followed by 40 cycles of 94�C for 30 s, 60�Cfor 30 s, and 72�C for 1 min. The final extension was at72�C for 10 min. The RT-PCR products were run on 2%agarose gels for 40 min at 120 V and 110 mA and visual-ized under UV after ethidium bromide staining.

Molecular discrimination of CTV strains

In order to find out whether lime samples developingcertain CTV symptoms can be discriminated using thebi-directional RT-PCR test of Huang et al. (2004), thefollowing protocol modified from Huang et al. (2004)and Jiang et al. (2008) was used. A total of 21 randomlyselected CTV-positive lime samples from field surveysand one randomly selected CTV-positive lime samplefrom a nursery were included in the test. The 22 samplesconsisted of two samples from seedlings developing leafcurling symptoms, six samples from symptomless seed-lings, 12 samples from lime trees developing stem pitting


symptoms and two samples from lime trees developingleaf curling symptoms. DNA extraction from leaf midribsand petioles of the 22 samples was achieved using RNeasyPlant Mini Kit (QIAGEN) according to the manufac-turer’s protocol.

Bi-directional RT-PCR was conducted for each sampleusing the primers HCP1 (5¢-ATG GAC GAC GAA ACAAAG AA-3¢), HCP2 (5 ¢-TCA ACG TGT GTT GAA TTTCC-3 ¢), CP3 (5 ¢-TTT GGA CTG ACG TCG TGT T-3 ¢)and CP4 (5 ¢-TTA CCA ATA CCC TTA GAA TTA T-3 ¢)(Huang et al., 2004). QIAGEN One-Step RT-PCR mastermix was used. The master mix for each sample was asbefore, using 0Æ6 lM of each of the above primers. TheRT-PCR conditions were: reverse transcription at 50�Cfor 50 min, initial PCR activation at 95�C for 15 min, 35cycles of denaturation at 94�C for 1 min, annealing at50�C for 2 min and extension at 72�C for 2 min. The finalextension step was at 72�C for 20 min.

Successful amplification of PCR products was checkedby running 10 lL of the PCR product on 2% agarose gels.According to Huang et al. (2004) and Jiang et al. (2008),the CTV-positive samples are expected to produce a frag-ment of 672 bp (HCP1 ⁄ HCP2). However, fragment sizesof 320 (HCP1 ⁄ CP3) and 392 bp (HCP2 ⁄ CP4) indicate thepotential presence of different haplotypes similar to thosefound in severe and mild isolates in other studies (Huanget al., 2004).

Phylogenetic analysis of CTV from Oman

In order to compare the coat protein sequence of the CTVisolates from Oman with CTV isolates from other partsof the world, the following experimental approach wasfollowed. Six randomly selected CTV-positive samples,which produced a 320 bp band in the previous test, wereused in the study (Table 1). The coat protein gene of thesix CTV isolates was amplified using primers HCP1 andHCP2 (Huang et al., 2004). The RT-PCR mixture andconditions were as described previously, using 0Æ6 lM ofprimers HCP1 and HCP2. Successful amplification of thecoat protein was checked on 2% agarose gels. The RT-PCR products of the CP gene for the six samples werepurified from dNTPs using QIAquick PCR Purificationkit (QIAGEN) following the manufacturer’s protocol.

Table 1 Characteristics of Omani Citrus tristeza virus isolates used in the

phylogenetic analysis

Isolate

accession

no.a

GenBank

accession

no.

Year of

isolation District

Lime

tree age

(years)

Field

symptoms

W001-4B JF310691 2010 Dibba 6 Stem pitting

W071-3 JF310692 2009 Barka 5 Stem pitting

W093-3 JF310693 2009 Madha 12 Stem pitting

W121-1 JF310694 2009 Sohar 2 Symptomless

W140-1 JF310695 2010 Suwaiq 2 Symptomless

W140-2 JF310696 2010 Suwaiq 2 Symptomless

aAll isolates were obtained from acid lime (Citrus aurantifolia).

The purified samples were then cloned using MGTMpTOP TA V2 vector and sequenced at Macrogen Inc.Sequencing of the CP gene for each isolate was done fromthree randomly selected bacterial colonies containing theinsert.

The forward and backward CP gene sequences for eachisolate were first aligned together and edited usingCHROMASPRO. The resulting CP gene sequences were thencompared to each other and to worldwide collections ofsequences deposited at the National Center for Biotech-nology Information (NCBI) using BLAST searches. Searchfor evidence of recombination events between these andother GenBank available sequences was performed withthe RDP software (Martin et al., 2005). The six sequenceswere aligned with 144 CP gene sequences from world-wide origins referred to in Nolasco et al. (2009) usingCLUSTALW (Thompson et al., 1994). A neighbour-joiningtree was constructed based on the matrix of pairwise dis-tances obtained using the Kimura 2 parameter evolution-ary model (MEGA5) (Tamura et al., 2011). Bootstrap 80%majority-rule consensus trees were generated using 1000replications.

The coefficient of differentiation was estimatedaccording to Nei & Kumar (2000). The coefficient of dif-ferentiation is the ratio of the mean inter-group diversityover mean diversity for the entire population. If close tozero, the population is not structured (all diversity is out-side the groups); if 1, all diversity falls within the specifiedgroups.

Results

Distribution of CTV

Assessing the presence of Citrus tristeza virus usingELISA in 526 lime samples collected from 22 differentdistricts showed that the virus was associated with 102(19Æ4%) lime samples (Table 2). CTV in lime wasdetected in all the surveyed districts, except in Taqaand Marbat. Recovery of CTV from different districtsvaried from 0 to 63% (Table 2). Moderate to high lev-els of infection (15–63%) were observed in districtsthat are located in the northern part of Oman (Table 2,Fig. 1). Limes in districts that are located in the south-ern part of the country were found to have low levelsof infection (0–12%). The virus was also detected atdifferent frequencies in sweet lime, citron, mandarin,sweet lemon (C. limetta), sweet orange and sourorange (Table 3).

Symptoms typical of CTV infection were detected insome of the citrus species that were surveyed. Stem pittingwas found to be the most common symptom in the field.Other symptoms included yellowing, curling and veinclearing of leaves of different citrus species as well as stun-ting, especially in sweet orange grafted on sour orangerootstocks and in lime trees (Table 3). Association ofCTV with these symptoms in the affected trees was con-firmed using ELISA tests.


Table 2 Distribution of Citrus tristeza virus (CTV)-infected acid lime trees

(C. aurantifolia) in different districts in Oman

Locations

Sample

size (farms)

Sample

size (trees)

Infected

treesa

% trees

infected

Barka 6 26 10 38

Rustaq 6 37 9 24

Shinas 9 30 14 47

Sohar 6 22 4 18

Suwaiq 10 40 10 25

Boushar 2 10 1 10

Seeb 7 16 10 63

Qurayat 6 40 4 10

Dhank 1 4 1 25

Yanqul 7 17 6 35

Ibri 6 17 4 24

Mahadha 6 13 4 31

Bahla 5 29 5 17

Nizwa 5 19 1 5

Samael 8 10 4 40

Dibba 9 36 7 19

Madha 13 33 5 15

Ibra 5 31 1 3

Mudhaibi 6 30 1 3

Marbat 4 15 0 0

Salalah 5 31 1 3

Taqa 5 20 0 0

Total 137 526 102

aPresence and absence of CTV was based on ELISA test. A

positive sample is considered a sample with absorbance of at least

double the absorbance recorded for the negative control

(absorbance measured at 405 nm).


Occurrence of CTV in nursery seedlings

RT-PCR analysis of 119 citrus samples from nurseryseedlings from different countries showed that 53 werepositive for CTV. The positive samples produced a frag-ment that was 131 bp following amplification with PIN1

and PIN2 primers. The virus was detected in acid lime,lemon, sweet orange, mandarin, grapefruit, kumquat,

Table 3 Citrus species infected with Citrus tristeza virus (CTV) in Oman

Host Sample size No. (%) infected trees

Field symptom

SP VC

Lime 526 102 (19Æ4) M R

Sweet lime 29 4 (13Æ8) L R

Sweet orange 23 4 (17Æ4) L –

Citron 10 3 (30Æ0) L –

Mandarin 6 2 (33Æ3) – R

Lemon 5 0 – –

Sour orange 5 1 (20Æ0) – –

Sweet lemon 3 1 (33Æ3) – –

Total 607 117 (19Æ3)

SP, stem pitting; VC, vein clearing; ST, stunting; LC, leaf curling; R, rare (

not detected ⁄ quantified.a1: Bahla, 2: Bousher, 3: Barka, 4: Dhank, 5: Dibba, 6: Ibra, 7: Ibri, 8: Ma

14: Rustaq, 15: Salalah, 16: Samael, 17: Seeb, 18: Shinas, 19: Sohar, 20:bDistricts that are in bold indicate presence of CTV.


sweet lime and Tahiti lime, with frequencies of infectionranging from 0 to 100% (mean 45%; Table 4). CTV wasdetected in citrus seedlings coming from Egypt, Pakistan,Syria, Lebanon and India as well as from citrus seedlingsoriginating from Oman. The virus was detected in seed-lings at the time of arrival from the country of origin aswell as in seedlings that had been imported from abroadbut kept in nurseries for 2–12 weeks.

Molecular discrimination of CTV strains

Analysis of 22 CTV-positive citrus samples using RT-PCR with HCP1, HCP2, CP3 and CP4 primers produced afragment 672 bp in length, representing the coat protein(CP) gene of CTV. All lime samples (12) that wereobtained from lime trees showing stem pitting symptomsproduced a 320 bp fragment (Table 5). Four samples thatwere obtained from symptomless lime seedlings and twosamples from a tree and a seedling showing leaf curlingsymptoms produced two fragments of 320 and 392 bp.Four other samples produced a 320 bp fragment(Table 5).

Phylogenetic analysis of CTV isolates

Amplification of the coat protein gene for six Omani iso-lates produced a 672 bp fragment that was sequenced.Sequences were deposited in GenBank with the accessionnumbers JF310691 to JF310696. Evidence for recombi-nation between these and other available GenBanksequences was not found. Three of the isolates, W071-3,W93-3 and W121-1 showed an identical nucleotidesequence. However, isolate W001-4B coming from Dib-ba showed two nucleotide mismatches from the rest ofthe Omani isolates. Isolates W140-1 and W140-2showed one base pair mismatch from each other butshowed 91Æ1% similarity to the other Omani isolates.These two isolates came from seedlings showing noapparent symptoms of CTV infection.

s

Locationa,bST LC

L L 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22

R R 1,3,5,8,12,16,17

M R 1,3,5,8,12,16,20

R L 2,4,5,8,16,17

R – 5,12

– R 1,5

– L 3,16,17

– – 12,16

0–5%); L, low (6–15%); M, moderate (16–40%); H, high (>40%); –,

dha, 9: Mahadha, 10: Marbat, 11: Mudhaibi, 12: Nizwa, 13: Qurayat,

Suwaiq, 21: Taqa, 22: Yanqul.

Table 4 Detection of Citrus tristeza virus (CTV) in nursery seedlings originating from different countries

Citrus species Origin Sample size (no. seedlings) No. (%) of infected seedlingsa

Seedlings positive for CTVb

Nursery Just arriving

Lime Oman 18 9 (50%) + +

India 3 3 (100%) ? +

Lemon Lebanon 11 5 (45%) + +

Syria 4 4 (100%) ? +

Egypt 2 0 ) ?

Sweet orange Egypt 14 3 (21%) + ?

Pakistan 2 2 (100%) + ?

Syria 13 7 (54%) + +

Jordon 2 0 ) ?

Mandarin Egypt 13 4 (31%) + ?

Syria 11 5 (45%) + +

Grapefruit Syria 3 1 (33%) ) +

Kumquat Syria 6 3 (50%) + +

Sour orange Oman 4 0 ) ?

Sweet lime Oman 6 4 (67%) + +

Lebanon 2 2 (100%) + ?

Tahiti lime Oman 5 1 (20%) + ?

aBased on RT-PCR analysis using primers PIN1 and PIN2.bDetection of CTV in seedlings from nurseries or at the time of arrival where (+) means positive for CTV, ()) means not detected and (?)

means not tested.

Table 5 RT-PCR analysis of Citrus tristeza virus (CTV) strains associated

with 22 lime trees and seedlings

Growth

stage Field symptoms

Sample

size

RT-PCR product (bp)a

320 392 320 and 392

Seedling Leaf curling ⁄ yellowing 2 1 0 1

Symptomless 6 2 0 4

Fruiting

trees

Stem pitting 12 12 0 0

Leaf curling ⁄ yellowing 2 1 0 1

aBased on RT-PCR analysis using HCP1, HCP2, CP3 and CP4

primers. Samples with a fragment size of 320 bp indicate the

potential presence of a severe strain; samples with fragment sizes

of 320 bp and 392 bp indicate the potential presence of at least two

different strains of CTV, mild and severe (Huang et al., 2004).


When compared to sequences in GenBank, the CP genesequences of the Omani isolates clustered in differentpositions (Fig. 2). Isolates W001-4B, W071-3, W93-3and W121-1 clustered, according to the grouping schemeproposed by Nolasco et al. (2009), in Group 4 along withthe Florida decline-inducing isolate T3. Isolates W140-1and W140-2 appear with a very good bootstrap supportas a separate clade in the branch leading to Group 5. Inthe collapsed tree obtained from 144 worldwidesequences it can be seen that the W140 cluster separatesfrom the closest group (Group 5) at a distance compara-ble to the divergence of some other groups (Fig. 2). Toinvestigate whether this could be considered a differentgroup, the coefficient of differentiation was estimatedwith the W140 haplotypes either included or not includedin Group 5. The coefficient rises from 0Æ775 to 0Æ809 ifW140 haplotypes are considered as a different groupinstead of part of Group 5.

Discussion

Citrus tristeza virus was detected in most of the surveyeddistricts in Oman, with frequencies of infection rangingfrom 0 to 63% (mean 19Æ4%). Moderate to high levels ofCTV infection (15–63%) were detected in districtslocated in the northern part of Oman compared to thesouth (0–12%). The highest levels of CTV infection in thenorth could be related to the proximity of these districtsto the entry sites of citrus seedlings into Oman as well asto the main nurseries that distribute and sell citrus seed-lings. Support for this hypothesis is evident from the highlevel (45%) of infection observed in this study in citrusseedlings imported by nurseries from abroad. Previousstudies have emphasized the role of nurseries in dissemi-nating many citrus diseases, including CTV (Bove, 1995).The contribution of nurseries towards spread of CTV inOman, as shown in this study, may necessitate futuremanagement programmes looking at obtaining cleanplanting material, as well as proper inspection of citrusmaterial imported from abroad.

Occurrence of CTV in most of the surveyed districtsin Oman may indicate that CTV has been introducedinto Oman and disseminated to different districts over along period of time, or that multiple introductions areoccurring. These hypotheses are supported by the occur-rence of different CTV haplotypes with different CPsequences as well as of different CTV strains, as evidentfrom the bi-directional RT-PCR test. Since no previoussurveys have been undertaken to characterize occur-rence of CTV in Oman, except a recent and limited sur-vey by the Ministry of Agriculture in 2008 (MA, 2009),it is difficult to determine when CTV was first intro-duced into the country. However, detection of CTV in


(c)(a)C270-3-12 Argentina (Gp 5)C270-3-7 Argentina (Gp 5)442-2 Croatia (Gp 5)

Gp 1

Gp 2

2-98 Madeira (Gp 5)PeralAC-31 Brazil (Gp 5)Col4 Colombia (Gp 5)

Gp M

Gp 5

W140-1W Col1 Colombia (Gp 5)

PeralAC-42 Brazil (Gp 5)ST15—10 S Tome (Gp 5)134B Reunion (Gp 5)

Gp 4

Gp 3b

Gp 3a

W140-2

0·04 0·02 0·01 0·00

443-13 Croatia (Gp 4)

2-1-05 Madeira (Gp 5)BaraoB-4 Brazil (Gp 5)15-118 Madeira (Gp 5)

(b)

86

8698 441-3 Croatia (Gp 4)

441-2 Croatia (Gp 4)443-4 Croatia (Gp 4)15-8S Tome (Gp 4)

010-8 Venezuela (Gp 5)CY98-30-1 Cyprus (Gp 5)CY98-33-61 Cyprus (Gp 5)CY94-39-1 Cyprus (Gp 5)

99

15-11 S Tome (Gp 4)CY89-60-36 Cyprus (Gp 4)CY89-60-38 Cyprus (Gp 4)

047-16 Venezuela (Gp 5)C320-5-21 Argentina (Gp 5)B249 Venezuela (Gp 5)C257-7-2 Argentina (Gp 5)

10098Oman

100

134A Reunion (Gp4)W001-4bW071-3W121-1W93-3

C320-5-22 Argentina (Gp 5)C257-2-27 Argentina (Gp 5)S78- 4 Jamaica (Gp 5)

99100

100100Oman

T3 Florida (Gp 4)AN0-1 Egypt (Gp 4)K1-76 Egypt (Gp 4)K1-77 Egypt (Gp 4)

S78-35 Jamaica (Gp 5)S133-31 Jamaica (Gp 5)

W140-1W140-2

0·03

0·03

0·02 0·01 0·00 0·04 0·02 0·01 0·000·03

100

9489

100

100100

100

100

9999

96

Figure 2 Phylogenetic tree showing relationship of CP gene sequences of six Citrus tristeza virus (CTV) isolates from Oman to haplotypes

belonging to all groups (a), Group 4 (b) and Group 5 (c) as defined by Nolasco et al. (2009). The tree was constructed by the neighbour-

joining method. Numbers close to the nodes represent the bootstrap values obtained from 1000 replications (values above 80 are shown).


nursery citrus seedlings imported from India in 1986(Bove, 1995) supports the hypothesis that CTV couldhave been continuously introduced into Oman sincethat time or even before. Detection of CTV in citrusseedlings imported by nurseries from different countriesis further evidence that CTV has been continuouslyintroduced into Oman.

The wide distribution of CTV across Oman couldalso be attributed to the use of layering in citrus propa-gation. Layering is commonly practised by farmers inOman for propagation of citrus trees, especially lime.Previous studies have provided evidence for transmis-sion of CTV via seedlings propagated vegetatively (Bar-Joseph & Lee, 1989). Because CTV is not known to betransmitted via citrus seeds (Bar-Joseph & Lee, 1989),detection of CTV in some lime trees originating fromseeds (data not provided) may provide evidence thatCTV could have been transmitted among some farms inOman via aphids.

A range of typical CTV symptoms was detected in cit-rus seedlings and trees that tested positive for CTV. Stempitting was found to be the most common, especially inlime trees. Stunting was found to be common in sweetorange grafted on sour orange and in some affected limetrees. Leaf cupping of lime and vein clearing were also


detected and found to be common, especially in seedlings.This appears to be the first record of association of CTVwith these symptoms and with sweet orange, sour orange,sweet lime, sweet lemon, citron and mandarin in Oman.However, future studies may consider quantifying theamount of economic loss due to infection of citrus specieswith CTV.

Molecular-based characterization of CTV strains inOman provided evidence that there are at least two differ-ent CTV strains present. This was evident from RT-PCRanalysis of the CP gene with the use of HCP1, HCP2, CP3

and CP4 primers for 22 CTV-positive samples. RT-PCRanalysis of 12 samples obtained from lime trees showingstem pitting symptoms provided evidence that all thesamples have severe CTV strains. This shows the effi-ciency of the bi-directional RT-PCR test in discriminatingsevere strains of CTV, at least those that are causing stempitting symptoms in Oman, which is in agreement withfindings obtained by Huang et al. (2004). A mixture of atleast two strains (potentially severe and mild) wasdetected in samples obtained from symptomless limeseedlings and a tree and a seedling with mild symptoms,but not in trees developing stem pitting symptoms. Thismay suggest that the presence of a mild strain confers pro-tection against the severe strain (cross-protection) and


therefore masks or reduces severity of symptoms; ahypothesis that deserves further investigation.

The six sequenced Omani isolates are in differentgroups (clades) with high bootstrap values which sug-gests that at least two introductions of divergent isolateshave occurred in Oman. Also, the genetic similarity ofCTV isolates from Oman and other countries confirmslong distance movement by traffic of propagative mate-rial through nurseries. This is supported by detection ofCTV in nursery seedlings imported from different coun-tries. Variability of the Omani CTV isolates supports pre-vious studies of existence of variation in CTV isolatesinfecting citrus in a particular location (Biswas, 2010;Melzer et al., 2010).

Interestingly, two haplotypes that were sequenced(W140) clustered in a particular position in relation tothe other CP groups. This suggests the existence of a newphylogenetic group separating from the Group 5 branch.The position of this clade could not be explained byrecombination events (which were not detected) and thelevel of divergence at which it separates is comparable tothe level of divergence of individualization of othergroups. Additionally, the coefficient of differentiationincreases when these haplotypes are considered apartfrom Group 5. This evidence supports the view that thesesequences may constitute a new phylogenetic group.Given that these isolates were obtained from two symp-tomless lime seedlings, it remains to be seen if these kindsof haplotypes are widespread in Oman and associatedwith any particular symptomatology, or just representsome atypical CTV isolates.

This study has provided evidence for widespread distri-bution of CTV in different regions of Oman and the asso-ciation with several citrus species and field symptoms.Due to the high level of infection with CTV (45%) ofimported citrus seedlings, strict quarantine measuresneed to be introduced and implemented in order to savethe citrus industry in Oman. In-depth analysis of CTVdiversity in Oman and neighbouring countries in the Ara-bian Peninsula is required, especially after uncovering theexistence of CTV haplotypes from Oman creating a newphylogenetic group. Biological indexing will be requiredto confirm association of CTV with many of the symp-toms observed. Seedlings from which the W140 haplo-types were recovered are under close observation for fieldsymptoms, and pathogenicity tests are planned touncover their biological characteristics.

Acknowledgements

The authors would like to acknowledge Dalia Al-Kha-tib, Aisha Al-Ghaithi, Hanan Al-Moqbali, Safa Al-Mazroui, Amna Al-Jabri, staff from the Ministry ofAgriculture and farmers for their help during surveysand laboratory work. Special thanks are due to SultanQaboos University for funding the study through theStrategic Research Project: Rejuvenating lime produc-tion in Oman; resolving current challenges (SR ⁄ AGR ⁄CROP ⁄ 08 ⁄ 01).

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