Plant Pathology (2011)

Doi: 10.1111/j.1365-3059.2011.02553.x

Molecular characterization and potential sources of Citrus tristeza 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
a

Department 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, 8005139 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 (1563%) compared to the south (012%). 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 rst 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 evidence that severe strains of CTV exist in Oman. Cloning and sequencing the CP gene of six isolates showed that they have 91100% nucleotide identity with each other and 9699% with representative isolates from other parts of the world. Phylogenetic 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 coefcient of differentiation values suggest that the two isolates from Oman constitute a new CTV phylogenetic group. Keywords: CTV phylogeny, lime, Tristeza viroids. Citrus tristeza virus (CTV), a member of the genus Closterovirus of the Closteroviridae family, is the most 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 on sour orange rootstocks (Moreno et al., 2008). CTV can cause different symptoms on citrus trees depending on the strain of the virus, citrus species, environmental conditions and propagation method. These include decline of citrus trees grafted on sour orange rootstocks, stem pitting symptoms on acid lime, pummelo (C. grandis), grapefruit (C. paradise) and sweet orange, and seedling yellows (McClean, 1960; Garnsey & Lee, 1988; Lee & Bar-Joseph, 2000; Brlansky et al., 2002). CTV can be transmitted by propagation of infected buds, import of diseased planting material and several aphid species such as Toxoptera citricida, Aphis gossypii and A. spiraecola (Roistacher & Bar-Joseph, 1987). Citrus tristeza virus was rst detected in Oman in 1986 in seedlings of lemon, mandarin and grapefruit imported from India (Bove, 1995). However, little attention has been given to the disease. A preliminary survey by the Ministry of Agriculture in 2008 in 10 different farms in Oman showed that the disease was present in four of them (MA, 2009). However, because that

Introduction
Citrus is an important genus of fruit crops in the world. In Oman, acid limes (Citrus aurantifolia) are the fourth major fruit crop in terms of production after date palms, bananas and mangoes (FAO, 2010). There are over 350 000 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 in all regions in Oman, with production being concentrated in the Al-Batinah region. Most citrus species are grown on their own, with grafting being a very limited practice. However, citrus propagation is commonly practised in Oman using seeds and layering. Most lime and sweet lime plants are propagated locally, while many other citrus species are imported via nurseries from countries such as India, Pakistan, Syria, Lebanon and Egypt. Most species of citrus are susceptible to several plant pathogenic fungi, prokaryotes, viruses, nematodes and

*E-mail: alsadi@squ.edu.om

2011 The Authors Plant Pathology 2011 BSPP

A. M. Al-Sadi et al.

survey was very limited, there is still little known about the distribution of CTV in Oman, or which citrus species are affected. In addition, there is a lack of knowledge about the most common symptoms associated with CTV infection and the potential role of commercial nurseries in disseminating CTV infected citrus seedlings. This establishes a barrier towards development and implementation of effective management strategies for CTV in the country. Biological indexing has been used in the past for strain differentiation in CTV. However, because indexing is time consuming (up to 1215 months) (Mukhopadhyay, 2004; Moreno et al., 2008), several serological and molecular methods have been developed over the past two decades for characterization of CTV strains. The nding that severe isolates of CTV (decline or stem pitting inducing isolates) differ from the mild isolates at position 371 of the coat protein (CP) gene (Niblett et al., 2000) has helped develop selective primers, which through bi-directional reverse transcription PCR (RT-PCR) has helped in accurate, sensitive and fast characterization of CTV strains (Cevik et al., 1996; Niblett et al., 2000; Huang et al., 2004). Although this test has proved to be efcient in differentiating CTV strains from Florida and some other places into two types, severe and mild (Huang et al., 2004; Jiang et al., 2008), it is not clear whether Omani lime trees developing severe CTV symptoms can be identied using the test. Restriction analysis as well as phylogenetic analysis of the CP gene of CTV isolates has helped group CTV haplotypes from different parts of the world into seven different groups (Mawassi et al., 1993; Zemzami et al., 2002; Nolasco et al., 2009). The grouping was found to be related to biological activities of the haplotypes (Nolasco et al., 2009). A recent study by Nolasco et al. (2009) on more than 140 CTV haplotypes from 20 different parts of the world has shown that all belong to one of the seven CTV groups. Oman lies on the far eastern part of the Arabian Peninsula, a place which has been considered the transition point through which lime and some other citrus species have been moved from India and China to the west (Davies & Albrigo, 1994). Because no isolates were included in the study by Nolasco et al. (2009) from Oman or other parts of the Arabian Peninsula, it is not known if isolates from this part of the world belong to any of the previously described seven groups. In addition, there is a lack of knowledge concerning molecular variability of CTV from Oman, so that it is not known whether CTV in Oman originates from common or multiple sources. This study was therefore conducted to characterize occurrence, phylogeny and sources of CTV in Oman. Specic objectives include: (i) to characterize the distribution of CTV in Oman; (ii) to nd out whether commercial nurseries could play a role in spreading CTV infected seedlings; and (iii) to characterize the phylogenetic relationship of CTV isolates from Oman with CTV from other parts of the world. Investigations into these aspects of the disease may help establish a solid background about distribution and molecular variability of CTV in

Oman, which will be important in delineating a disease control strategy.

Materials and methods

Distribution of CTV
In order to characterize distribution of CTV in Oman, a survey was conducted from January 2009 to July 2010. The survey covered 22 districts located in eight different geographical regions in the Sultanate of Oman (Fig. 1). A total of 137 farms were visited, with at least ve randomly surveyed farms per district, except for four districts which are limited in the number of citrus farms. Because acid lime is the dominant species of citrus in the country (MAF, 2005), it was the main focus of the survey. Sample collection was done from a total of 526 lime trees; at least ve lime trees per farm, except for farms with limited numbers of lime trees. During the course of the survey, samples were also collected from other citrus trees which were present in the surveyed farms. Samples representing the young developing leaves and twigs were randomly collected from trees, with each sample consisting of 15 30 leaves. Samples were collected from trees seedlings showing one or more of the following symptoms: seedling yellowing, stem pitting, stunting, leaf curling, vein clearing or necrosis. In farms where these symptoms were not detected, the collection of samples was from randomly selected trees. Samples were placed in sealed plastic bags and kept in an ice box during transport to the Plant Pathology Research Laboratory at Sultan Qaboos University, and then stored at )80C for further analysis. Double antibody sandwich procedure-enzyme linked immunosorbent assay (DAS-ELISA; CTV-IDENTIKIT, Adgen) was used for detection of CTV in all the citrus samples, following the manufacturers protocol. CTV positive and negative samples provided with the kits were used as controls in every ELISA test. The colour reaction was monitored by measuring absorbance at 405 nm using MultiskanEx spectrophotometer (ThermoLabsystems). Citrus samples were considered CTV positive if the absorbance reading of the samples was at least double the absorbance of the negative control (Garnsey & Cambra, 1991).

Occurrence of CTV in nursery seedlings

In order to nd out whether citrus seedlings imported from abroad could act as potential sources of CTV into Oman, the following experimental approach was followed. Three separate surveys were carried out in the summer, autumn and winter of 2010. Each survey covered ve different nurseries located in Seeb district, Oman. A total of 119 different citrus samples were collected from nurseries, 314 samples per nursery. These samples represented different citrus species which included acid lime (21), Tahiti lime (C. latifolia) (5), sweet lime (8), sweet orange (31), sour orange (4), mandarin (24), grapefruit (3), lemon (17) and kumquat (ForPlant Pathology (2011)

Molecular characterization of CTV from Oman

Iran Dibba Oman Mahadha Yanqul Dhank Rustaq Samael Nizwa Ibri Ibra Bahla Mudhaibi Madha

UAE KSA

Salalah

Taqa Marbat

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

tunala margarite) (6). Out of the 119 samples, 86 seedlings were imported from Syria, Egypt, Lebanon, Jordon, Pakistan or India, while 33 seedlings were propagated locally. The collection of samples from nurseries was from seedlings which had just arrived from the places of origin (27) and also from imported seedlings which had been kept in nurseries for 212 weeks (92).

RT-PCR for CTV detection

Detection of CTV in the nursery samples was achieved using RT-PCR. Midribs and petioles of the citrus leaves collected from nurseries were cut and then ground using liquid nitrogen. RNA was extracted from 01 g of the ground midribs and petioles using RNeasy Mini Kit (QIAGEN) according to the manufacturers protocol. RT-PCR was performed using QIAGEN One-Step RT-PCR master mix and primers PIN1 (5-GGT TCA CGC ATA CGT TAA GCC TCA CTT-3) and PIN2 (5TAT CAC TAG ACA ATA ACC GGA TGG GTA-3) (Olmos et al., 1999). The master mix for each sample consisted of: 10 lL of 5 QIAGEN One-step RT-PCR buffer, 2 lL dNTP mix, 10 lL of 5 Q-solution, 06 lM of each
Plant Pathology (2011)

primer, 2 lL of QIAGEN One-step RT-PCR Enzyme Mix, 10 lL of the RNA sample and RNase-free water up to 50 lL. The cycling conditions were as follows: reverse transcription at 50C for 30 min, then one cycle at 95C for 15 min, followed by 40 cycles of 94C for 30 s, 60C for 30 s, and 72C for 1 min. The nal extension was at 72C for 10 min. The RT-PCR products were run on 2% agarose gels for 40 min at 120 V and 110 mA and visualized under UV after ethidium bromide staining.

Molecular discrimination of CTV strains

In order to nd out whether lime samples developing certain CTV symptoms can be discriminated using the bi-directional RT-PCR test of Huang et al. (2004), the following protocol modied from Huang et al. (2004) and Jiang et al. (2008) was used. A total of 21 randomly selected CTV-positive lime samples from eld surveys and one randomly selected CTV-positive lime sample from a nursery were included in the test. The 22 samples consisted of two samples from seedlings developing leaf curling symptoms, six samples from symptomless seedlings, 12 samples from lime trees developing stem pitting

A. M. Al-Sadi et al.

symptoms and two samples from lime trees developing leaf curling symptoms. DNA extraction from leaf midribs and petioles of the 22 samples was achieved using RNeasy Plant Mini Kit (QIAGEN) according to the manufacturers protocol. Bi-directional RT-PCR was conducted for each sample using the primers HCP1 (5-ATG GAC GAC GAA ACA AAG AA-3), HCP2 (5 -TCA ACG TGT GTT GAA TTT CC-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 master mix was used. The master mix for each sample was as before, using 06 lM of each of the above primers. The RT-PCR conditions were: reverse transcription at 50C for 50 min, initial PCR activation at 95C for 15 min, 35 cycles of denaturation at 94C for 1 min, annealing at 50C for 2 min and extension at 72C for 2 min. The nal extension step was at 72C for 20 min. Successful amplication of PCR products was checked by 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 fragment of 672 bp (HCP1 HCP2). However, fragment sizes of 320 (HCP1 CP3) and 392 bp (HCP2 CP4) indicate the potential presence of different haplotypes similar to those found in severe and mild isolates in other studies (Huang et al., 2004).

Phylogenetic analysis of CTV from Oman

In order to compare the coat protein sequence of the CTV isolates from Oman with CTV isolates from other parts of the world, the following experimental approach was followed. Six randomly selected CTV-positive samples, which produced a 320 bp band in the previous test, were used in the study (Table 1). The coat protein gene of the six CTV isolates was amplied using primers HCP1 and HCP2 (Huang et al., 2004). The RT-PCR mixture and conditions were as described previously, using 06 lM of primers HCP1 and HCP2. Successful amplication of the coat protein was checked on 2% agarose gels. The RTPCR products of the CP gene for the six samples were puried from dNTPs using QIAquick PCR Purication kit (QIAGEN) following the manufacturers protocol.

The puried samples were then cloned using MGTM pTOP TA V2 vector and sequenced at Macrogen Inc. Sequencing of the CP gene for each isolate was done from three randomly selected bacterial colonies containing the insert. The forward and backward CP gene sequences for each isolate were rst aligned together and edited using CHROMASPRO. The resulting CP gene sequences were then compared to each other and to worldwide collections of sequences deposited at the National Center for Biotechnology Information (NCBI) using BLAST searches. Search for evidence of recombination events between these and other GenBank available sequences was performed with the RDP software (Martin et al., 2005). The six sequences were aligned with 144 CP gene sequences from worldwide origins referred to in Nolasco et al. (2009) using CLUSTALW (Thompson et al., 1994). A neighbour-joining tree was constructed based on the matrix of pairwise distances obtained using the Kimura 2 parameter evolutionary model (MEGA5) (Tamura et al., 2011). Bootstrap 80% majority-rule consensus trees were generated using 1000 replications. The coefcient of differentiation was estimated according to Nei & Kumar (2000). The coefcient of differentiation is the ratio of the mean inter-group diversity over mean diversity for the entire population. If close to zero, the population is not structured (all diversity is outside the groups); if 1, all diversity falls within the specied groups.

Results
Distribution of CTV
Assessing the presence of Citrus tristeza virus using ELISA in 526 lime samples collected from 22 different districts showed that the virus was associated with 102 (194%) lime samples (Table 2). CTV in lime was detected in all the surveyed districts, except in Taqa and Marbat. Recovery of CTV from different districts varied from 0 to 63% (Table 2). Moderate to high levels of infection (1563%) were observed in districts that are located in the northern part of Oman (Table 2, Fig. 1). Limes in districts that are located in the southern part of the country were found to have low levels of infection (012%). The virus was also detected at different frequencies in sweet lime, citron, mandarin, sweet lemon (C. limetta), sweet orange and sour orange (Table 3). Symptoms typical of CTV infection were detected in some of the citrus species that were surveyed. Stem pitting was found to be the most common symptom in the eld. Other symptoms included yellowing, curling and vein clearing of leaves of different citrus species as well as stunting, especially in sweet orange grafted on sour orange rootstocks and in lime trees (Table 3). Association of CTV with these symptoms in the affected trees was conrmed using ELISA tests.

Table 1 Characteristics of Omani Citrus tristeza virus isolates used in the phylogenetic analysis Isolate accession no.a W001-4B W071-3 W093-3 W121-1 W140-1 W140-2
a

GenBank accession no. JF310691 JF310692 JF310693 JF310694 JF310695 JF310696

Year of isolation 2010 2009 2009 2009 2010 2010

District Dibba Barka Madha Sohar Suwaiq Suwaiq

Lime tree age (years) 6 5 12 2 2 2

Field symptoms Stem pitting Stem pitting Stem pitting Symptomless Symptomless Symptomless

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

Plant Pathology (2011)

Molecular characterization of CTV from Oman

Table 2 Distribution of Citrus tristeza virus (CTV)-infected acid lime trees (C. aurantifolia) in different districts in Oman Sample size (farms) 6 6 9 6 10 2 7 6 1 7 6 6 5 5 8 9 13 5 6 4 5 5 137 Sample size (trees) 26 37 30 22 40 10 16 40 4 17 17 13 29 19 10 36 33 31 30 15 31 20 526 Infected treesa 10 9 14 4 10 1 10 4 1 6 4 4 5 1 4 7 5 1 1 0 1 0 102 % trees infected 38 24 47 18 25 10 63 10 25 35 24 31 17 5 40 19 15 3 3 0 3 0

Locations Barka Rustaq Shinas Sohar Suwaiq Boushar Seeb Qurayat Dhank Yanqul Ibri Mahadha Bahla Nizwa Samael Dibba Madha Ibra Mudhaibi Marbat Salalah Taqa Total

sweet lime and Tahiti lime, with frequencies of infection ranging from 0 to 100% (mean 45%; Table 4). CTV was detected in citrus seedlings coming from Egypt, Pakistan, Syria, Lebanon and India as well as from citrus seedlings originating from Oman. The virus was detected in seedlings at the time of arrival from the country of origin as well as in seedlings that had been imported from abroad but kept in nurseries for 212 weeks.

Molecular discrimination of CTV strains

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

Phylogenetic analysis of CTV isolates

Amplication of the coat protein gene for six Omani isolates produced a 672 bp fragment that was sequenced. Sequences were deposited in GenBank with the accession numbers JF310691 to JF310696. Evidence for recombination between these and other available GenBank sequences was not found. Three of the isolates, W071-3, W93-3 and W121-1 showed an identical nucleotide sequence. However, isolate W001-4B coming from Dibba showed two nucleotide mismatches from the rest of the Omani isolates. Isolates W140-1 and W140-2 showed one base pair mismatch from each other but showed 911% similarity to the other Omani isolates. These two isolates came from seedlings showing no apparent symptoms of CTV infection.

a Presence 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 nursery seedlings from different countries showed that 53 were positive for CTV. The positive samples produced a fragment that was 131 bp following amplication 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 Field symptoms Host Lime Sweet lime Sweet orange Citron Mandarin Lemon Sour orange Sweet lemon Total Sample size 526 29 23 10 6 5 5 3 607 No. (%) infected trees 102 (194) 4 (138) 4 (174) 3 (300) 2 (333) 0 1 (200) 1 (333) 117 (193) SP M L L L VC R R R ST L R M R R LC L R R L R L Locationa,b 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22 1,3,5,8,12,16,17 1,3,5,8,12,16,20 2,4,5,8,16,17 5,12 1,5 3,16,17 12,16

SP, stem pitting; VC, vein clearing; ST, stunting; LC, leaf curling; R, rare (05%); L, low (615%); M, moderate (1640%); H, high (>40%); , not detected quantied. a 1: Bahla, 2: Bousher, 3: Barka, 4: Dhank, 5: Dibba, 6: Ibra, 7: Ibri, 8: Madha, 9: Mahadha, 10: Marbat, 11: Mudhaibi, 12: Nizwa, 13: Qurayat, 14: Rustaq, 15: Salalah, 16: Samael, 17: Seeb, 18: Shinas, 19: Sohar, 20: Suwaiq, 21: Taqa, 22: Yanqul. b Districts that are in bold indicate presence of CTV.

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A. M. Al-Sadi et al.

Table 4 Detection of Citrus tristeza virus (CTV) in nursery seedlings originating from different countries Seedlings positive for CTVb Citrus species Lime Lemon Origin Oman India Lebanon Syria Egypt Egypt Pakistan Syria Jordon Egypt Syria Syria Syria Oman Oman Lebanon Oman Sample size (no. seedlings) 18 3 11 4 2 14 2 13 2 13 11 3 6 4 6 2 5 No. (%) of infected seedlingsa 9 3 5 4 0 3 2 7 0 4 5 1 3 0 4 2 1 (50%) (100%) (45%) (100%) (21%) (100%) (54%) (31%) (45%) (33%) (50%) (67%) (100%) (20%) Nursery + ? + ? ) + + + ) + + ) + ) + + + Just arriving + + + + ? ? ? + ? ? + + + ? + ? ?

Sweet orange

Mandarin Grapefruit Kumquat Sour orange Sweet lime Tahiti lime

a

Based on RT-PCR analysis using primers PIN1 and PIN2. Detection 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 RT-PCR product (bp)a

Discussion
Citrus tristeza virus was detected in most of the surveyed districts in Oman, with frequencies of infection ranging from 0 to 63% (mean 194%). Moderate to high levels of CTV infection (1563%) were detected in districts located in the northern part of Oman compared to the south (012%). The highest levels of CTV infection in the north could be related to the proximity of these districts to the entry sites of citrus seedlings into Oman as well as to the main nurseries that distribute and sell citrus seedlings. Support for this hypothesis is evident from the high level (45%) of infection observed in this study in citrus seedlings imported by nurseries from abroad. Previous studies have emphasized the role of nurseries in disseminating many citrus diseases, including CTV (Bove, 1995). The contribution of nurseries towards spread of CTV in Oman, as shown in this study, may necessitate future management programmes looking at obtaining clean planting material, as well as proper inspection of citrus material imported from abroad. Occurrence of CTV in most of the surveyed districts in Oman may indicate that CTV has been introduced into Oman and disseminated to different districts over a long period of time, or that multiple introductions are occurring. These hypotheses are supported by the occurrence of different CTV haplotypes with different CP sequences as well as of different CTV strains, as evident from the bi-directional RT-PCR test. Since no previous surveys have been undertaken to characterize occurrence of CTV in Oman, except a recent and limited survey by the Ministry of Agriculture in 2008 (MA, 2009), it is difcult to determine when CTV was rst introduced into the country. However, detection of CTV in
Plant Pathology (2011)

Growth stage

Field symptoms

Sample 320 392 320 and 392 size 1 2 12 1 0 0 0 0 1 4 0 1

Seedling Leaf curling yellowing 2 Symptomless 6 Fruiting Stem pitting 12 trees Leaf curling yellowing 2
a

Based 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 gene sequences of the Omani isolates clustered in different positions (Fig. 2). Isolates W001-4B, W071-3, W93-3 and W121-1 clustered, according to the grouping scheme proposed by Nolasco et al. (2009), in Group 4 along with the Florida decline-inducing isolate T3. Isolates W140-1 and W140-2 appear with a very good bootstrap support as a separate clade in the branch leading to Group 5. In the collapsed tree obtained from 144 worldwide sequences it can be seen that the W140 cluster separates from the closest group (Group 5) at a distance comparable to the divergence of some other groups (Fig. 2). To investigate whether this could be considered a different group, the coefcient of differentiation was estimated with the W140 haplotypes either included or not included in Group 5. The coefcient rises from 0775 to 0809 if W140 haplotypes are considered as a different group instead of part of Group 5.

Molecular characterization of CTV from Oman

(a)

100 89 94 100 100 100 100 100 99 99 96

Gp 1 Gp 2 Gp M Gp 5 W140-1 W W140-2 Gp 4 Gp 3b Gp 3a

(c)
C270-3-12 Argentina (Gp 5) C270-3-7 Argentina (Gp 5) 442-2 Croatia (Gp 5) 2-98 Madeira (Gp 5) PeralAC-31 Brazil (Gp 5) Col4 Colombia (Gp 5) Col1 Colombia (Gp 5) PeralAC-42 Brazil (Gp 5) ST1510 S Tome (Gp 5) 134B Reunion (Gp 5) 2-1-05 Madeira (Gp 5) BaraoB-4 Brazil (Gp 5) 15-118 Madeira (Gp 5) 010-8 Venezuela (Gp 5) CY98-30-1 Cyprus (Gp 5) 86 CY98-33-61 Cyprus (Gp 5) 86 CY94-39-1 Cyprus (Gp 5) 047-16 Venezuela (Gp 5) C320-5-21 Argentina (Gp 5) B249 Venezuela (Gp 5) C257-7-2 Argentina (Gp 5) 100 C320-5-22 Argentina (Gp 5) C257-2-27 Argentina (Gp 5) S78- 4 Jamaica (Gp 5) 100 99 100 S78-35 Jamaica (Gp 5) S133-31 Jamaica (Gp 5) W140-1 W140-2

004

003

002

001

000

(b)
98

443-13 Croatia (Gp 4) 441-3 Croatia (Gp 4) 441-2 Croatia (Gp 4) 443-4 Croatia (Gp 4) 15-8S Tome (Gp 4) 15-11 S Tome (Gp 4) 99 CY89-60-36 Cyprus (Gp 4) CY89-60-38 Cyprus (Gp 4) 134A Reunion (Gp4)

100

98

100

W001-4b W071-3 Oman W121-1 W93-3 T3 Florida (Gp 4) AN0-1 Egypt (Gp 4) K1-76 Egypt (Gp 4) K1-77 Egypt (Gp 4)

Oman

003

002

001

000

004

003

002

001

000

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 dened by Nolasco et al. (2009). The tree was constructed by the neighbourjoining 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 could have been continuously introduced into Oman since that time or even before. Detection of CTV in citrus seedlings imported by nurseries from different countries is further evidence that CTV has been continuously introduced into Oman. The wide distribution of CTV across Oman could also be attributed to the use of layering in citrus propagation. Layering is commonly practised by farmers in Oman for propagation of citrus trees, especially lime. Previous studies have provided evidence for transmission of CTV via seedlings propagated vegetatively (BarJoseph & Lee, 1989). Because CTV is not known to be transmitted via citrus seeds (Bar-Joseph & Lee, 1989), detection of CTV in some lime trees originating from seeds (data not provided) may provide evidence that CTV could have been transmitted among some farms in Oman via aphids. A range of typical CTV symptoms was detected in citrus seedlings and trees that tested positive for CTV. Stem pitting was found to be the most common, especially in lime trees. Stunting was found to be common in sweet orange grafted on sour orange and in some affected lime trees. Leaf cupping of lime and vein clearing were also
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detected and found to be common, especially in seedlings. This appears to be the rst record of association of CTV with these symptoms and with sweet orange, sour orange, sweet lime, sweet lemon, citron and mandarin in Oman. However, future studies may consider quantifying the amount of economic loss due to infection of citrus species with CTV. Molecular-based characterization of CTV strains in Oman provided evidence that there are at least two different CTV strains present. This was evident from RT-PCR analysis of the CP gene with the use of HCP1, HCP2, CP3 and CP4 primers for 22 CTV-positive samples. RT-PCR analysis of 12 samples obtained from lime trees showing stem pitting symptoms provided evidence that all the samples have severe CTV strains. This shows the efciency of the bi-directional RT-PCR test in discriminating severe strains of CTV, at least those that are causing stem pitting symptoms in Oman, which is in agreement with ndings obtained by Huang et al. (2004). A mixture of at least two strains (potentially severe and mild) was detected in samples obtained from symptomless lime seedlings and a tree and a seedling with mild symptoms, but not in trees developing stem pitting symptoms. This may suggest that the presence of a mild strain confers protection against the severe strain (cross-protection) and

A. M. Al-Sadi et al.

therefore masks or reduces severity of symptoms; a hypothesis that deserves further investigation. The six sequenced Omani isolates are in different groups (clades) with high bootstrap values which suggests that at least two introductions of divergent isolates have occurred in Oman. Also, the genetic similarity of CTV isolates from Oman and other countries conrms long distance movement by trafc of propagative material through nurseries. This is supported by detection of CTV in nursery seedlings imported from different countries. Variability of the Omani CTV isolates supports previous studies of existence of variation in CTV isolates infecting 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 to the other CP groups. This suggests the existence of a new phylogenetic group separating from the Group 5 branch. The position of this clade could not be explained by recombination events (which were not detected) and the level of divergence at which it separates is comparable to the level of divergence of individualization of other groups. Additionally, the coefcient of differentiation increases when these haplotypes are considered apart from Group 5. This evidence supports the view that these sequences may constitute a new phylogenetic group. Given that these isolates were obtained from two symptomless lime seedlings, it remains to be seen if these kinds of haplotypes are widespread in Oman and associated with any particular symptomatology, or just represent some atypical CTV isolates. This study has provided evidence for widespread distribution of CTV in different regions of Oman and the association with several citrus species and eld symptoms. Due to the high level of infection with CTV (45%) of imported citrus seedlings, strict quarantine measures need to be introduced and implemented in order to save the citrus industry in Oman. In-depth analysis of CTV diversity in Oman and neighbouring countries in the Arabian Peninsula is required, especially after uncovering the existence of CTV haplotypes from Oman creating a new phylogenetic group. Biological indexing will be required to conrm association of CTV with many of the symptoms observed. Seedlings from which the W140 haplotypes were recovered are under close observation for eld symptoms, and pathogenicity tests are planned to uncover their biological characteristics.

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Acknowledgements
The authors would like to acknowledge Dalia Al-Khatib, Aisha Al-Ghaithi, Hanan Al-Moqbali, Safa AlMazroui, Amna Al-Jabri, staff from the Ministry of Agriculture and farmers for their help during surveys and laboratory work. Special thanks are due to Sultan Qaboos University for funding the study through the Strategic Research Project: Rejuvenating lime production in Oman; resolving current challenges (SR AGR CROP 08 01).

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Molecular characterization of CTV from Oman

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