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Keywords:

  • CTV phylogeny;
  • lime;
  • Tristeza

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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 evidence 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. 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 coefficient of differentiation values suggest that the two isolates from Oman constitute a new CTV phylogenetic group.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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 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 first 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 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 12–15 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 finding 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 efficient 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 identified 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. Specific objectives include: (i) to characterize the distribution of CTV in Oman; (ii) to find 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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 five 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 five 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 −80°C for further analysis.

image

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

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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 manufacturer’s 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 find 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 five different nurseries located in Seeb district, Oman. A total of 119 different citrus samples were collected from nurseries, 3–14 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 (Fortunala 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 2–12 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 0·1 g of the ground midribs and petioles using RNeasy Mini Kit (QIAGEN) according to the manufacturer’s 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 (5′-TAT CAC TAG ACA ATA ACC GGA TGG GTA-3′) (Olmos et al., 1999). The master mix for each sample consisted of: 10 μL of 5× QIAGEN One-step RT-PCR buffer, 2 μL dNTP mix, 10 μL of 5× Q-solution, 0·6 μm of each primer, 2 μL of QIAGEN One-step RT-PCR Enzyme Mix, 10 μL of the RNA sample and RNase-free water up to 50 μL. The cycling conditions were as follows: reverse transcription at 50°C for 30 min, then one cycle at 95°C for 15 min, followed by 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min. The final extension was at 72°C 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 find 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 modified from Huang et al. (2004) and Jiang et al. (2008) was used. A total of 21 randomly selected CTV-positive lime samples from field 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 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 manufacturer’s 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 0·6 μm of each of the above primers. The RT-PCR conditions were: reverse transcription at 50°C for 50 min, initial PCR activation at 95°C for 15 min, 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 2 min and extension at 72°C for 2 min. The final extension step was at 72°C for 20 min.

Successful amplification of PCR products was checked by running 10 μL 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 amplified using primers HCP1 and HCP2 (Huang et al., 2004). The RT-PCR mixture and conditions were as described previously, using 0·6 μm of primers HCP1 and HCP2. Successful amplification of the coat protein was checked on 2% agarose gels. The RT-PCR products of the CP gene for the six samples were purified from dNTPs using QIAquick PCR Purification kit (QIAGEN) following the manufacturer’s protocol. The purified 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.

Table 1.   Characteristics of Omani Citrus tristeza virus isolates used in the phylogenetic analysis
Isolate accession no.aGenBank accession no.Year of isolationDistrictLime tree age (years)Field symptoms
  1. aAll isolates were obtained from acid lime (Citrus aurantifolia).

W001-4BJF3106912010Dibba6Stem pitting
W071-3JF3106922009Barka5Stem pitting
W093-3JF3106932009Madha12Stem pitting
W121-1JF3106942009Sohar2Symptomless
W140-1JF3106952010Suwaiq2Symptomless
W140-2JF3106962010Suwaiq2Symptomless

The forward and backward CP gene sequences for each isolate were first 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 coefficient of differentiation was estimated according to Nei & Kumar (2000). The coefficient 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 specified groups.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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 (19·4%) 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 (15–63%) 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 (0–12%). The virus was also detected at different frequencies in sweet lime, citron, mandarin, sweet lemon (C. limetta), sweet orange and sour orange (Table 3).

Table 2.   Distribution of Citrus tristeza virus (CTV)-infected acid lime trees (C. aurantifolia) in different districts in Oman
LocationsSample size (farms)Sample size (trees)Infected treesa% trees infected
  1. 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).

Barka6261038
Rustaq637924
Shinas9301447
Sohar622418
Suwaiq10401025
Boushar210110
Seeb7161063
Qurayat640410
Dhank14125
Yanqul717635
Ibri617424
Mahadha613431
Bahla529517
Nizwa51915
Samael810440
Dibba936719
Madha1333515
Ibra53113
Mudhaibi63013
Marbat41500
Salalah53113
Taqa52000
Total137526102 
Table 3.   Citrus species infected with Citrus tristeza virus (CTV) in Oman
HostSample sizeNo. (%) infected treesField symptomsLocationa,b
SPVCSTLC
  1. SP, stem pitting; VC, vein clearing; ST, stunting; LC, leaf curling; R, rare (0–5%); L, low (6–15%); M, moderate (16–40%); H, high (>40%); –, not detected/quantified.

  2. a1: 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.

  3. bDistricts that are in bold indicate presence of CTV.

Lime526102 (19·4)MRLL1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22
Sweet lime294 (13·8)LRRR1,3,5,8,12,16,17
Sweet orange234 (17·4)LMR1,3,5,8,12,16,20
Citron103 (30·0)LRL2,4,5,8,16,17
Mandarin62 (33·3)RR5,12
Lemon50R1,5
Sour orange51 (20·0)L3,16,17
Sweet lemon31 (33·3)12,16
Total607117 (19·3)     

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 field. 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 confirmed using ELISA tests.

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 amplification with PIN1 and PIN2 primers. The virus was detected in acid lime, lemon, sweet orange, mandarin, grapefruit, kumquat, 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 2–12 weeks.

Table 4.   Detection of Citrus tristeza virus (CTV) in nursery seedlings originating from different countries
Citrus speciesOriginSample size (no. seedlings)No. (%) of infected seedlingsaSeedlings positive for CTVb
NurseryJust arriving
  1. aBased on RT-PCR analysis using primers PIN1 and PIN2.

  2. 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.

LimeOman189 (50%)++
India33 (100%)?+
LemonLebanon115 (45%)++
Syria44 (100%)?+
Egypt20?
Sweet orangeEgypt143 (21%)+?
Pakistan22 (100%)+?
Syria137 (54%)++
Jordon20?
MandarinEgypt134 (31%)+?
Syria115 (45%)++
GrapefruitSyria31 (33%)+
KumquatSyria63 (50%)++
Sour orangeOman40?
Sweet limeOman64 (67%)++
Lebanon22 (100%)+?
Tahiti limeOman51 (20%)+?

Molecular discrimination of CTV strains

Analysis of 22 CTV-positive citrus samples using RT-PCR 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).

Table 5.   RT-PCR analysis of Citrus tristeza virus (CTV) strains associated with 22 lime trees and seedlings
Growth stageField symptomsSample sizeRT-PCR product (bp)a
320392320 and 392
  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).

SeedlingLeaf curling/yellowing2101
Symptomless6204
Fruiting treesStem pitting121200
Leaf curling/yellowing2101

Phylogenetic analysis of CTV isolates

Amplification 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 91·1% similarity to the other Omani isolates. These two isolates came from seedlings showing no apparent symptoms of CTV infection.

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 coefficient of differentiation was estimated with the W140 haplotypes either included or not included in Group 5. The coefficient rises from 0·775 to 0·809 if W140 haplotypes are considered as a different group instead of part of Group 5.

image

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).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Citrus tristeza virus was detected in most of the surveyed districts in Oman, with frequencies of infection ranging from 0 to 63% (mean 19·4%). Moderate to high levels of CTV infection (15–63%) were detected in districts located in the northern part of Oman compared to the south (0–12%). 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 difficult to determine when CTV was first introduced into the country. However, detection of CTV in 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 (Bar-Joseph & 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 detected and found to be common, especially in seedlings. This appears to be the first 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 efficiency 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 findings 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 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 confirms long distance movement by traffic 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 coefficient 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 field 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 confirm association of CTV with many of the symptoms observed. Seedlings from which the W140 haplotypes were recovered are under close observation for field symptoms, and pathogenicity tests are planned to uncover their biological characteristics.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors would like to acknowledge Dalia Al-Khatib, Aisha Al-Ghaithi, Hanan Al-Moqbali, Safa Al-Mazroui, 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).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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