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Journal of Pathology (2013), 95 (1), 201-206 Edizioni ETS Pisa, 2013 201

SHORT COMMUNICATION DIVERSITY OF TRISTEZA VIRUS IN JAMAICA

L. Fisher1,2*, P. Tennant1,2 and W.A. McLaughlin3

1 Department of Life Sciences, 4 Anguilla Close, The University of the West Indies, Mona Campus, Kingston, Jamaica 2 The Biotechnology Centre, 2 St. John’s Close, The University of the West Indies, Mona Campus, Kingston, Jamaica 3 Department of Basic Medical Sciences, Biochemistry Section, The University of the West Indies, Mona Campus, Kingston, Jamaica * Current address: Sciences Department, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 32611, USA

SUMMARY Middle East and North and South America, but it is al- so common to find relatively benign isolates that cause (CTV), one of the most serious no visible symptoms or loss of tree vigor. CTV originat- pathogens affecting species, causes consider- ed in southeast Asia and was most likely introduced into able economic losses to worldwide. other regions through the exchange of infected prop- While mild pathotypes have been known for many years agative materials and locally dispersed by several aphid in Jamaica, decline outbreaks were only recently recog- species including Toxoptera citricidus (synonym T. citri- nized after the introduction of the vector Toxoptera cit- cida) and Aphis gossypii, in a semipersistent manner ricidus in 1993. In this study, coat protein (CP) gene se- (Moreno et al., 2008). Typically the virus exists in infect- quences of isolates from four major citrus-growing re- ed trees as heterogeneous populations, with variants gions in Jamaica were obtained and their molecular di- and defective RNAs (Vives et al., 2005). Persistent in- versity compared with reference CTV genotypes from fection, coupled with repeated horizontal transmission other regions. CP sequences from Jamaican isolates mediated by aphids, has also created opportunities for showed identities between 90 to 100% at the nucleotide recombination between genotypes resulting in extensive level and shared similar identities with comparable se- virus diversity (Roy and Brlansky, 2010). quences reported for other Jamaican isolates in 2009 Since the 1990s, there have been increasing reports and 2010. Phylogenetic analysis revealed segregation in- of new incidences of CTV in Central America and the to five of seven lineages. One third of the Jamaican iso- Caribbean along with the northward movement of T. lates clustered with Florida-T30 and Spain-T385 geno- citricidus (Rocha-Peña et al., 1998). The virus was first types, with 99 to 100% identity within the lineage. The recorded in Jamaica in the early 1960s (Stell, 1961). A remaining isolates grouped closely with Florida T36-like low incidence of tristeza was noted and attributed to the genotypes or with the and stem pit- presence of A. gossypii. T. citricidus was later detected in ting B249 from Venezuela and the New Zealand CTV- 1993 (Hoy et al., 2007). Island-wide surveys conducted resistance breaking genotypes. Identities among these in 2003 documented a more than 5-fold increase in groups ranged between 91 and 99%. The detection of CTV including isolates that react to the monoclonal an- genetic variants and potential recombination events in- tibody MCA-13 in more than half of the locations previ- volving parents from different lineages provide further ously assessed in 1999 shortly after the introduction of evidence of the diversity among Jamaican CTV isolates. T. citricidus (Fisher et al., 2005). Published reports, based on the analysis of a few isolates, indicated the Key words: CTV, phylogenetic analysis, nucleotide di- presence of both decline and stem pitting isolates (Fish- versity, citrus, tristeza. er et al., 2010; Nolasco et al., 2009). Although economic losses directly linked to CTV have only been document- Citrus tristeza virus (CTV) is the causal agent of de- ed in one of the main citrus-growing regions in Jamaica cline syndromes of scions grafted on sour orange and (Lee et al., 2002), an overall decline in citrus production stem pitting in citrus species, and hybrids. His- has been noted. Production of the Jamaican torically, CTV has been a devastating pathogen to citrus (Ugli) when compared to sweet orange and grapefruit, industries worldwide and is still of concern either from shows the greatest proportionate reduction. a disease or regulatory perspective (Albiach-Marti, Citrus is one of the important traditional crops in the 2012). Destructive isolates of CTV have caused epi- Jamaican agricultural sector, as it remains a major demics in citrus-growing regions of the Mediterranean, source of economic resilience. While the domestic mar- ket is the largest market for citrus fresh fruit and ac- counts for approximately 85% of production, fruits and Corresponding author: P. Tennant juices are exported to the United Kingdom, United Fax: +876.977.1075 E-mail: [email protected] States, Canada, and other countries in the Caribbean. 026_JPP1393SC(Tennant)_201 19-03-2013 13:21 Pagina 202

202 Genetic diversity of Jamaican CTV isolates Journal of Plant Pathology (2013), 95 (1), 201-206

Given that genetic diversity, studies of CTV in Jamaica ent SSCP patterns. Amplicons of 16 isolates were puri- are limited to a few isolates and since estimation of the fied with the QIAquick system (Roche Diagnostics, genetic variation of CTV is necessary for proper diagno- Germany), TA cloned into the pGEM-T Easy vector sis and the development of long-term management (Promega, USA) and sequenced (1-2 clones per loca- strategies, the molecular characterization of Jamaican tion) in both directions using the universal M13 forward CTV isolates was extended in this study by nucleotide and reverse primers (University at Albany, USA). Vector sequence analysis of the coat protein (CP) gene of iso- and primer sequences from the CTV p27 and p18 gene lates from four major citrus growing regions. Various regions were removed and multiple sequence align- CTV strain typing methods target the CP gene (Niblett ments with those of previous studies (Fisher et al., 2010; et al., 2000; Yokomi et al., 2010), the sequences of Nolasco et al., 2009) and reference CTV sequences were which parallel the genetic structure of the terminal 3 kb performed using the ClustalX 1.8 (Thompson et al., region of the genome and allow for strain differentia- 1997). MEGA4 was used to assess the phylogenetic re- tion. lationships among the isolates using the neighbor-join- Eleven of 16 CTV isolates from four main citrus- ing method after bootstrapping to 1000 replicates for growing Jamaican regions were characterized on Dun- tree construction and inter and intra nucleotide dis- can grapefruit (Citrus aurantifolia Macf.), sweet orange tances based on the Kimura 2-parameter model (Saitou (Citrus sinensis (L.) Osb.) or sweet orange grafted onto and Nei, 1987; Nei and Kumar, 2000; Tamura et al., sour orange (Citrus aurantium L.) (Fisher et al., 2010). 2007). The recombination detection program, RDP4 Samples were collected from sweet orange grafted version Beta 4.16, was used to assess and identify poten- onto the sour orange rootstock that exhibited signs of tial recombination events and possible parental se- decline including swelling at the bud union, sparse fo- quences in the CP gene (Martin et al., 2010). The algo- liage and heavy crops of small fruits, and tested by rithms RDP (Martin and Rybicki, 2000), GENECONV DAS-ELISA using polyclonal and MCA-13 monoclonal (Padidam et al., 1999), BOOTSCAN (Martin et al., antibodies to confirm infection with CTV (Fisher et al., 2005), MaxChi (Smith, 1992), CHIMAERA (Posada 2010). Amplicons were generated by RT-PCR using the and Crandall, 2001), SISCAN (Gibbs et al., 2000), primer pairs CTV-SF15851 and CTV-SR17262 or CTV- 3SEQ (Boni et al., 2007) were employed. By default a MF15920 and CTV-MR17236 (Fisher et al., 2010). Pre- Bonferroni correction highest acceptable P-value of liminary assessment of CP amplicons by single-strand 0.05 was used throughout. conformation polymorphism (SSCP) revealed 24 differ- More than half of the Jamaican CTV isolates induced

Table 1. Characteristics of Jamaican CTV isolates cloned in the study.

Symptoms on GenBank Field MCA-13 Isolate Origin DG, SwO or accession symptoms1 reactivity3 SwO+SO2 Nos Clarendon Park 10 Clarendon Clarendon park - SwO+SO-St + HM160505 Frankfield 11 Frankfield + SwO-St + HM160506 Montpelier 12 Hanover Montpelier - SwO-ns - GU983384 Montpelier 18 - nt + HM160508 DG-SP, Knockalva 1 Knockalva + + HM160517 SwO+SO-St&B DG-SP, Knockalva 11 - + HM160518 SwO+SO-St Arscott 13 Manchester Arscott - SwO-ns + HM160512 DG-ns, Arscott 28 + + HM160513 SwO+SO-B Arscott 38 + nt + HM160514 Mile Gully 9 Mile Gully + SwO-St + HM160516 Wakefield 21 St. Catherine Wakefield + nt - GU983389 Wakefield 24 - SwO-ns - GU983388 DG-ns, Bybrook 5 Bybrook - SwO-ns, - GU983387 SwO+SO-ns DG-SP, HM160502, Linstead 2 Linstead + - SwO+SO-St GU983386 Linstead 5 + nt + HM160500 Linstead 3 + nt + HM160501 1Symptoms characteristic of CTV, such as swelling at the bud union, twig dieback, sparse foliage and canopy, heavy crops of small fruit size and quality. 2DG ‘Duncan’ grapefruit, SwO sweet orange, SwO+SO sweet orange grafted onto sour orange rootstock, B bud union phloem necrosis, St stunting, SP severe stem pitting, ns no symptom expression, nt not tested. 3Reaction with monoclonal antibody MCA-13 in DAS-ELISA. 026_JPP1393SC(Tennant)_201 19-03-2013 13:21 Pagina 203

Journal of Plant Pathology (2013), 95 (1), 201-206 Fisher et al. 203

stunting or stem pitting symptoms on Duncan grape- fruit or sweet orange or bud union necrosis on sweet or- ange grafted on sour orange (Table 1). Symptoms typi- cal of seedling yellows were not observed. All isolates tested positive for universal CTV reactivity in DAS- ELISA (data not shown) and most, including those de- rived from apparently asymptomatic field trees, showed MCA-13 reactivity (Table 1). Other isolates were MCA- 13 nonreactive even though they induced severe symp- toms on grapefruit or sweet orange suggesting that MCA-13 does not detect all severe Jamaican CTV iso- lates. Sequence analysis revealed high similarity, ranging from 90 to 100%, among the Jamaican CTV isolates cloned in this study. Similar values were also observed in comparisons with Jamaican CTV sequences previous- ly published in 2009 (91-100%) and 2010 (90-99%) that are given in Fig. 1. Phylogenetic analysis indicated clustering of all Jamaican isolates, including those previ- ously published, into groups defined by reference CTV isolates (Fig. 1), not reflecting geographic speciation, but rather the exchange of infected planting material between citrus-producing countries (Martin et al., 2009). In one instance, an isolate (Linstead 2) contained sequences belonging to more than one group but this does not preclude the existence of other isolates harbor- ing a mixture of variants. Persistent and successive in- fections by aphids with a population of genetically-relat- ed variants could explain the presence of sequences from more than one group in the same isolate (D’Urso et al., 2003). Clustering of the Jamaican isolates within the respective groups supported local spread of the virus (Iglesias et al., 2008). Fig. 1. Phylogenetic analysis of CP nucleotide sequences of Approximately 29% of the Jamaican isolates in this Jamaican CTV isolates and reference CTV isolates using the study and those reported in 2009 and 2010 clustered neighbor-joining method. The optimal tree with the sum of with the asymptomatic T30- and T385-like genotypes branch length = 0.53716498 is given. The percentage of repli- (Fig. 1), with average identities of 99-100%. The re- cate trees in which the associated taxa clustered together in maining isolates segregated in at least one of four the bootstrap tests (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in groups comprised of isolates that cause symptoms of the same units as those of the evolutionary distances used to quick decline (T36) or stem pitting (NZRB, VT, and infer the phylogenetic tree. All positions containing gaps and B249). However, a greater number of the Jamaican iso- missing data were eliminated from the dataset (complete lates (50%) were aligned with the Florida decline T36 deletion option). Sequences obtained for the Jamaican CTV and T66 lineage of group 1 or the NZRB isolates of isolates analyzed in this study were submitted to the Gen- Bank and the accession numbers are listed in Table 1. These group 2 (Fig. 1). The recently described NZRB New isolates were named based on the location from which they Zealand CTV isolates have been established as pheno- were collected. Other Jamaican isolates (Fisher et al., 2010; typically and phylogenetically diverse CTV isolates ca- Nolasco et al., 2009) were obtained from the GenBank. Fish- pable of breaking CTV-resistance in Poncirus trifoliata er et al., 2010 isolates: Jamaica Blue Hole 1 (unpublished), Ja- (Harper et al., 2010). The Jamaican isolates in this study maica Campbell’s Castle 2 FJ792686, Jamaica Summerfield 5 FJ810850, Jamaica Wakefield 25 FJ810849. Nolasco et al., shared an average sequence similarity of 91-99% with 2009: isolates Jamaica Byb82 EU579398, Jamaica Byb87 the NZRB isolates. Average identities shared with T36, EU579397, Jamaica Byb90 EU579399, Jamaica S133-21 T66, and VT were 91-98%, 91-97% and 91-93%, re- EU579403, Jamaica S133-31 EU579405, Jamaica S133-32 spectively. EU579404, Jamaica S78-35 EU579401, Jamaica S78-37 In contrast, the Jamaican isolates of this study were EU579400, Jamaica S78-44 EU579402. CTV reference iso- lates within each group are highlighted in bold. M mild iso- not as closely related to the NUagA, T3, T318, SY658, lates, D decline isolates, SP stem pitting isolates, RB resist- and B165 genotypes with average identities of 91-93%, ance-breaking isolates and SY seedling yellows isolates. 91-93%, 90-96%, 91-97% and 90-96%, respectively. 026_JPP1393SC(Tennant)_201 19-03-2013 13:21 Pagina 204

204 Genetic diversity of Jamaican CTV isolates Journal of Plant Pathology (2013), 95 (1), 201-206

Previously reported Jamaican CTV isolates (Nolasco et al., 2009; Fisher et al., 2010) also occurred in the same b clades containing severe genotypes, except for the VT- like group. Average identities shared with T36 and T66 -02 -03 -04 -06 -04 -06 -04 -04 -09 were 92-98% and 91-97%, respectively. NZRB-like genotypes were not found among the Jamaican isolates described in 2009 (Nolasco et al., 2009). On the other Average P-value 9.137 × 10 4.740 × 10 1.401 × 10 7.039 × 10 5.157 × 10 4.886 × 10 2.458 × 10 3.780 × 10 6.915 × 10 hand, Jamaican CTV isolates described in Nolasco et al. (2009), Fisher et al. (2010) and this study grouped with the B249 sweet orange and grapefruit stem pitting-in- ducing isolate from Venezuela (Febres et al., 2003). Identities were between 91and 98%. No Jamaican isolates formed a separate clade as is found with some isolates from citrus-growing regions of India (Biswas et al., 2012) and southwest Asia (Al-Sadi et al., 2012). All virus groups were supported by high bootstrap values (Fig. 1). Within- and between-group nucleotide diversity analyses indicated that isolates in group M had very low average intra-group genetic di- Methods detecting recombination events BOOTSCAN SISCAN 3Seq RDP GENECONV BOOTSCAN MaxChi Chimaera 3Seq versity (0.006±0.001) while the isolates grouping with the severe genotypes showed values up to three times a higher than the mild isolates (0.020 ± 0.003). The inter- group distances were always higher than within groups supporting separation into the seven lineages. The high- est divergence was observed among groups 4 and 5 (0.073 ± 0.009), groups 5 and M (0.074 ± 0.010) and groups 1 and 4 (0.067 ± 0.009). Of note, severe geno- types identified in the 2010 study and this study were distributed across the four surveyed regions in spite of Recombination site sequence identified in the 391-450 the absence of symptoms in field trees. Mild genotypes were found in two regions, St Catherine and Hanover. Two recombination events were detected (Table 2). The same recombination events were detected by three or more algorithms and are therefore considered evi- dence of recombination. The isolate Wakefield 25 was the major parent in both events (Table 2). Wakefield 25 was found to induce stem pitting on sweet orange (Fish- er et al., 2010). The recombinant Knockalva 1 induced stem pitting on Duncan grapefruit and bud union necrosis on sweet orange grafted on sour orange; Lin- stead 5 was not characterized on these indicator (Table 1). Genome sequencing could explain the biolog- ical significance of Wakefield 25 in pathogenesis and provide insight into the evolutionary history. Although it has been suggested that recombination hotspots are Donor isolates (major x minor) involved in recombination Wakefield 24Jamaica Wakefield 25 x Jamaica 224-299 Clarendon Jamaica Wakefield 25 x Jamaica Park 10 generally distributed throughout the 5’ ORFs of the CTV genome (Biswas et al., 2012; Roy and Brlansky, 2010), recombination events in CP gene sequences have been found to give rise to variants, albeit at a lower fre- quency (Sharma et al., 2012). Multiple recombination events resulting in isolates derived from two or more CTV genotypes have been reported in various citrus- producing regions in South America (Martin et al., Detection of recombination events in the CP gene Jamaican CTV isolates using RDP4 detection program. 2009), USA (Vives et al., 2005) and India (Roy and Br- lansky, 2010). Recombinant events with P values <0.05 was considered significant. Recombination sites represent the breakpoints based on nucleotide alignments of CP sequence. Recombinant CTV isolate identified Jamaica Linstead 5 1 Jamaica Knockalva a b The results of the phylogenetic analysis corroborated Table 2. Table 026_JPP1393SC(Tennant)_201 19-03-2013 13:21 Pagina 205

Journal of Plant Pathology (2013), 95 (1), 201-206 Fisher et al. 205

the biological characterization (Fig. 1, Table 1). Samples B249 were kindly provided by Dr. V. Febres and those infected with isolates of group M did not induce symp- of T3 by Dr. M. Hilf. tom expression on grapefruit, sweet orange, whereas those of groups 2 (Portugal 19-21- and NZRB-like) and 5 (Venezuela B249-like) caused no symptom develop- REFERENCES ment, stunting on sweet orange or bud union necrosis on sweet orange grafted on sour orange. Bud union Albiach-Marti M., 2012. Molecular virology and pathogenici- phloem necrosis associated with decline was observed ty of Citrus tristeza virus. In: Garcia M., Romanowski V. with group 1 (T36- and T66-like and intermediates) iso- (eds). Viral Genomes - Molecular Structure, Diversity, lates inoculated on sweet orange grafted on sour orange. Gene Expression Mechanisms and Host-Virus Interac- tions. pp. 275-302. IntechOpen, Croatia. 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206 Genetic diversity of Jamaican CTV isolates Journal of Plant Pathology (2013), 95 (1), 201-206

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Received September 4, 2012 Accepted November 14, 2012