Detection and characterization of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> stem pitting isolates in Jamaica

An island wide survey for Citrus tristeza virus (CTV) in citrus orchards across Jamaica (13 regions) was conducted over 2 years. Trees (1, 885) showing virus-like symptoms as well as asymptomatic trees were randomly sampled for testing by ELISA and 55 samples from the 6 major citrus growing regions were graft inoculated on indicator plants. Most samples (74%) reacted to polyclonal antibodies against CTV in ELISA, while 20% were positive in tests using monoclonal antibodies specific to severe CTV strains. Samples collected from the 6 major citrus growing regions produced vein clearing and stem pitting symptoms on Mexican lime indicator plants (87%). In addition, stem pitting symptoms were induced on Duncan grapefruit, sweet orange, sour orange or sweet orange grafted on sour orange. Nucleotide sequencing of the coat protein gene sequences isolated from these samples indicated high identities (88 to 95.5%) among the Jamaican isolates and previously reported stem pitting strains from Central and North America and Eurasia (88 to 100%). The results suggest a shared ancestry with isolates from other geographical locations, rather than geographical speciation, and presumably separate CTV introductions into Jamaica.     

Biological characterization of citrus tristeza virus isolates by in vitro tissue cultures

Stem segments from Mexican lime, sweet orange, grapefruit, Citrus excelsa and alemow, infected with five citrus tristeza virus (CTV) isolates, were cultured in vitro . Regeneration of roots and shoots were modified as a result of infection. The effect of CTV on the morphogenesis of stem segments cultured in vitro depended on the CTV isolate and the plant host, and showed a correlation with the in vivo effects observed in biological indexing. Evaluation of the morphogenic response of stem segments of Mexican lime and grapefruit can be used as an additional tool for the biological characterization of CTV isolates. The symptoms on sweet orange plants obtained from regenerated shoots indicated that CTV is unevenly distributed in the host plant cells and that the regeneration process may be utilized as a tool to separate strains from complex field isolates.     

Selective transmission of the seedling yellows-type genotypic variants of Citrus tristeza virus by Aphis gossypii in northern Iran

Citrus tristeza virus (CTV) has existed in northern Iran for more than five decades. The long-time interaction of different virus genotypes with Aphis gossypii, as the only aphid vector of CTV in northern Iran, has led to the emergence of highly virulent subpopulations, among others, in the established foci. Here, we studied the population structure of the originally established CTV isolates present in Satsuma mandarin (Citrus unshiu) trees imported from Japan, and subisolates thereof, formed following experimental transmission by A. gossypii, as well as those evolved through natural transmission by this aphid species in the groves. Symptoms of the naturally spread and the experimentally aphid-transmitted isolates were similar to those of the Satsuma CTV source isolates for all indicator plants except for sour orange (Citrus aurantium) and grapefruit (Citrus paradisi), with the aphid-transmitted isolates additionally inducing severe seedling yellows and stunting in these two indicators. Studies on the population heterogeneity of these isolates through comparison of their single-strand conformational polymorphism profiles and nucleotide sequences of the 25 kDa capsid protein gene from the predominant haplotypes, and dot-blot hybridization signals, revealed the presence of two major T36- and SY568- (or NUagA-) like genotypes along with a minor poorly characterized one in the originally infected Satsuma trees; in contrast, only a certain genomic variant having the highest similarity to the isolate SY568 (and NUagA) was predominant both in the naturally infected trees and in those infected experimentally by A. gossypii. It seems that transmission by A. gossypii to sweet orange (Citrus sinensis) has led to the preponderance of the CTV genomic variants inducing severe seedling yellows in northern Iran.     

Characterization of Citrus tristeza virus isolates in northern Iran

The biological and molecular properties of four Citrus tristeza virus (CTV) isolates isolated from infected Satsuma trees imported from Japan, and growing in citrus groves in northern Iran (Mahdasht orchards, Mazandaran Province), were investigated. CTV-infected samples were collected from sweet orange trees and grafted onto Alemow (Citrus macrophylla Wester) seedlings. On indicator plants, these isolates produced various symptoms including vein clearing and stem pitting on Mexican lime, Alemow, and Citrus hystrix, and yellowing and stunting on sour orange and grapefruit seedlings. Citrus samples were also surveyed for CTV using serological tests. The coat protein (CP) gene of these isolates was amplified using specific primers, yielding an amplicon of 672 bp for all isolates. Sequence analysis showed 98%–99% sequence homology of Iranian isolates with the Californian CTV severe stem-pitting isolate SY568 and 97%–98% homology with the Japanese seedling yellows isolate NUagA. The Iranian isolates were compared by restriction fragment length polymorphism (RFLP) analysis of the CP amplicon for further classification.     

Increase and Patterns of Spread of Citrus Tristeza Virus Infections in Costa Rica and the Dominican Republic in the Presence of the Brown Citrus Aphid, Toxoptera citricida

ABSTRACT Citrus tristeza virus (CTV) was monitored for 4 years by monoclonal antibody probes via enzyme-linked immunosorbent assay in four citrus orchards in northern Costa Rica and four orchards in the Dominican Republic following the introduction of the brown citrus aphid, Toxoptera citricida. The Gompertz nonlinear model was selected as the most appropriate in most cases to describe temporal increase of CTV. Ordinary runs analysis for association of CTV-positive trees failed to show a spatial relationship of virus status among immediately adjacent trees within or across rows. The beta-binomial index of dispersion for various quadrat sizes suggested aggregations of CTV-positive trees for all plots within the quadrat sizes tested. Spatial autocorrelation analysis of proximity patterns suggested that aggregation often existed among quadrats of various sizes up to four lag distances; however, significant lag positions discontinuous from the main proximity pattern were rare. Some asymmetry was also detected for some spatial autocorrelation proximity patterns. These results were interpreted to mean that, although CTV-positive trees did not often influence immediately adjacent trees, virus transmission was common within a local area of influence that extended two to eight trees in all directions. Where asymmetry was indicated, this area of influence was somewhat elliptical. The spatial and temporal analyses gave some insight into possible underlying processes of CTV spread in the presence of T. citricida and suggested CTV spread was predominantly to trees within a local area. Patterns of longer-distance spread were not detected within the confines of the plot sizes tested. Longer-distance spread probably exists, but may well be of a complexity beyond the detection ability of the spatial analysis methods employed, or perhaps is on a scale larger than the dimensions of the plots studied.     

Spatial and temporal analyses of citrus sudden death as a tool to generate hypotheses concerning its etiology

ABSTRACT Citrus sudden death (CSD), a new disease of unknown etiology that affects sweet orange grafted on Rangpur lime, was visually monitored for 14 months in 41 groves in Brazil. Ordinary runs analysis of CSD-symptomatic trees indicated a departure from randomness of symptomatic trees status among immediately adjacent trees mainly within rows. The binomial index of dispersion (D) and the intraclass correlation (k) for various quadrat sizes suggested aggregation of CSD-symptomatic trees for almost all plots within the quadrat sizes tested. Estimated parameters of the binary form of Taylor's power law provided an overall measure of aggregation of CSD-symptomatic trees for all quadrat sizes tested. Aggregation in each plot was dependent on disease incidence. Spatial autocorrelation analysis of proximity patterns suggested that aggregation often existed among quadrats of various sizes up to three lag distances; however, significant lag positions discontinuous from main proximity patterns were rare, indicating a lack of spatial association among discrete foci. Some asymmetry was also detected for some spatial autocorrelation proximity patterns, indicating that within-row versus across-row distributions are not necessarily equivalent. These results were interpreted to mean that the cause of the disease was most likely biotic and its dissemination was common within a local area of influence that extended to approximately six trees in all directions, including adjacent trees. Where asymmetry was indicated, this area of influence was somewhat elliptical. Longer-distance patterns were not detected within the confines of the plot sizes tested. Annual rates of CSD progress based on the Gompertz model ranged from 0.37 to 2.02. Numerous similarities were found between the spatial patterns of CSD and Citrus tristeza virus (CTV) described in the literature, both in the presence of the aphid vector, Toxoptera citricida. CSD differs from CTV in that symptoms occur in sweet orange grafted on Rangpur lime. Based on the symptoms of CSD and on its spatial and temporal patterns, our hypothesis is that CSD may be caused by a similar but undescribed pathogen such as a virus and probably vectored by insects such as aphids by similar spatial processes to those affecting CTV.     

Evidence for resistance to Citrus tristeza virus in pomelo (Citrus maxima Merr.) grown in Darjeeling and Sikkim hills of India

Darjeeling and Sikkim hills of India are well known for production of mandarin orange (Citrus reticulata). The recent spread of Citrus tristeza virus (CTV) has threatened the citrus cultivation in this region. During a survey in Darjeeling and Sikkim hills, pomelo trees were recorded as CTV free. Since pomelo trees did not show any disease appearance, a study was undertaken to ascertain whether they are resistant to CTV infection or resistant to aphid feeding or both. Toxoptera citricida, the most efficient aphid vector and which is abundantly present in this region, did not feed on pomelo when other Citrus species such as mandarin, kagzi lime and rough lemon were available. Additionally, CTV isolates of Darjeeling and Sikkim hills were not transmissible to pomelo either by T. citricida or grafting. We report for the first time that pomelo is resistant to isolates of CTV present in this region.     

Comparison of the minor coat protein gene sequences of aphid-transmissible and -nontransmissible isolates of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis>

The nucleotide sequences for the minor coat protein (CPm) gene and its deduced amino acid sequences for two aphid-transmissible and two nontransmissible isolates of Citrus tristeza virus (CTV) from symptomless orchard trees of Miyagawa satsuma [Citrus unshiu (Macf.) Marc.] on trifoliate orange [Poncirus trifoliate (L.) Raf.] and declining Washington navel [C. sinensis (L.) Osb.] trees on sour orange (C. aurantium L.) rootstocks were analyzed and compared with those of highly transmissible CTV strains available in GenBank. The isolates produced severe symptoms on indicator plants and their aphid transmissibility was assayed through acquisition by A. gossypii of CTV and subsequent inoculation feeding on young Mexican lime seedlings. The CPm gene nucleotides and coded amino acid sequences were very similar among the nontransmissible isolates and among the transmissible. Five of 73 nucleotide substitutions that existed between CPm gene nucleotide sequence of nontransmissible and transmissible isolates caused changes in the deduced amino acid sequences of the nontransmissible isolates. Two nucleotide substitutions yielded new amino acids with similar properties. However, the three remaining mutations led to substitution of new amino acids with a different charge and polarity at positions 14, 238 and 239. The last two mutations occurred at the C-terminal region of the CPm, which is implicated in the formation of a salt bridge that helps to maintain the protein’s tertiary structure. Amino acid substitutions can affect aphid transmission efficiency by altering the conformation of the proteins or masking motifs involved in the interaction between CPm and aphid stylets.     

柑橘衰退病毒含量对其蚜传效率的影响

为明确毒源植株和蚜虫中柑橘衰退病毒(Citrus tristeza virus,CTV)的发生情况与蚜虫传播CTV能力的关系,将褐色橘蚜、棉蚜、橘二叉蚜和绣线菊蚜置于分别感染了4个CTV分离株的锦橙上取食24 h后,运用巢式RT-PCR和实时RT-PCR检测蚜虫和锦橙的带毒情况,并分析蚜虫的传毒能力。结果显示,蚜虫中CTV的平均带毒率为0.76~0.84,其中棉蚜的最高,其次为绣线菊蚜、褐色橘蚜和橘二叉蚜。锦橙中各CTV分离株的含量差异不大,与蚜虫传毒效率间无显著相关性;也未发现蚜虫带毒率与其传播CTV能力之间存在相关性。蚜虫体内CTV的含量为5.36×103±2.33×103~2.01×106±3.67×105拷贝,褐色橘蚜中含量最高,其次为棉蚜、橘二叉蚜和绣线菊蚜;且高蚜传能力CTV分离株在褐色橘蚜体内的含量远高于低蚜传能力分离株。表明蚜虫体内CTV的含量可能与蚜虫传毒能力有关。     

Separation and interference of strains from a citrus tristeza virus isolate evidenced by biological activity and double-stranded RNA (dsRNA) analysis

Separation of strains of citrus tristeza virus (CTV), differentiated by their double-stranded RNA (dsRNA) profiles, was obtained by graft-inoculating citron plants from a Mexican lime that had been recently aphid- or graft-inoculated with a mild CTV isolate (T-385). Up to 24 sub-isolates with differing dsRNA profiles were obtained from the aphid-inoculated lime. Some of these sub-isolates induced stronger symptoms in several citrus species than the original T-385 isolate. One sub-isolate, T-385-33, was mild in Mexican lime, but induced stem pitting on sweet orange. Inoculation of this isolate on Mexican lime, sour orange and Eureka lemon induced mild or no symptoms when inoculum was taken from citron, but very severe symptoms when the inoculum was from sweet orange. Mexican lime and sweet orange plants co-inoculated with T-385-33 from sweet orange in combination with the other 23 sub-isolates showed mild symptoms. The results obtained suggest that there is natural cross-protection among sub-isolates in the original T-385 isolate.     

Biological, serological and molecular characterization of three isolates of Citrus tristeza closterovirus introduced into Morocco

The biological, serological and genomic diversity of three Citrus tristeza virus (CTV) isolates from various geographical regions was studied: isolate P1 from lemon cv. 'Meyer' in a field near Marrakech (MA) in 1983, and isolates P2 and R1 detected in imported Spanish clementine germplasm by the Moroccan NPPO in 1998 and 2000. P1 induced severe vein clearing on Mexican lime and grapefruit, mild stem pitting on Mexican lime and moderate stem pitting on grapefruit. P2 and R1 only induced mild vein clearing on Mexican lime and caused no stem pitting or other symptoms on indicator plants used as controls. Only isolate P1 reacted with monoclonal antibody MCA-13, whereas all isolates reacted positively with the 3DF1+3CA5 mixture. The Moroccan clones P1–3 and P1–5, and all other severe isolates obtained from GenBank, showed a phenylalanine at amino acid position 124 of their coat protein sequences. This epitope confers MCA13 reactivity. The Spanish clones had tyrosine instead at this position. The deduced amino-acid sequence of coat protein of P1 clones clusters close to severe strains CB3–104 and FL7, respectively from Brazil and USA (Florida) (Group 5), whereas the sequences from P2 and R1 cluster close to typical strains from Portugal 25–120 and USA (Florida) T30 (Group M). The three techniques for distinguishing CTV isolates were clearly correlated.     

Examination of the effect of aphid vector population composition on the spatial dynamics of citrus tristeza virus spread by stochastic modeling

ABSTRACT Aphid vector species population composition is known to affect the spatial patterns of citrus tristeza virus (CTV) and the changes in these patterns over time. However, the biological processes that are associated with virus spread have not been well defined. The spatiotemporal dynamics of CTV were examined using data collected from research plots in the Dominican Republic and Costa Rica, where the brown citrus aphid (BCA), Toxoptera citricida, was the predominant species, and in Florida, where the BCA was absent and the melon aphid, Aphis gossypii, was the predominant vector. Data were analyzed using a spatiotemporal stochastic model for disease spread, and parameter values were evaluated using Markov chain Monte Carlo stochastic integration methods. Where the melon aphid was the dominant species, the model parameter likelihood values supported the hypothesis that the disease was spread through a combination of random background transmission (transmission originating from inoculum sources outside the plot) and a local interaction (transmission from inoculum sources within the plot) operating over short distances. Conversely, when BCA was present, results often suggested a local short-range transmission interaction that was not restricted to nearest-neighbor interactions and that the presence of background infection was not necessary to explain the observations.     

Survey of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> (CTV) diversity in pigmented <Emphasis Type="Italic">Citrus x paradisi</Emphasis> (Macfad.) (Grapefruit) trees in north-western Argentina

Citrus tristeza virus (CTV) is the most severe viral pathogen of citrus and elicits a wide range of devastating disease symptoms. Grapefruit cultivars (Citrus x paradisi) are the most sensitive among citrus to the effects of CTV infections. Grapefruit is an important crop within the north-western Argentine citrus industry; however, production has been affected by CTV stem-pitting. In general, CTV diversity within South America is poorly studied, with data on grapefruit CTV populations being particularly limited. In this study, 50 samples were collected from Star Ruby, Henninger’s Ruby and Ruben Pink cultivars, within the provinces of Tucumán, Salta and Jujuy in north-western Argentina. The CTV p33 gene was PCR amplified and the resulting amplicons sequenced with Sanger sequencing. A subset of these amplicons was sequenced with Illumina MiSeq sequencing. AT-1-like sequences were dominant within the majority of populations, as determined by Sanger sequencing, followed by sequences clustering within the unresolved Kpg3/SP/T3 and RB clades. Sequencing by Illumina MiSeq confirmed this, as well as detecting minor sequence types within the HA 16–5, VT, B165 and A18 clades.     

Aphid Transmission Alters the Genomic and Defective RNA Populations of Citrus tristeza virus Isolates

ABSTRACT A total of 14 Spanish isolates of Citrus tristeza virus (CTV) and 1 isolate from Japan were transmitted by Aphis gossypii, and the subisolates obtained were compared with the source isolates for symptom expression and double-stranded RNA (dsRNA) pattern. Of the 14 Spanish isolates, 9 showed altered dsRNA patterns after aphid transmission but only minor variations in the intensity of symptoms induced on Mexican lime. Northern blot hybridization with complementary DNA (cDNA) probes corresponding to both the 5' and the 3' termini of the CTV genomic RNA (gRNA) showed that the dsRNA bands that could be used to discriminate between the dsRNA pattern of the source and the aphid-transmitted isolates were the replicative forms of defective RNAs (D-RNAs). Conversely, the Japanese isolate and two subisolates obtained from it by aphid transmission had the same dsRNA pattern, but one of the subisolates induced milder symptoms in several hosts. Dot-blot hybridization with cDNA probes representing several regions of the gRNA showed that most of the aphid-transmitted isolates differed from the corresponding source isolate by their hybridization pattern. Our results indicate that aphid transmission often sorts the populations of gRNA variants and D-RNAs present in CTV isolates.     

Biological diversity of citrus tristeza virus (CTV) isolates in Spain

A survey of citrus tristeza virus (CTV) isolates was carried out in most citrus-growing areas in Spain. Twenty-two isolates were selected by geographical origin, cultivar of source tree, and symptoms observed on the host or in preliminary tests, and were biologically characterized.
A wide range of variation in transmissibility by aphids and symptom intensity on nine different indicator species or scion-rootstock combinations was observed among CTV isolates. Mexican lime. Citrus macrophylla , and to a lesser extent citron were the most useful hosts for characterizing these isolates, and leaf symptoms and stem pitting were the most discriminating traits. Positive correlation was observed between symptoms induced on Mexican lime and C. macrophylla , but not between the symptoms induced on these indicators under greenhouse conditions and the homologous symptoms on plants grown in the screenhouse. Some of the traits studied enabled us to establish relatively well-defined groups of isolates, but in most cases a continuous range of variation was obtained and no clear group could be defined.     

湖南柑橘衰退病调查分析及弱毒系筛选

 The investigation showed that stem-pitting Citrus tristeza virus (CTV)occurred commonly in citrus production areas in several varieties of Hunan Province. Accurate detection of CTV strains was performed by p23/PCR method, PCR and the results indicated that the most samples were infected with several CTV isolates. Three mild strains were isolated and their pathogenicity was identified by biological identification, it indicated that p23/PCR groups had uniformity with the pathogenicity of CTV isolates. Furthermore, three mild isolates were tested in the cross protection by analysis of biological symptoms and composition of p23 gene. Different protecting effects were observed among these strains and W17 mild isolate was effective.     

Diversity of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> populations in commercial grapefruit orchards in Southern Africa,determined using Illumina MiSeq technology

Grapefruit cultivars are highly sensitive to CTV infections and in order to increase their productive lifespan, the Southern African citrus industry makes use of cross-protection. However, the breakdown of cross-protection is commonly observed in commercial grapefruit plantings. In order to determine which genotypes of CTV are associated with commercial Citrus x paradisi (Macfad.) cv. ‘Star Ruby’ in Southern Africa, 192 samples, pre-immunised with the GFMS 12 population, were collected from the grapefruit production areas of Hoedspruit, Malelane, Swaziland, Northern Cape, Sundays River Valley and Nkwalini Valley and six samples from non-pre-immunised plants in Letsitele as well as three samples from greenhouse maintained plants harbouring populations derived from the original GFMS 12 source. The p33 gene was amplified with direct Sanger sequencing performed on the resulting amplicons. A subset of 92 samples randomly selected and p33 gene amplicons subjected to Illumina MiSeq amplicon sequencing. High levels of CTV diversity were observed between and within orchards. Most populations were made up of a dominant component with several minor sequence types. Resistance Breaking (RB) sequences were most numerous, especially in recently planted orchards and present within all of the populations. The Kpg3/SP/T3 group appeared to be the second most prevalent, with increased prevalence in older orchards. Sequences mapping to HA 16–5, VT, AT-1, T36, Taiwan-Pum/M/T5 and T30, were represented sporadically within numerous collection sites.     

Molecular analysis suggests that recent <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> outbreaks in Italy were originated by at least two independent introductions

Citrus tristeza virus (CTV) is the causal agent of the most important virus disease of citrus. Numerous CTV isolates differing in biological and molecular characteristics have been reported worldwide. Recently, CTV was detected in Italy in several citrus crops from three separate areas: (1) Cassibile, province of Syracuse; (2) Massafra, province of Taranto; and (3) Belpasso, province of Catania. CTV isolates from Massafra and Cassibile were mild, whereas isolates from Belpasso induced severe symptoms. To study the genetic variation of CTV populations of these areas, 150 samples per area were examined by single strand conformation polymorphism (SSCP) and nucleotide sequence analysis of CTV gene p20. All isolates from the same area showed the same SSCP pattern whereas for each area a different SSCP pattern was obtained. The Massafra and the Cassibile isolates had a nucleotide identity higher than 99% with a mild isolate from Spain and about 92% with the Belpasso isolates, which were similar (identity higher than 99%) to severe isolates from California and Japan. These results suggest at least two independent introductions of CTV in Italy, probably by import of CTV-infected budwoods. Within each area, the virus population was homogeneous suggesting diffusion of CTV by aphid transmission. The GenBank accession numbers of the sequences reported in this paper are: AY262000, AY263360 and AY263361 corresponding to gene p20 of CTV isolates from Massafra, Cassibile and Belpasso (Italy), respectively.     

Evolutionary analysis of Citrus tristeza virus outbreaks in Calabria,Italy: two rapidly spreading and independent introductions of mild and severe isolates

The evolution of citrus tristeza virus (CTV) from outbreaks occurred in Calabria, Italy, was compared with that of CTV outbreaks reported previously in another two proximal Italian regions, Sicily and Apulia. Examination of four genomic regions (genes p20, p25 and p23, and one fragment of open reading frame 1) showed two recombination events, and phylogenetic analysis disclosed two divergent CTV groups in Calabria: one formed by severe and the other by mild isolates. This analysis, together with others involving population genetic parameters, revealed a low migration rate of CTV between the three Italian regions, as well as significant differences in selective pressures, epidemiology and demography, all affecting the genetic structure of CTV populations.