Citrus Tristeza Virus Genotypes Research Articles

Molecular and biological characterization of a novel citrus tristeza virus isolate that causes severe symptoms in Citrus junos cv. Ziyangxiangcheng.

The complete genomic sequence of a novel citrus tristeza virus (CTV) isolate, CT91-A1, from Orah tangor grafted on Citrus junos cv. Ziyangxiangcheng rootstock in China was determined by transcriptome sequencing. Sequence alignments showed that isolate CT91-A1 shared 83.3 to 95.5% nucleotide sequence identity with extant CTV genotypes at the whole-genome level, with the highest similarity to the S1 genotype. Phylogenetic analysis revealed that CT91-A1 clustered in a unique subclade with the S1 genotype. Isolate CT91-A1 induced severe stem pitting in Mexican lime and C. junos cv. Ziyangxiangcheng and moderate stem pitting in Guanximiyou pummelo and Duncan grapefruit. It was successfully transmitted by Aphis citricidus, and it can potentially cause significant damage to the citrus industry in China.

Population dynamics of Citrus tristeza virus (CTV) in single aphid-transmitted sub-isolates of the South African GFMS12 isolate.

Grapefruit trees in South Africa have been cross protected against severe stem pitting genotypes of Citrus tristeza virus (CTV) since the 1920s using a mild strain initially called 'Nartia' but later referred to as grapefruit mild strain 12 (GFMS12).In the current study, the GFMS12 isolate was used as the source for single aphid transmissions (SAT) using Toxoptera citricida, commonly called the brown citrus aphid (BrCA). The BrCA-transmitted CTV sub-isolates were analyzed by the heteroduplex mobility assay (HMA), serological assays, genetic marker analysis (GMA), and selected sub-isolates were biologically indexed. Reverse transcription PCR of genomic regions was conducted using universal primers followed by cloning the PCR products, HMA and sequence analysis; nine genotypes of CTV were identified in the complex of GFMS12, including both severe and mild genotypes. A singleBrCAtransmitted up to six CTV genotypes simultaneously in one sub-isolate. The HMA was found to be a rapid, reliable tool for the identification of genotypes and can be useful in the development of CTV management strategies and budwood certification programs.

Amino acid, sugar, phenolic, and terpenoid profiles are capable of distinguishing Citrus tristeza virus infection status in citrus cultivars: Grapefruit, lemon, mandarin, and sweet orange.

Citrus tristeza virus (CTV) is the most severe viral disease for citrus production. Many strains of CTV have been characterized and their symptomology widely varies, ranging from asymptomatic or mild infections to severe symptomology that results in substantial yield loss or host death. The capacity of the different CTV strains to affect the biochemistry of different citrus species has remained largely unstudied, despite that associated metabolomic shifts would be relevant toward symptom development. Thus, amino acid, sugar, phenolic, and terpenoid levels were assessed in leaves of healthy and CTV-infected grapefruit, lemon, mandarin, and two different sweet orange cultivars. Both mild [VT-negative (VT-)] and severe [VT-positive (VT+)] CTV genotype strains were utilized. When looking at overall totals of these metabolite classes, only amino acid levels were significantly increased by infection of citrus with severe CTV strains, relative to mild CTV strains or healthy plants. No significant trends of CTV infection on summed amounts of all sugar, phenolic, or terpenoid compounds were observed. However, individual compound levels were affected by CTV infections. Subsequent canonical discriminant analysis (CDA) that utilized profiles of individual amino acids, terpenoids, or phenolics successfully distinguished leaf samples to specific citrus varieties and identified infection status with good accuracy. Collectively, this study reveals biochemical patterns associated with severity of CTV infections that can potentially be utilized to help identify in-field CTV infections of economic relevance.

Genotype variation of citrus tristeza virus after passage on different hosts, and changes in the virus genotype populations by the vector Aphis gossypii

Phylogenetic analyses categorize seven genotypes of citrus tristeza virus (CTV). The symptoms caused by this pathogen, their expression and severity are influenced by CTV genotypes, host species, cultivars, and infected host rootstocks. This study aimed to verify how populations of Chilean CTV isolates changed following inoculation from infected sweet orange to Mexican lime trees, and to determine if CTV genotype populations influenced transmission efficiency via Aphis gossypii. Reverse transcription polymerase chain reaction showed variation in genotypes of populations of CTV in Mexican lime, after graft inoculations using infected sweet orange chip-buds. Severe genotypes (VT) were detected after inoculation of mild isolate CTV populations (T30). The T30 donor populations also reduced transmissibility via A. gossypii; however, these results may not be conclusive due to mixture with the VT genotype. There is evidence of high rates of virus acquisition by this aphid species, but also low transmission efficiency, which may partially explain the historical absence of tristeza epidemics in Chile.

Citrus Tristeza Virus Genotype Detection Using High-Throughput Sequencing.

The application of high-throughput sequencing (HTS) has successfully been used for virus discovery to resolve disease etiology in many agricultural crops. The greatest advantage of HTS is that it can provide a complete viral status of a plant, including information on mixed infections of viral species or virus variants. This provides insight into the virus population structure, ecology, or evolution and can be used to differentiate among virus variants that may contribute differently toward disease etiology. In this study, the use of HTS for citrus tristeza virus (CTV) genotype detection was evaluated. A bioinformatic pipeline for CTV genotype detection was constructed and evaluated using simulated and real data sets to determine the parameters to discriminate between false positive read mappings and true genotype-specific genome coverage. A 50% genome coverage cut-off was identified for non-target read mappings. HTS with the associated bioinformatic pipeline was validated and proposed as a CTV genotyping assay.

First comprehensive sanitary report of citrus-infecting viruses and viroids in Uruguay

Citrus is the highest-value fruit crop in terms of international trade. However, citrus species are susceptible to several diseases caused by different pathogens which directly cause a decrease in production leading to economic losses. In the last half-century, the citrus industry in Uruguay has had a strong socio-economic impact and is also constantly evolving to stay competitive in world markets, by introduction of new varieties and improvement of production practices to obtain high yielding orchards. Nevertheless, despite the existence since 2014 of the Uruguayan National Citrus Sanitary and Certification Program, scarce information is available regarding the virus and viroid status of commercial citrus in Uruguay. The incidence of citrus tristeza virus (CTV), citrus psorosis virus (CPsV), satsuma dwarf virus (SDV), citrus exocortis viroid (CEVd), hop stunt viroid (HSVd), citrus dwarfing viroid (CDVd), citrus bark cracking viroid (CBCVd) and citrus bent leaf viroid (CBLVd) was investigated in this study, as well as CTV genotypes prevalent in the country. Molecular diagnostic assays were used to test 1175 samples including Valencia and Navel sweet oranges, Mandarin hybrids, Clementines and lemons, which were randomly collected from seven citrus-producing provinces. Only 6% of the samples were negative for the pathogens screened, while 93% of them were CTV positive. SDV, CBLVd and CBCVd were not detected. Co-infections were frequently detected, finding plants with up to four simultaneous pathogens, including CTV in all co-infected plants. This is the first comprehensive survey of several citrus-infecting viruses and viroids in Uruguay, as well as a determination of the CTV genotypes prevalent in the country.

Complete genome sequences of two novel genotypes of Citrus tristeza virus infecting Poncirus trifoliata in China

Two Chinese isolates of Citrus tristeza virus (CTV) from Poncirus trifoliata, designated as CN-L1-ZT1 and CN-M1-ZT1, were completely sequenced. The phylogenetic analysis of these isolates suggested that two novel CTV genotypes L1 and M1 should be considered. In addition, the recombination analysis indicated that most of the 3′ terminal p33 to p23 open reading frames (ORFs) of these isolates were derived from different unknown CTV isolates. Thus, the L1 and M1 genotypes were two recombinants distinct from known CTV genotypes. RT-PCR protocols developed based on the p33 regions facilitated genotyping of L1 and M1 from other major CTV genotypes in China. These results could play an important role in further study of global CTV diversity and evolution.

A rapid detection tool for VT isolates of Citrus tristeza virus by immunocapture-reverse transcriptase loop-mediated isothermal amplification assay.

Severe strains of Citrus tristeza virus (CTV) cause quick decline and stem pitting resulting in significant economic losses in citrus production. A immunocapture reverse-transcriptase loop-mediated amplification (IC-RT-LAMP) assay was developed in this study to detect the severe VT strains that are typically associated with severe CTV symptoms. The sensitivity of RT-LAMP assay was determined by ten-fold serial dilutions of CA-VT-AT39 RNA, in comparison to one-step RT-droplet digital (dd) PCR. RT-LAMP detected up to 0.002 ng RNA with an amplification time of 10:35 (min:sec.), equivalent to 11.3 copies as determined by one step RT-ddPCR. The RT-LAMP assay specifically detected CA-VT-AT39 RNA and did not cross react with other CTV genotypes tested (T36, T30, RB, S1 and T68). To facilitate rapid on-site detection, the RT-LAMP assay was improved by first capturing the CTV virions from citrus crude leaf sap using CTV-IgG (IC-RT-LAMP), thereby eliminating nucleic acid extraction steps. IC-RT-LAMP assay was optimized with two-fold dilutions of CTV-IgG ranging from 1:500 to 1:16,000. The IC-RT-LAMP assay detected the CA-VT-AT39 virions in all dilutions tested. The minimum amplification time was 6:45 (min:sec) with 1:500 and 1:1000 of CTV-IgG dilutions. The limit of detection of IC-RT-LAMP assay with crude leaf sap of CA-VT-AT39 was 1:320 with a maximum amplification time of 9:08 (min:sec). The IC-RT-LAMP assay was validated for VT genotype by comparing to IC-RT-qPCR using the CTV from 40 field tree samples. A 100% agreement was observed between tests, regardless of single or mixed infections of CTV VT with other genotypes. Therefore, the IC-RT-LAMP assay can serve as a useful tool in the management of potentially severe strains of CTV.

Nucleotide heterogeneity at the terminal ends of the genomes of two California Citrus tristeza virus strains and their complete genome sequence analysis

BackgroundThe non-translated regions at the genome ends of RNA viruses serve diverse functions and can exhibit various levels of nucleotide (nt) heterogeneity. However, the extent of nt heterogeneity at the extreme termini of Citrus tristeza virus (CTV) genomes has not been comprehensively documented. This study aimed to characterize two widely prevalent CTV genotypes, T36-CA and T30-CA, from California that have not been sequenced or analyzed substantially. The information obtained will be used in our ongoing effort to construct the infectious complementary (c) DNA clones of these viruses.MethodsThe terminal nts of the viral genomes were identified by sequencing cDNA clones of the plus- and/or minus-strand of the viral double-stranded (ds) RNAs generated using 5′ and 3′ rapid amplification of cDNA ends. Cloned cDNAs corresponding to the complete genome sequences of both viruses were generated using reverse transcription-polymerase chain reactions, sequenced, and subjected to phylogenetic analysis.ResultsAmong the predominant terminal nts identified, some were identical to the consensus sequences in GenBank, while others were different or unique. Remarkably, one of the predominant 5′ nt variants of T36-CA contained the consensus nts “AATTTCAAA” in which a highly conserved cytidylate, seen in all other full-length T36 sequences, was absent. As expected, but never systematically verified before, unique variants with additional nt (s) incorporated upstream of the 5′ terminal consensus nts of T36-CA and T30-CA were also identified. In contrast to the extreme 5′ terminal nts, those at the extreme 3′ termini of T36-CA and T30-CA were more conserved compared to the reference sequences, although nt variants were also found. Notably, an additional thymidylate at the extreme 3′ end was identified in many T36-CA sequences. Finally, based on pairwise comparisons and phylogenetic analysis with multiple reference sequences, the complete sequences of both viruses were found to be highly conserved with those of the respective genotypes.ConclusionsThe extreme terminal nts in the T36-CA and T30-CA genomes were identified, revealing new insights on the heterogeneity of these CTV genomic regions. T36-CA and T30-CA were the first and the second genotypes, respectively, of CTV originating from California to be completely sequenced and analyzed.

Bottlenecks and complementation in the aphid transmission of citrus tristeza virus populations.

Aphid transmission is a major factor in the formation of citrus tristeza virus (CTV) populations. Here, we examined the effect of population interaction on aphid transmissibility of different CTV genotypes. We found that there was no correlation between the proportion of viral genotypes in the source population and what was transmitted. We next examined the transmission of a poorly transmitted infectious cDNA clone (T36) in mixture with other CTV genotypes. T36 transmission increased from 0.5% alone, to up to 35.7%, depending on the coinfecting genotype. These results suggest that interaction between CTV genotypes affects the transmission of this virus.

Understanding superinfection exclusion by complex populations of Citrus tristeza virus

Superinfection exclusion (SIE) is a phenomenon in which a primary viral infection restricts a secondary infection with the same or closely related virus. Previously we showed that SIE by Citrus tristeza virus (CTV) occurs only between isolates of the same virus genotype. This work, however, was done using single genotype-containing isolates, while most field citrus trees harbor complex populations composed of different virus genotypes. Here we examined SIE in plants simultaneously infected with several CTV genotypes. The experiments showed that exclusion of a secondary infection by a CTV variant was triggered by the presence of another variant of the same genotype in the primary population, even under the conditions of its low-level accumulation, and was not affected by co-occurrence of additional heterologous genotypes. The same rule appeared to be in effect when SIE by mixed populations was tested in a series of different citrus varieties.

Simultaneous visualization of two Citrus tristeza virus genotypes provides new insights into the structure of multi-component virus populations in a host

Complex Citrus tristeza virus (CTV) populations composed of mixtures of different strains of the virus are commonly found in citrus trees in the field. At present, little is known about how these populations are formed, maintained, and how they are structured within a host. Here we used a novel in situ hybridization approach allowing simultaneous visualization of two different RNA targets with high sensitivity and specificity to examine the distribution of two isolates, T36 and T68-1, representing phylogenetically distinct strains of CTV, in a citrus host in single and mixed infections. Remarkably, in doubly inoculated plants the two virus variants appeared to be well mixed within the infected tissue and showed no spatial segregation. In addition, both CTV variants were often found occupying the same cells. Possible mechanisms involved in shaping CTV populations and the biological significance of the observed lack of structural separation of the individual components are discussed.

Genotypic diversity of Citrus tristeza virus within red grapefruit, in a field trial site in South Africa

Grapefruit cultivars (Citrus paradisi Macfad.) are extremely sensitive to Citrus tristeza virus (CTV) infections and are pre-immunized with mild-strain cross-protecting sources not containing components that elicit symptoms such as stem-pitting and decline, to ensure longer periods of productivity. However, pre-immunizing sources often lose their efficiency and for this reason the previously commercially applied grapefruit cross-protecting source GFMS (grapefruit mild-strain) 12 has been replaced by GFMS 35. This study was undertaken to determine the diversity of CTV genotypes within trees that were inoculated with either GFMS 12 or GFMS 35. Samples were collected from a number of different trees of two red grapefruit cultivars (cv. Star Ruby and cv. Flame), planted 10 years prior to sampling in the Malelane production area of South Africa. Reverse transcription-polymerase chain reaction amplification of a 5’ variable region (A-region) and a 3’ conserved region (p23 gene) was followed by cloning, sequencing of multiple clones, and phylogenetic analyses. The genotypic identities of clones were determined based on their relatedness to reference CTV strains. Sequence types within the VT genotypic group dominated in all of the samples, with T30-like sequence types being a minor component in some populations of the field collected samples. The original pre-immunising populations of GFMS 12 and GFMS 35 were characterised on greenhouse maintained plants and compared with the populations exposed to field infections by aphids. While the methodology employed only allows a coarse representation of the genotype composition of the CTV population, this study provides insight into which genotypes of CTV must be incorporated within a mild-strain cross-protecting source within the South African Citrus Improvement Scheme (SACIS).

Characterization of a novel citrus tristeza virus genotype within three cross-protecting source GFMS12 sub-isolates in South Africa by means of Illumina sequencing

Tristeza disease (caused by citrus tristeza virus, CTV) is currently controlled in South Africa by means of cross-protection. In this study, we characterized the CTV populations of three grapefruit mild strain 12 (GFMS12) single-aphid-transmission-derived sub-isolates at the whole-genome level using Illumina sequencing technology. A novel South African isolate (CT-ZA3, of the T68 genotype) was shown to be the dominant genotype in all GFMS12 sub-isolates tested, along with reads unique to various other genotypes occurring as minor components. Uncertainty remains as to the significance of these minor components.

The Prevalence of the Citrus tristeza virus Trifoliate Resistance Breaking Genotype Among Puerto Rican Isolates.

Citrus tristeza virus (CTV) isolates have been grouped into six genotypes: T3, T30, T36, VT, B165, and resistance breaking (RB) based on symptoms, host range, and genomic sequence data. The RB genotype has recently been identified with the novel property of replicating in trifoliate orange trees, a resistant host for the other five genotypes. Puerto Rican CTV isolate B301 caused mild vein clearing symptoms in Mexican lime but did not induce seedling yellows or stem pitting reactions in appropriate indicator Citrus spp., which are typical host reactions of the isolate T30. The isolate B301 was not detected by the genotype specific primer (GSP), which identifies the CTV-T3, -T30, -T36, -VT, and B165 genotypes. A primer pair for reverse transcription polymerase chain reaction (RT-PCR) amplification of the CTV-RB genotype was designed from the heat shock protein (p65) region based on the complete genomic sequences of trifoliate RB isolates from New Zealand available in the GenBank databases. The amplicon sequence from isolate B301 was 98% identical to that of the other trifoliate RB isolates. In addition, B301 was successfully inoculated into 'Carrizo citrange' (a trifoliate hybrid) but did not induce any symptoms. Furthermore, the complete genome sequence of B301 followed by the phylogenetic analysis revealed that the isolate is part of the RB clade with other CTV-RB isolates from New Zealand and Hawaii. Additional CTV isolates obtained from Puerto Rico were tested with the RB-GSP and confirmed the presence of trifoliate RB isolates in mixed infection with known CTV genotypes. Although this is the first report of a CTV trifoliate RB genotype from Puerto Rico, this genotype was present there prior to 1992.

Diversity of Citrus tristeza virus in Jamaica.

Citrus tristeza virus (CTV), one of the most serious pathogens affecting Rutaceae species, causes considerable economic losses to citrus production worldwide. While mild pathotypes have been known for many years in Jamaica, decline outbreaks were only recently recognized after the introduction of the vector Toxoptera citricidus in 1993. In this study, coat protein (CP) gene sequences of isolates from four major citrus-growing regions in Jamaica were obtained and their molecular diversity compared with reference CTV genotypes from other regions. CP sequences from Jamaican isolates showed identities between 90 to 100% at the nucleotide level and shared similar identities with comparable sequences reported for other Jamaican isolates in 2009 and 2010. Phylogenetic analysis revealed segregation into five of seven lineages. One third of the Jamaican isolates clustered with Florida-T30 and Spain-T385 genotypes, with 99 to 100% identity within the lineage. The remaining isolates grouped closely with Florida T36-like genotypes or with the grapefruit and orange stem pitting B249 from Venezuela and the New Zealand CTV-resistance breaking genotypes. Identities among these groups ranged between 91 and 99%. The detection of genetic variants and potential recombination events involving parents from different lineages provide further evidence of the diversity among Jamaican CTV isolates.

Citrus tristeza virus: evolution of complex and varied genotypic groups

Amongst the Closteroviridae, Citrus tristeza virus (CTV) is almost unique in possessing a number of distinct and characterized strains, isolates of which produce a wide range of phenotype combinations among its different hosts. There is little understanding to connect genotypes to phenotypes, and to complicate matters more, these genotypes are found throughout the world as members of mixed populations within a single host plant. There is essentially no understanding of how combinations of genotypes affect symptom expression and disease severity. We know little about the evolution of the genotypes that have been characterized to date, little about the biological role of their diversity and particularly, about the effects of recombination. Additionally, genotype grouping has not been standardized. In this study we utilized an extensive array of CTV genomic information to classify the major genotypes, and to determine the major evolutionary processes that led to their formation and subsequent retention. Our analyses suggest that three major processes act on these genotypes: (1) ancestral diversification of the major CTV lineages, followed by (2) conservation and co-evolution of the major functional domains within, though not between CTV genotypes, and (3) extensive recombination between lineages that have given rise to new genotypes that have subsequently been retained within the global population. The effects of genotype diversity and host-interaction are discussed, as is a proposal for standardizing the classification of existing and novel CTV genotypes.

A genetic system for Citrus Tristeza Virus using the non-natural host Nicotiana benthamiana: an update

In nature Citrus tristeza virus (CTV), genus Closterovirus, infects only the phloem cells of species of Citrus and related genera. Finding that the CTV T36 strain replicated in Nicotiana benthamiana (NB) protoplasts and produced normal virions allowed development of the first genetic system based on protoplast transfection with RNA transcribed from a full-genome cDNA clone, a laborious and uncertain system requiring several months for each experiment. We developed a more efficient system based on agroinfiltration of NB leaves with CTV-T36-based binary plasmids, which caused systemic infection in this non-natural host within a few weeks yielding in the upper leaves enough CTV virions to readily infect citrus by slash inoculation. Stem agroinoculation of citrus and NB plants with oncogenic strains of Agrobacterium tumefaciens carrying a CTV-T36 binary vector with a GUS marker, induced GUS positive galls in both species. However, while most NB tumors were CTV positive and many plants became systemically infected, no coat protein or viral RNA was detected in citrus tumors, even though CTV cDNA was readily detected by PCR in the same galls. This finding suggests (1) strong silencing or CTV RNA processing in transformed cells impairing infection progress, and (2) the need for using NB as an intermediate host in the genetic system. To maintain CTV-T36 in NB or assay other CTV genotypes in this host, we also tried to graft-transmit the virus from infected to healthy NB, or to mechanically inoculate NB leaves with virion extracts. While these trials were mostly unsuccessful on non-treated NB plants, agroinfiltration with silencing suppressors enabled for the first time infecting NB plants by side-grafting and by mechanical inoculation with virions, indicating that previous failure to infect NB was likely due to virus silencing in early infection steps. Using NB as a CTV host provides new possibilities to study virus-host interactions with a simple and reliable system.

Past and future of a century old Citrus tristeza virus collection: a California citrus germplasm tale

Citrus tristeza virus (CTV) isolates collected from citrus germplasm, dooryard and field trees in California from 1914 have been maintained in planta under quarantine in the Citrus Clonal Protection Program (CCPP), Riverside, California. This collection, therefore, represents populations of CTV isolates obtained over time and space in California. To determine CTV genetic diversity in this context, genotypes of CTV isolates from the CCPP collection were characterized using multiple molecular markers (MMM). Genotypes T30, VT, and T36 were found at high frequencies with T30 and T30+VT genotypes being the most abundant. The MMM analysis did not identify T3 and B165/T68 genotypes; however, biological and phylogenetic analysis suggested some relationships of CCPP CTV isolates with these two genotypes. Phylogenetic analysis of the CTV coat protein (CP) gene sequences classified the tested isolates into seven distinct clades. Five clades were in association with the standard CTV genotypes T30, T36, T3, VT, and B165/T68. The remaining two identified clades were not related to any standard CTV genotypes. Spatiotemporal analysis indicated a trend of reduced genotype and phylogenetic diversity as well as virulence from southern California (SC) at early (1907–1957) in comparison to that of central California (CC) isolates collected from later (1957–2009) time periods. CTV biological characterization also indicated a reduced number and less virulent stem pitting (SP) CTV isolates compared to seedling yellows isolates introduced to California. This data provides a historical insight of the introduction, movement, and genetic diversity of CTV in California and provides genetic and biological information useful for CTV quarantine, eradication, and disease management strategies such as CTV-SP cross protection.

Genotype composition of populations of grapefruit-cross-protecting citrus tristeza virus strain GFMS12 in different host plants and aphid-transmitted sub-isolates

Citrus tristeza virus (CTV) causes severe losses in grapefruit production in South Africa and requires mild-strain cross-protection to maintain production. Unfortunately, cross-protection breakdown of the pre-immunizing CTV grapefruit mild source GFMS12 is prevalent in grapefruit in South Africa. The CTV genotype composition of the GFMS12 population inoculated onto different hosts was determined by sequencing part of ORF1a and the p23 gene of multiple clones from each plant. Analysis of the GFMS12 population in Mexican lime and Marsh and Star Ruby grapefruit varieties revealed that at least four genotypes occur in the GFMS12 population and that genotype compositions differed amongst the populations in different host plants. Single-aphid-transmitted sub-isolates derived from the GFMS12 mother population on Mexican lime appeared to contain three populations of a mixture of VT-like and recombinant B165/VT-like genotypes; a mixture of recombinant RB/VT- and B165/VT-like genotypes; and a single recombinant B165/VT-like genotype. This study underlines the importance of determining the genotype composition of a potential CTV pre-immunizing source on a range of inoculated host species before utilization.