Citrus Tristeza Virus Isolates Research Articles

Identification of an Isolate of Citrus Tristeza Virus by Nanopore Sequencing in Korea and Development of a CRISPR/Cas12a-Based Assay for Rapid Visual Detection of the Virus.

Citrus tristeza virus (CTV) is a highly destructive viral pathogen posing a significant threat to citrus crops worldwide. Disease management and crop protection strategies necessitate the development of rapid and accurate detection methods. In this study, we employed Oxford Nanopore sequencing to detect CTV in Citrus unshiu samples. Subsequently, we developed a specific and sensitive detection assay combining CRISPR/Cas12a with reverse transcription-recombinase polymerase amplification. The CRISPR-Cas12a assay exhibited exceptional specificity for CTV, surpassing conventional RT-PCR by at least 10-fold in sensitivity. Remarkably, the developed assay detected CTV in field samples, with zero false negatives. This diagnostic approach is user-friendly, cost-effective, and offers tremendous potential for rapid onsite detection of CTV. Therefore, the CRISPR-Cas12a assay plays a significant role in managing and preserving citrus trees that are free from viruses in the industry.

Increased Diversity of Citrus Tristeza Virus in Europe.

This study investigated the genetic diversity of Citrus tristeza virus (CTV) isolates from Montenegro and Croatia, European countries with the northernmost citrus-growing regions situated on the Eastern Adriatic coast. Fifteen complete or nearly complete CTV genomes were reconstructed by high-throughput sequencing of samples collected in distinct municipalities in Montenegro and Opuzen municipality in Croatia. Phylogenetic analyses assigned some of the sequences to VT and T30 strains, previously recorded in Europe, while remarkably other isolates were placed in S1 and RB groups, which have not been reported in Europe so far. In addition, a new phylogenetic lineage comprising only isolates from Montenegro was delineated and tentatively proposed as the MNE cluster. Recombination analysis revealed evidence of 11 recombination events in the sequences obtained in this study, between isolates of related strains, within isolates of the same strain, and between distant strains. These findings show that CTV diversity in Europe is higher than reported before and calls for the reevaluation of management strategies.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Transmission of isolates of Citrus tristeza virus by single aphids (Toxoptera citricida)

Citrus tristeza virus (CTV) can be transmitted by several aphid species in a semi-persistent mode, with Toxoptera citricida being the most efficient vector. In Brazil, mild CTV isolates are used for pre-immunization of citrus trees against severe isolates. We aimed to determine the capacity and efficiency of T. citricida in separating the viral complex into haplotypes from three well-characterized CTV isolates (PIAC, CS1, and Forte Rolândia). Single-aphid transmission assays were conducted to determine CTV transmission efficiency. The results showed that T. citricida transmitted only haplotypes from the PIAC and CS1 isolates, with efficiencies of 8 and 4%, respectively. Both isolates caused mild CTV symptoms in Brazil. However, isolate Forte Rolândia, which causes severe symptoms in citrus trees, was not transmitted by T. citricida. The detection of CTV haplotypes from PIAC and CS1 isolates in sweet orange (Citrus sinensis) plants after a single aphid transmission occurred at different time points. The first haplotype observed was from PIAC 150 days after citrus was challenged by a single aphid, followed by CS1 at day 210 after transmission. In addition, differences in the single-strand conformation polymorphism patterns between the CTV isolates and CTV haplotypes were determined, suggesting that an aphid can acquire and transmit only one CTV haplotype to citrus plants during phloem feeding. The study of the mechanism of transmission by the vector can increase our knowledge of the interactions among hosts, vectors, and pathogens, which are often neglected.

Minor Variants of Orf1a, p33, and p23 Genes of VT Strain Citrus Tristeza Virus Isolates Show Symptomless Reactions on Sour Orange and Prevent Superinfection of Severe VT Isolates.

The control of tristeza quick decline (QD) of citrus is based on the use of rootstocks that are tolerant or resistant to the Citrus tristeza virus (CTV), but some of them show bio-agronomic limits. The application of cross-protection (CP) has been insufficiently explored. The present study examined the possibility of QD control by cross-protection (CP) following reports showing the dependence of the CP strategy on the close genetic relationships between the protective and challenging CTV isolates. Taking advantage of deep sequencing technologies, we located six naturally infected trees harboring no-seedling yellow (no-SY) and no QD decline (mild) VT isolates and used these for challenge inoculation with three QD VT isolates. Symptom monitoring showed that all six Sicilian mild no-SY isolates, based on their genomic relatedness and mild symptoms reactions, provide effective protection against the three severe local VT isolates. The differences between the six mild and three severe isolates were confined to just a few nucleotide variations conserved in eight positions of three CTV genes (p23, p33, and Orf1a). These results confirm that the superinfection exclusion (SIE mechanism) depends on close genetic relatedness between the protective and challenging severe VT strain isolates. Ten years of investigation suggest that CP could turn into an efficient strategy to contain CTV QD infections of sweet orange trees on SO rootstock.

High throughput sequencing of a stem pitting citrus tristeza virus isolate from Hunan Province P.R. China

High throughput sequencing of a stem pitting citrus tristeza virus isolate from Hunan Province P.R. China

Complete genome sequencing and characterization of a potential new genotype of Citrus tristeza virus in Iran.

Citrus tristeza virus (CTV) is one of the economically destructive viruses affecting citrus trees worldwide, causing significant losses in fruit production. Comparative genomic studies have shown genetic diversity in various regions of the genome of CTV isolates, which has classified the virus into several genotypes. In recent years, some orange citrumelo-tolerant rootstocks showed yellowing, decline, and vein clearing in northern Iran (Mazandaran province, Sari). We confirmed the presence of CTV in the symptomatic trees by reverse transcription PCR (RT-PCR). The complete genome of a Sari isolate of CTV (Sari isolate) was sequenced using next-generation sequencing (NGS) technology. In addition, phylogenetic analysis, differential gene expression of the virus and identification of its variants in a population were studied. We obtained the final contigs of the virus (nt) and annotated all genomes to viral ORFs, untranslated regions (UTRs), intergenic regions, and 5' and 3' ends of the genome. Phylogenetic analysis of the Sari isolate and other genotypes of CTV showed that the Sari isolates were placed in a distinct cluster without a sister group. Based on the number of specific transcripts (TPM) in CTV RNA -Seq, P13 was the most highly expressed gene related to the host range of the virus and its systemic infection. The ORFs of the polyprotein, P33, and P18 showed variation in a single population of the sari isolate. The CTV has a potential for variation in a population in a host, and these variations may contribute to the best fit of the CTV in different situations. In Iran, whole genome sequencing of the CTV was performed for the first time, and we gained new insights into CTV variation in a population.

A Review On Citrus tristeza virus (CTV) And Its Management Approaches

Citrus tristeza virus (CTV), one of the casual agents of citrus decline, is responsible for the death of millions of citrus trees and reduced production and productivity of citrus orchards worldwide. CTV epidemics has been recorded from several parts of the world where mainly steam pitting (SP) and quick decline (QD) strains of CTV have induced severe disease reactions. Identification and characterization of CTV isolates primarily has been focused on the biological assaying in indicator plants, serology-based ELISA and molecular PCR tests. Controlling the presence and spread of CTV where it is absent or establishment is limited heavily relies upon preventive measures, quarantine and legislations. Cross protection is an appealing technique especially for controlling CTV – Stem Pitting strains and use of CTV resistant rootstocks largely prevent infection by CTV – Quick Decline strains. More reliable and effective way to control CTV is breeding for resistant or tolerant cultivars. Advances in molecular biology have lead scientists to find out genes and map genetic loci of CTV resistant citrus and related species that could be exploited in breeding. However incorporation of resistant ability offered by a handful of citrus and its related species into the susceptible cultivars containing other desirable agronomical traits is challenging through classical plant breeding approaches. The following review work is based on Citrus tristza virus and its management practices.

Callose synthase and xyloglucan endotransglucosylase gene expression over time in Citrus × clementina and Citrus × sinensis infected with citrus tristeza virus

Citrus tristeza virus (CTV) is a virus that already caused great losses in citrus producing regions. The cell wall of plant cells plays an important role in the defence response to viruses. Following several studies indicating that cell wall enzyme transcripts of callose synthase 7 (calS7) and xyloglucan endotransglucosylase 9 (xth9) are modified during a viral infection, transcript expression of calS7 isoform x5 (calS7x5) and xth9 was evaluated over time in Citrus × sinensis ‘Valencia Late’ (VL) and Citrus × clementina ‘Fina’ (CL), infected with the severe CTV isolate T318A, by quantitative (q) PCR. qPCR analysis of healthy and CTV infected citrus was performed at 15 days, 10 months and at 31 months post-inoculation (dpi/mpi), respectively. The CTV titer, evaluated at the three time-points by qPCR, increased over time in bark tissues, with VL plants exhibiting a titer about 5 times higher than CL 31 mpi. CTV infection did not cause significant changes in calS7x5 gene expression over time in both citrus cultivars. However, CTV infection was associated with significant up-regulation of xth9 in VL compared to controls 31 mpi. This study highlights that CTV infection can affect the expression of specific cell wall-associated genes over time and that this influence was distinct for VL and CL. This study provides further insight into the CTV-citrus host interaction, with the long-term response of VL to a severe CTV isolate involving a high expression of the xth9 gene.

Biological and molecular characterization of a resistance-breaking isolate of citrus tristeza virus from Uruguay and its effects on Poncirus trifoliata growth performance

Resistance-breaking (RB) isolates of citrus tristeza virus (CTV) can replicate and move systemically in Poncirus trifoliata, a rootstock widely used for management of decline caused by CTV and other purposes. In Uruguay, severe CTV isolates are prevalent, and an RB isolate (designated as RB-UY1) was identified. In order to predict the implications of this genotype circulating in citrus crops grafted on trifoliate rootstocks, the aim of this work was to determine the biological and molecular characteristics of this isolate, the efficiency of its transmission by Toxoptera citricida, and its effects on plant growth performance of P. trifoliata. Our results show that RB-UY1 can be classified as a mild isolate, that it is phylogenetically associated with the RB1 group, and that it is efficiently transmitted by T. citrida. They also suggest that the RB-UY1 isolate should not affect the performance of citrus crops grafted on trifoliate rootstocks, although some growth parameters of P. trifoliata seedlings were affected four years after inoculation.

Quick Decline and Stem Pitting Citrus tristeza virus Isolates Induce a Distinct Metabolomic Profile and Antioxidant Enzyme Activity in the Phloem Sap of Two Citrus Species

Susceptibility to the severe Citrus tristeza virus (CTV), T36, is higher for Citrus macrophylla (CM) than for C. aurantium (CA). How host-virus interactions are reflected in host physiology is largely unknown. In this study, the profile of metabolites and the antioxidant activity in the phloem sap of healthy and infected CA and CM plants were evaluated. The phloem sap of quick decline (T36) and stem pitting (T318A) infected citrus, and control plants was collected by centrifugation, and the enzymes and metabolites analyzed. The activity of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), in infected plants increased significantly in CM and decreased in CA, compared to the healthy controls. Using LC-HRMS2 a metabolic profile rich in secondary metabolites was assigned to healthy CA, compared to healthy CM. CTV infection of CA caused a drastic reduction in secondary metabolites, but not in CM. In conclusion, CA and CM have a different response to severe CTV isolates and we propose that the low susceptibility of CA to T36 may be related to the interaction of the virus with the host's metabolism, which reduces significantly the synthesis of flavonoids and antioxidant enzyme activity.

PM 7/31 (2) Citrus tristeza virus

<scp>PM</scp> 7/31 (2) Citrus tristeza virus

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.

Resposta fotossintética de citros a isolados de Citrus tristeza virus com potencial para proteção cruzada

Abstract The objective of this work was to evaluate the photosynthetic response of citrus varieties to the inoculation of isolates T30 and T3 of Citrus tristeza virus (CTV) characterized as having potential to provide cross-protection against severe CTV isolates in citrus. Citrus plants of 'Campbell' orange, 'Persian' lime, and 'Key' lime were subjected to inoculations by both isolates by patch. Noninoculated plants were used as the control. Symptom expression, photosynthetic and transpiration rates, and stomatal conductance were evaluated by IRGA. Stomatal density and size were evaluated by epidermal impressions. The CTV isolates were also evaluated and molecularly characterized by RT-PCR and Sanger sequencing. 'Campbell' orange and 'Persian' lime plants remained asymptomatic after the inoculations. The inoculation treatments with both CTV isolates did not reduce photosynthetic capacity, transpiration rate, and stomatal conductance, in comparison with the control. Stomatal density and size varied according to the citrus species. T30 and T3 do not affect the photosynthetic responses of 'Campbell' orange and 'Persian' lime; therefore, these isolates have the potential to provide cross-protection to citrus varieties against severe isolates of CTV.

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.

From the smallest to the largest subcellular plant pathogen: Citrus tristeza virus and its unique p23 protein

Knowledge on diseases caused by Citrus tristeza virus (CTV) has greatly increased in last decades after their etiology was demonstrated in the past seventies. Professor Ricardo Flores substantially contributed to these advances in topics like: i) improvement of virus purification to obtain biologically active virions, ii) sequencing mild CTV isolates for genetic comparisons with sequences of moderate or severe isolates and genetic engineering, iii) analysis of genetic variation of both CTV genomic RNA ends and features of the highly variable 5′ end that allow accommodating this variation within a conserved secondary structure, iv) studies on the structure, subcellular localization and biological functions of the CTV-unique p23 protein, and v) potential use of p23 and other 3′-proximal regions of the CTV genome to develop transgenic citrus resistant to the virus. Here we review his main achievements on these topics and how they contributed to deeper understanding of CTV biology and to new potential measures for disease control.

Molecular characterization of four citrus tristeza virus isolates that cause severe symptoms in navel orange in Ganzhou, China

Molecular characterization of four citrus tristeza virus isolates that cause severe symptoms in navel orange in Ganzhou, China

Citrus Diseases Exotic to Florida: Citrus Tristeza Virus–Stem Pitting (CTV-SP)

This document is one in a series designed to provide important information on the causal agent, symptoms, and transmission of exotic citrus diseases. This information can be used as an educational tool to raise awareness about these diseases and for scouting and identification efforts. Disseminating information about the diseases to the citrus industry may prevent their introduction and spread in Florida. This document will focus on the exotic viral disease caused by isolates of citrus tristeza virus–stem pitting (CTV-SP). Original version: Chung, Kuang-Ren, and Ronald Brlansky. 2006. “Citrus Diseases Exotic to Florida: Citrus Tristeza Virus– Stem Pitting (CTV-SP)”. EDIS 2006 (7). https://doi.org/10.32473/edis-pp149-2006.

Effects of graft and aphid transmission on the genetic diversity and population structure of Turkish citrus tristeza virus isolates

Effects of graft and aphid transmission on the genetic diversity and population structure of Turkish citrus tristeza virus isolates

Location matters: from changing a presumption about the Citrus tristeza virus tissue tropism to understanding the stem pitting disease.

Stem pitting is a common virus-induced disease phenotype that tremendously impacts growth of perennial woody plants. How stem pitting develops in the infected trees remains unclear. Here, we assessed the development of stem pits upon infection of citrus by Citrus tristeza virus (CTV), which has been regarded as 'phloem-limited'. By taking advantage of a highly susceptible virus host - Citrus macrophylla - and a CTV isolate lacking a viral effector - the p33 protein, the development pattern of stem pitting was revealed via time-course observations and histological analyses. The stem pits result from the virus-colonized nonlignified 'gumming' malformations which are initiated by virus invasion into multiple spatially separated tissue layers - protophloem, metaphloem, and, unexpectedly, metaxylem. Notably, invasion of CTV into the unspecialized metaxylem cells interrupted the differentiation of the xylem tracheary elements. With the radial spread of CTV into the adjacent cells towards the stem periphery, the clusters of virus-colonized immature metaxylem cells extended in size, merging, at a certain stage, with virus-bearing cells in the protophloem and metaphloem layers. Collectively, our data provide a new insight into the process of the stem pitting development and the role of the xylem tissue in the virus pathogenicity.

Effects of Two Citrus Tristeza Virus Isolates on Sweet Orange (Citrus sinensis) Propagated on a Citrus Tristeza Virus Tolerant Rootstock: A Metabolomics and Transcriptomics Approach

Citrus tristeza virus (CTV) was once the most economically important citrus pathogen in the world. In regions where CTV is endemic, CTV tolerant rootstocks have been widely adopted. Despite these changes, CTV can still affect plant health and fruit quality. To assess the impact of CTV infection on scions grown on CTV tolerant rootstock, Washington navel [Citrus sinensis (L.) Osbeck] scions grafted onto Carrizo rootstock (Poncirus trifoliate × Citrus sinensis) received grafts from citrus infected with a mild isolate (T30 isolate 07539) or a stem-pitting isolate (CCTEA-107) of CTV, and metabolomics and transcriptomics analyses were performed over a period of 10 months after grafting of CTV-infected material. The phenotypic response to either isolate was weak. The largest difference in the leaf metabolome was seen 10 months postgrafting for both isolates, with a stronger response observed for plants infected with the stem-pitting isolate. Analysis of differentially expressed genes revealed that plants infected with the milder isolate exhibited a difference in gene expression 4 months after infection, whereas plants infected with the stem-pitting isolate exhibited a difference in gene expression 10 months after infection. This study highlights differences and similarities in the metabolome and transcriptome response of Washington navel trees on Carrizo rootstock to two CTV isolates.