Evolution and Molecular Epidemiology of Citrus tristeza virus on Crete: Recent Introduction of a Severe Strain

Sporadic incidences of Citrus tristeza virus (CTV) in western Crete resulting from the introduction of a mild strain (Spanish isolate T385) have been reported previously. Further analysis within this region has identified an emerging second CTV strain with minimal genetic divergence, sharing 99% nucleotide identity with the severe stem‐pitting isolate Taiwan‐Pum/SP/T1. Other severe isolates from the Mediterranean region appear in the same phylogenetic cluster, indicating movement or new introductions and the need for targeted control actions and improved phytosanitary measures in this area.     

Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem‐associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression

Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23‐kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N‐terminal 157‐amino‐acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV‐like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23‐derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem‐specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV‐specific symptoms (vein clearing and necrosis, and stem pitting), but not the non‐specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem‐specific accumulation of the p23Δ158–209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N‐terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host.     

Expanded Strain‐Specific RT‐PCR Assay for Differential Detection of Currently Known Citrus Tristeza Virus Strains: a Useful Screening Tool

Genotypic characterization of Citrus tristeza virus (CTV) strains has progressed significantly, but their phenotypic expression is poorly established as CTV naturally occurs as mixed‐strain populations. A screening system for the analysis of mixed‐strain populations is required for population studies and the correlation with symptom expression. In this study, a published CTV strain‐specific detection assay was expanded and improved to facilitate detection of currently known CTV strains. Supplementary RT‐PCR assays were developed for two variant groups of the RB strain and the HA16‐5 strain, and assays for the T36 strain and generic CTV detection were improved. The value of the strain‐specific assays was shown by the ability to identify the strain components of two CTV cross‐protecting sources, GFMS35 and LMS6, used in the South African budwood certification scheme and to demonstrate the segregation of strains in budwood source trees.     

Overview of Strain Characterization in Relation to Serological and Molecular Detection of Citrus tristeza <i>Closterovirus</i>

Tristeza is a devastating viral disease in all the citrus growing countries throughout the world and has killed millions of citrus trees in severely affected orchards. The citrus species grafted on sour orange rootstock are affected by this disease. Predominantly, the sweet orange, grapefruit and lime trees grafted on sour orange exhibit severe symptoms like quick decline, vein clearing, pin holing, bark scaling and degeneration leading to variable symptoms. Symptomatic expression of Citrus tristeza virus (CTV) in different hosts has been attributed to virus isolates which are from severe to mild. Different serological and molecular assays have been deployed to differentiate the strains of CTV. Citrus tristeza virus is diversified towards its strains on the basis of biological, serological and molecular characterization. Phenotypic expression is due to genetic alteration and different molecular basis have now been adopted for strain differentiation. This review will give a brief idea about the different CTV isolates, their characterization based on nucleic acid and serological assays. Different methods along with salient features for strain characterization has also been reviewed. This review will also open the new aspects towards formulation of management strategies through different detection techniques.     

Production of additional allotetraploid somatic hybrids combining mandarings and sweet orange with pre-selected pummelos as potential candidates to replace sour orange rootstock

Summary Sour orange (Citrus aurantium L.) rootstock has historically been a widely utilized eitrus rootstock throughout the world due to its wide soil adaptability and superior horticultural performance. However, quick-decline isolates of citrus tristeza virus (CTV) have demolished entire industries of sour orange rootstock in some countries, including Brazil and Venezuela. CTV is presently destroying millions of trees of sour orange rootstock in Florida and threatens the citrus industries of Texas and Mexico, where sour orange is the predominant rootstock. Efforts to replace sour orange rootstock are combining traditional breeding and biotechnology approaches, including somatic hybridization and transformation. Molecular techniques have confirmed that sour orange is probably a hybrid of mandarin and pummelo. A major focus of our program continues to be the somatic hybridization of superior mandarins with pre-selected pummelo parents. Here, we report the regeneration of allotetraploid somatic hybrid plants from seven new mandarin+pummelo combinations and one new sweet orange+pummelo combination. All new somatic hybrids were confirmed by leaf morphology, ploidy analysis via flow cytometry, and random amplified polymorphic DNA analysis to show nuclear contributions from both parents in corresponding hybrids. These new somatic hybrids are being propagated by tissue culture and/or rooted cuttings for further evaluation of disease resistance and horticultural performance in field trials.     

Sscp analysis of variations in haplotypes of citrus tristeza virus isolated from yuzu (Citrus junos) in geographically separate regions of Korea

Yuzu (Cittus junos) trees were examined from six geographically separate provinces in the Republic of Korea, including four islands (Geoje, Namhae, Wan, and Jeju), 1 peninsula (Goheung), and 1 inland area (Boseong). The population of sequence variants of citrus tristeza virus (CTV) was isolated and analyzed by single-strand conformation polymorphism (SSCP) analysis of cDNA from thep20 gene. SSCP profiles of 65 PCR products showed different band patterns but with similar intensities. Sixteen haplotypes were subgrouped according to their SSCP profiles and severity of symptoms. Their genomes were sequenced and compared. DNA analysis of thep20 genes revealed nucleotide identities ranging from 88-99.8%. Based on SSCP analysis, the pathologically mild isolates of CTV yielded two to three DNA bands, whereas the most virulent isolates contained more than two bands. Comparisons of these physically separate haplotypes suggest that CTV isolates with multiple SSCP profiles could have arisen as a result of a mixed infection and genetic recombination of two divergent isolates. Plants with severe disease symptoms, such as stem pitting, closely corresponded to a CTV strain showing typical SSCP profiles in Florida (USA) and Japan.     

Enhancement or attenuation of disease by deletion of genes from Citrus tristeza virus

Stem pitting is a common virus-induced disease of perennial woody plants induced by a range of different viruses. The phenotype results from sporadic areas of the stem in which normal xylem and phloem development is prevented during growth of stems. These alterations interfere with carbohydrate transport, resulting in reduced plant growth and yield. Citrus tristeza virus (CTV), a phloem-limited closterovirus, induces economically important stem-pitting diseases of citrus. CTV has three nonconserved genes (p33, p18, and p13) that are not related to genes of other viruses and that are not required for systemic infection of some species of citrus, which allowed us to examine the effect of deletions of these genes on symptom phenotypes. In the most susceptible experimental host, Citrus macrophylla, the full-length virus causes only very mild stem-pitting symptoms. Surprisingly, we found that certain deletion combinations (p33 and p18 and/or p13) induced greatly increased stem-pitting symptoms, while other combinations (p13 or p13 plus p18) resulted in reduced stem pitting. These results suggest that the stem-pitting phenotype, which is one of more economically important disease phenotypes, can result not from a specific sequence or protein but from a balance between the expression of different viral genes. Unexpectedly, using green fluorescent protein-tagged full-length virus and deletion mutants (CTV9Δp33 and CTV9Δp33Δp18Δp13), the virus was found at pitted areas in abnormal locations outside the normal ring of phloem. Thus, increased stem pitting was associated not only with a prevention of xylem production but also with a proliferation of cells that supported viral replication, suggesting that at random areas of stems the virus can elicit changes in cellular differentiation and development.     

A severe citrus tristeza virus isolate causing the collapse of trees of sour orange before virus is detectable throughout the canopy§

A rapidly spreading decline of ‘Minneola’ tangelos, ‘Shamouti’ and ‘Valencia’ sweet oranges grafted on sour orange rootstock in the Morasha area, in the coastal plain of Israel, was found to be caused by a severe ‘seedling yellows’ strain of the citrus tristeza virus (CTV). Repeated ELISA tests revealed great variation in distribution of CTV throughout the canopies, even in declining trees. In a substantial number of the declining trees, samples of up to 10 twigs per tree were not always sufficient for CTV detection. The ELISA values (O.D. 405 nm) in the parts found infected were high, whereas in most of the twigs showing negative ELISA results the virus was absent as indicated by biological indexing. The Morasha strain of CTV was also characterised by rapid annual spread rates. The ratio D/E (the proportion of Declining trees found among ELISA-positive ones) is proposed as a simple index of strain severity. The epidemiological consequences of the uneven distribution of CTV and rapid decline are discussed.     

Acquisition and transmission of selected CTV isolates by Aphis gossypii

Citrus tristeza virus (CTV) is a severe threat to the citrus industry. Disease symptoms and severity may vary depending on the CTV isolates. These are responsible for the decline of trees grafted on sour orange rootstock, or stem pitting on some citrus commercial cultivars regardless of rootstock. In the Calabria region (Italy), CTV was first reported on cultivars imported from other countries. However, recent observations suggested that natural spread of CTV was occurring and a study was needed to determine the epidemiological status and aphid transmission of CTV in Calabria. The role played by local A. gossypii in the spread of CTV was analyzed in the laboratory using various viral acquisition, inoculation periods with three different CTV isolates. Single aphid vectors acquired CTV after a minimum of 30 min acquisition access period (AAP) and were able to transmit the virus after a 60 min inoculation access period (IAP) to healthy plants. A minimum of four aphid vectors were needed to reach 50% transmission probability. The results suggested that the three tested strains are transmitted by A. gossypii in a semi-persistent mode. The results demonstrated that local A. gossypii population can acquire and transmit efficiently the tested virus isolates with serious implications on the virus spread.     

Characterization of grapefruit plants (<Emphasis Type="Italic">Citrus paradisi</Emphasis> Macf.) transformed with citrus tristeza closterovirus genes

Grapefruit (Citrus paradisi Macf. cv Duncan) plants were transformed with several sequences from citrus tristeza closterovirus (CTV) that varied in terms of position in the CTV genome and virus strain origin in an attempt to obtain resistant plants. The sequences included the capsid protein gene from three different strains, a nontranslatable version of the capsid protein gene, the replicase (RdRp), the minor capsid protein (p27), a highly transcribed gene of unknown function (p20) and the more conserved 3' end of the genomic RNA. Transgenic plants were generated from all of the constructs, except from the p20 and p27 genes. Southern and Western blot analyses demonstrated that stably transformed grapefruit plants were obtained and that at least some transgenes were expressed. In a first effort at virus challenge, 25 transgenic lines were graft inoculated with a severe strain of CTV. Although some transgenic plants averaged lower titers of virus than controls, there was great variability in titer in both controls and transgenic plants, and all were apparently susceptible to the virus.     

The resistance of sour orange to Citrus tristeza virus is mediated by both the salicylic acid and RNA silencing defence pathways

Citrus tristeza virus (CTV) induces in the field the decline and death of citrus varieties grafted on sour orange (SO) rootstock, which has forced the use of alternative decline‐tolerant rootstocks in affected countries, despite the highly desirable agronomic features of the SO rootstock. Declining citrus plants display phloem necrosis below the bud union. In addition, SO is minimally susceptible to CTV compared with other citrus varieties, suggesting partial resistance of SO to CTV. Here, by silencing different citrus genes with a Citrus leaf blotch virus‐based vector, we have examined the implication of the RNA silencing and salicylic acid (SA) defence pathways in the resistance of SO to CTV. Silencing of the genes RDR1, NPR1 and DCL2/DCL4, associated with these defence pathways, enhanced virus spread and accumulation in SO plants in comparison with non‐silenced controls, whereas silencing of the genes NPR3/NPR4, associated with the hypersensitive response, produced a slight decrease in CTV accumulation and reduced stunting of SO grafted on CTV‐infected rough lemon plants. We also found that the CTV RNA silencing suppressors p20 and p23 also suppress the SA signalling defence, with the suppressor activity being higher in the most virulent isolates.     

Antigenic presentation of heterologous epitopes engineered into the outer surface-exposed helix 4 loop region of human papillomavirus L1 capsomeres

The molecular characterization of isolates of citrus tristeza virus (CTV) from eight locations in Mexico was undertaken by analyzing five regions located at the opposite ends of the virus genome. Two regions have been previously used to study CTV variability (coat protein and p23), while the other three correspond to other genomic segments (p349-B, p349-C and p13). Our comparative nucleotide analyses included CTV sequences from different geographical origins already deposited in the GenBank databases. The largest nucleotide differences were located in two fragments located at the 5' end of the genome (p349-B and p349-C). Phylogenetic analyses on those five regions showed that the degree of nucleotide divergence among strains tended to correlate with their pathogenicity. Two main groups were defined: mild, with almost no noticeable effects on the indicator plants and severe, with drastic symptoms. Mild isolates clustered together in every analyzed ORF sharing a genetic distance below 0.022, in contrast with the severe isolates, which showed a more disperse distribution and a genetic distance of 0.276. Analyses of the p349-B and p349-C regions evidenced two lineages within the severe group: severe common subgroup (most of severe isolates) and severe divergent subgroup (T36-like isolates). This study represents the first attempt to analyze the genetic variability of CTV in Mexico by constructing phylogenetic trees based on new genomic regions that use group-specific nucleotide and amino acid sequences. These results may be useful to implement specific assays for strain discrimination. Moreover, it would be an excellent reference for the CTV situation in México to face the recent arrival of brown citrus aphid.     

Serological characterisation of citrus tristeza virus isolates from Israel

Seven monoclonal antibodies (MAbs) showing homologous reactions with the VT strain of citrus tristeza virus (CTV) were tested against 21 CTV strains or isolates, representing the range of biological diversity and the geographical distribution of CTV in Israel. All the CTV strains gave positive reactions in ELISA with polyclonal antibodies and with one MAb (#25#2). Two MAbs; #13#21 and #3/7 reacted with 19 and 12 out of the 21 CTV strains, respectively. Seventeen CTV strains, including all those previously assigned by sequencing their coat protein gene (CPG) to the CPG-VT group (Mawassi, Gafny & Bar-Joseph, 1993), reacted with five or more MAbs. Four CTV strains of minor epidemiological importance, including two members of the CPG-MT group, did not react with five or more of the MAbs. These results indicated the existence of two serogroups of Israeli CTV strains that can be differentiated by MAbs and which closely correlate with and extend the previous CPG grouping. The extensive biological variation within each CPG group confirms recent analyses suggesting that the CTV pathogenic traits are not necessarily associated with a sequence or antigenic variation of the CTV-CPG.     

Reassortant DS‐1‐like G1P[4] Rotavirus A strains generated from co‐circulating strains in Vietnam, 2012/2013

One major mechanism by which Rotavirus A (RVA) evolves is genetic reassortment between strains with different genotype constellations. However, the parental strains of the reassortants generated have seldom been identified. Here, the whole genome of two suspected reassortants, RVA/Human‐wt/VNM/SP127/2013/G1P[4] and RVA/Human‐wt/VNM/SP193/2013/G1P[4], with short RNA electropherotypes were examined by Illumina MiSeq sequencing and their ancestral phylogenies reconstructed. Their genotype constellation, G1‐P[4]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2, indicated that they were G1 VP7 mono‐reassortants possessing DS‐1‐like genetic backbones. The two strains were ≧99.7% identical across the genome. While their VP7 genes were ≧99.7 identical to that of a Wa‐like strain RVA/Human‐wt/VNM/SP110/2012/G1P[8] which co‐circulated during the 2012/2013 season, 10 genes were ≧99.8% identical to that of the DS‐1‐like strains RVA/Human‐wt/VNM/SP015/2012/G2P[4] (and SP108) that co‐circulated during the season. The identities were consistent with the phylogenetic relationships observed between the genes of the reassortants and those of the afore‐mentioned strains. Consequently, the G1P[4] strains appear to have been generated by genetic reassortment between SP110‐like and SP015‐like strains. In conclusion, this study provides robust molecular evidence for the first time that G1P[4] strains detected in Hanoi Vietnam were generated by inter‐genogroup reassortment between co‐circulating G1P[8] and G2P[4] strains within the same place and season.

     

Detection and Inoculation of Peanut Witches' Broom Phytoplasma (16SrII‐A) and Periwinkle Leaf Yellowing Phytoplasma (16SrI‐B) in Citrus Cultivars in Taiwan

To clarify the phytoplasma associated with Huanglongbing (HLB), a detection survey of phytoplasma in field citrus trees was performed using the standardized nested PCR assay with primer set P1/16S‐Sr and R16F2n/R16R2. The HLB‐diseased citrus trees with typical HLB symptoms showed a high detection of 89.7% (322/359) of HLB‐Las, while a low detection of phytoplasma at 1.1% (4/359) was examined in an HLB‐affected Wentan pummelo (Citrus grandis) tree (1/63) and Tahiti lime (C. latifolia) trees (3/53) that were co‐infected with HLB‐Las. The phytoplasma alone was also detected in a healthy Wentan pummelo tree (1/60) at a low incidence total of 0.3% (1/347). Healthy citrus plants were inoculated with the citrus phytoplasma (WP‐DL) by graft inoculation with phytoplasma‐infected pummelo scions. Positive detections of phytoplasma were monitored only in the Wentan pummelo plant 4 months and 3.5 years after inoculation, and no symptoms developed. The citrus phytoplasma infected and persistently survived in a low titre and at a very uneven distribution in citrus plants. Peanut witches' broom (PnWB) phytoplasma (16SrII‐A) and periwinkle leaf yellowing (PLY) phytoplasma belonging to the aster yellows group (16SrI‐B) maintained in periwinkle plants were inoculated into healthy citrus plants by dodder transmission. The PnWB phytoplasma showed infection through positive detection of the nested PCR assay in citrus plants and persistently survived without symptom expression up to 4 years after inoculation. Positive detections of the phytoplasma were found in a low titre and several incidences in the other inoculated citrus plants including Ponkan mandarin, Liucheng sweet orange, Eureka lemon and Hirami lemon. None of the phytoplasma‐infected citrus plants developed symptoms. Furthermore, artificial inoculation of PLY phytoplasma (16SrI‐B) into the healthy citrus plants demonstrated no infection. The citrus symptomless phytoplasma was identified to belong to the PnWB phytoplasma group (16SrII‐A).