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.     

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.     

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.     

Citrus tristeza virus: survival at the edge of the movement continuum

Systemic invasion of plants by viruses is thought to involve two processes: cell-to-cell movement between adjacent cells and long-distance movement that allows the virus to rapidly move through sieve elements and unload at the growing parts of the plant. There is a continuum of proportions of these processes that determines the degrees of systemic infection of different plants by different viruses. We examined the systemic distribution of Citrus tristeza virus (CTV) in citrus species with a range of susceptibilities. By using a "pure" culture of CTV from a cDNA clone and green fluorescent protein-labeled virus we show that both cell-to-cell and long-distance movement are unusually limited, and the degree of limitation varies depending on the citrus host. In the more-susceptible hosts CTV infected only a small portion of phloem-associated cells, and moreover, the number of infection sites in less-susceptible citrus species was substantially decreased further, indicating that long-distance movement was reduced in those hosts. Analysis of infection foci in the two most differential citrus species, Citrus macrophylla and sour orange, revealed that in the more-susceptible host the infection foci were composed of a cluster of multiple cells, while in the less-susceptible host infection foci were usually single cells, suggesting that essentially no cell-to-cell movement occurred in the latter host. Thus, CTV in sour orange represents a pattern of systemic infection in which the virus appears to function with only the long-distance movement mechanism, yet is able to survive in nature.     

Etiology of three recent diseases of citrus in São Paulo State: sudden death, variegated chlorosis and huanglongbing

The state of S?o Paulo (SSP) is the first sweet orange growing region in the world. Yet, the SSP citrus industry has been, and still is, under constant attack from various diseases. In the 1940s, tristeza-quick decline (T-QD) was responsible for the death of 9 million trees in SSP. The causal agent was a new virus, citrus tristeza virus (CTV). The virus was efficiently spread by aphid vectors, and killed most of the trees grafted on sour orange rootstock. Control of the disease resided in replacing sour orange by alternative rootstocks giving tolerant combinations with scions such as sweet orange. Because of its drought resistance, Rangpur lime became the favourite alternative rootstock, and, by 1995, 85% of the SSP sweet orange trees were grafted on this rootstock. Therefore, when in 1999, many trees grafted on Rangpur lime started to decline and suddenly died, the spectre of T-QD seemed to hang over SSP again. By 2003, the total number of dead or affected trees was estimated to be over one million. The new disease, citrus sudden death (CSD), resembles T-QD in several aspects. The two diseases have almost the same symptoms, they spread in time and space in a manner strikingly similar, and the pathological anatomy of the bark at the bud union is alike. Transmission of the CSD agent by graft-inoculation has been obtained with budwood inoculum taken not only on CSD-affected trees (grafted on Rangpur lime), but also on symptomless trees (grafted on Cleopatra mandarin) from the same citrus block. This result shows that symptomless trees on Cleopatra mandarin are tolerant to the CSD agent. Trees on rootstocks such as Sunki mandarin or Swingle citrumelo are also tolerant. Thus, in the CSD-affected region, control consists in replacing Rangpur lime with compatible rootstocks, or in approach-grafting compatible rootstock seedlings to the scions of trees on Rangpur lime (inarching). More than 5 million trees have been inarched in this way. A new disease of sweet orange, citrus variegated chlorosis (CVC), was observed in 1987 in the Triangulo Mineiro of Minas Gerais State and the northern and north-eastern parts of SSP. By 2000, the disease affected already 34% of the 200 million sweet orange trees in SSP. By 2005, the percentage had increased to 43%, and CVC was present in all citrus growing regions of Brazil. Electron microscopy showed that xylem-limited bacteria were present in all symptomatic sweet orange leaves and fruit tissues tested, but not in similar materials from healthy, symptomless trees. Bacteria were consistently cultured from twigs of CVC-affected sweet orange trees but not from twigs of healthy trees. Serological analyses showed the CVC bacterium to be a strain of Xylella fastidiosa. The disease could be reproduced and Koch's postulates fulfilled, by mechanically inoculating a pure culture of X. fastidiosa isolate 8.1.b into sweet orange seedlings. The genome of a CVC strain of X. fastidiosa was sequenced in SSP in the frame of a project supported by FAPESP and Fundecitrus. X. fastidiosa is the first plant pathogenic bacterium, the genome of which has been sequenced. Until recently, America was free of huanglongbing (HLB), but in March 2004 and August 2005, symptoms of the disease were recognized, respectively in the State of S?o Paulo (SSP) and in Florida, USA. HLB was known in China since 1870 and in South Africa since 1928. Because of its destructiveness and its rapid spread by efficient psyllid insect-vectors, HLB is probably the most serious citrus disease. HLB is caused by a phloem sieve tube-restricted Gram negative bacterium, not yet available in culture. In the 1990s, the bacterium was characterized by molecular techniques as a member of the alpha proteobacteria designated Candidatus Liberibacter africanus for the disease in Africa, and Candidatus Liberibacter asiaticus for HLB in Asia. In SSP, Ca. L. asiaticus is also present, but most of the trees are infected with a new species, Candidatus Liberibacter americanus.     

Production of Virus-tree Nucellar Plantlets from Unfertilized Ovules of Citrus in vitro

On a series of Murashige and Tucker (MT) media supplemented with different growth regulators, the 8-week-old unfertilized ovules of Washington navel orange (Citrus sinensis) were able to regenerate perfect plantlets via somatic embryogenesis or organogenesis. The sorts and combinations of exogenous hormones had remarkable effects on the induction, growth and differentiation of its callus. It was found that the most suitable induction medium was MT medium supplemented with 1.0 mg/l BA and 0.5mg/l 1AA. The most suitable differentiation medium was MT medium supplemented with 1.0 mg/l IBA. It was proved by indicator plant examination that the nucellar plantlets free of citrus exocortis virus (CEV) and citrus tristeza virus (CTV) had been obtained from infected trees.     

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.     

Detection of Citrus Tristeza and Greening in Pakistan Through Electron Microscopy

An electron microscopic investigation was carried out on citrus fruits and leaves collected during a survey in Pakistan. Thread-like particles of citrus tristeza virus (CTV) were ascertained in phloem tissues of the columclla, whereas the bacterium associated with greening was detected in sieve tubes of the columella and in leaf midribs of trees showing typical yellow vein, fruit maiformation an decline. CTV was also confirmed by ELISA tests.     

The movement and distribution of citrus tristeza virus and citrus exocortis viroid in citrus seedlings1

The systemic movement of citrus tristeza virus (CTV) in sour orange (Citrus aurantium) seedlings and of citrus exocortis viroid (CEVd) in Etrog citron (C. medica) seedlings was studied. The movement of the two pathogens was analysed by detection in sections of roots and stems at different time intervals. Both pathogens were detected initially in the basal parts and the roots and subsequently spread to the shoot. CTV and CEVd moved in young citrus seedlings at similar rates. The findings are consistent with long distance phloem transport of the virus and the viroid. The practical implications of the pattern of systemic movement for diagnosis of infected trees are discussed.     

Detection of Australian Field Isolates of Citrus Tristeza Virus by Double-Stranded RNA Analysis

Double-stranded RNA analysis indicated that widespread latent infection by citrus tristeza virus (CTV) occurs naturally in a citrus variety collection at Merbein, Australia. Detection was based on the presence of the putative replicative form (RF) of the virus (ca 19. 5 kilobase pairs) and the presence or absence of three previously known putative subgenomic dsRNAs with molecular sizes of ca 3. 1,1.7 and 0.8 kilobase pairs. With the exception of some citrus relatives normally used as rootstocks, the dsRNAs were readily detectable in all the trees tested. Variability was noticed among the dsRNAs profiles of individual seedling trees of Microcitrus australasica var. sanguinea. By contrast, dsRNA patterns of trees of open-pollinated Ellendale tangor were indistinguishable. A number of dsRNA bands found in West Indian lime infected with CTV were not detected when this isolate was graft-inoculated to healthy sweet orange seedlings. A simple method for enhancing the sensitivity of detection of the CTV dsRNA bands by silver staining is described which involves incubating the gels in RNase A prior to staining; this allowed detection of the putative CTV RF in only 40 mg of green hark material.     

Expressed sequence enrichment for candidate gene analysis of citrus tristeza virus resistance

Several studies have reported markers linked to a putative resistance gene from Poncirus trifoliata (Ctv-R) located at linkage group 4 that confers resistance against one of the most important citrus pathogens, citrus tristeza virus (CTV). To be successful in both marker-assisted selection and transformation experiments, its accurate mapping is needed. Several factors may affect its localization, among them two are considered here: the definition of resistance and the genetic background of progeny.Two progenies derived from P. trifoliata, by self-pollination and by crossing with sour orange (Citrus aurantium), a citrus rootstock well-adapted to arid and semi-arid areas, were used for linkage group-4 marker enrichment. Two new methodologies were used to enrich this region with expressed sequences. The enrichment of group 4 resulted in the fusion of several C. aurantium linkage groups. The new one A(7+3+4) is now saturated with 48 markers including expressed sequences. Surprisingly, sour orange was as resistant to the CTV isolate tested as was P. trifoliata, and three hybrids that carry Ctv-R, as deduced from its flanking markers, are susceptible to CTV. The new linkage maps were used to map Ctv-R under the hypothesis of monogenic inheritance. Its position on linkage group 4 of P. trifoliata differs from the location previously reported in other progenies. The genetic analysis of virus-plant interaction in the family derived from C. aurantium after a CTV chronic infection showed the segregation of five types of interaction, which is not compatible with the hypothesis of a single gene controlling resistance. Two major issues are discussed: another type of genetic analysis of CTV resistance is needed to avoid the assumption of monogenic inheritance, and transferring Ctv-R from P. trifoliata to sour orange might not avoid the CTV decline of sweet orange trees.Communicated by C. Möllers     

The pathogenicity determinant of Citrus tristeza virus causing the seedling yellows syndrome maps at the 3'-terminal region of the viral genome

Citrus tristeza virus (CTV) (genus Closterovirus , family Closteroviridae ) causes some of the more important viral diseases of citrus worldwide. The ability to map disease-inducing determinants of CTV is needed to develop better diagnostic and disease control procedures. A distinctive phenotype of some isolates of CTV is the ability to induce seedling yellows (SY) in sour orange, lemon and grapefruit seedlings. In Florida, the decline isolate of CTV, T36, induces SY, whereas a widely distributed mild isolate, T30, does not. To delimit the viral sequences associated with the SY syndrome, we created a number of T36/T30 hybrids by substituting T30 sequences into different regions of the 3' half of the genome of an infectious cDNA of T36. Eleven T36/T30 hybrids replicated in Nicotiana benthamiana protoplasts. Five of these hybrids formed viable virions that were mechanically transmitted to Citrus macrophylla , a permissive host for CTV. All induced systemic infections, similar to that of the parental T36 clone. Tissues from these C. macrophylla source plants were then used to graft inoculate sour orange and grapefruit seedlings. Inoculation with three of the T30/T36 hybrid constructs induced SY symptoms identical to those of T36; however, two hybrids with T30 substitutions in the p23-3' nontranslated region (NTR) (nucleotides 18 394–19 296) failed to induce SY. Sour orange seedlings infected with a recombinant non-SY p23-3' NTR hybrid also remained symptomless when challenged with the parental virus (T36), demonstrating the potential feasibility of using engineered constructs of CTV to mitigate disease.     

Intra-farm diversity and evidence of genetic recombination of Citrus tristeza virus in Delhi region of India

Infection of Citrus tristeza virus (CTV) in different citrus orchards of New Delhi was detected by direct antigen coated-ELISA and RT-PCR. Sweet orange (Citrus sinensis) orchards were found to be susceptible to CTV with estimated disease incidence up to 39%. Kagzi kalan (C. lemon), Pumello (C. paradisi) and Kinnow mandarin (C. reticulata) orchards did not show CTV infection. Three CTV isolates, D1, D7 and D15 randomly selected from infected sweet orange orchards were considered for biological and molecular characterization. In the host range study, all the Delhi isolates infected Darjeeling mandarin (C. reticulata), Kagzi lime (C. aurantifolia), sour orange (C. aurantium) and sweet orange but not Kinnow mandarin. A fragment of 5??ORF1a and complete coat protein (CP) gene of these three isolates were cloned, sequenced and compared with other Indian and international CTV isolates. Delhi isolates shared 85?C92% sequence identity for 5??ORF1a fragment and 89?C91% for CP gene among them. Phylogenetic analysis segregated three Delhi isolates into three genogroups for each of 5??ORF1a fragment and CP gene, however phylogenetic relationships for both the genomic regions was incongruent. Recombination detecting program RDP3 detected CTV isolate D7 as recombinant, indicating genetic variability in CTV isolates might be the outcome of recombination events between divergent CTV sequences. An attempt was made in present study to characterize CTV isolates biologically and at genetic level, and to determine genetic diversity at farm level and study the recombination of CTV isolates in Delhi region.     

Sensitive and Specific Digoxigenin-labelled RNA Probes for Routine Detection of Citrus tristeza virus by Dot-blot Hybridization

A non‐radioactive dot‐blot hybridization assay for the successful detection of Citrus tristeza virus (CTV) RNA in total nucleic acid extracts of infected citrus was developed. Two digoxigenin (DIG)‐labelled minus‐sense riboprobes, complementary to the coat protein gene sequence of a Chinese and an Apulian CTV isolate were synthesized. Several citrus tissues were evaluated as optimal virus source and leaf petioles were found appropriate material for reliable detection. The hybridization assay showed a detection limit corresponding to 0.2 mg of fresh infected tissue. The riboprobes allowed CTV detection in isolates from different geographical areas, grown in the screenhouse or in the field, resulting in similar hybridization patterns. The infected trees were tested during different seasons with positive results, although from July to August most of the samples gave a weaker hybridization signal, compared to other seasons. The high sensitivity and reliability of the molecular hybridization assay described make it a good alternative to serological methods for CTV detection.     

Symptomatic expression of tristeza-infected citrus plants in Pakistan

Variable symptoms were recorded during a survey in the citrus trees infected or suspected to be infected with citrus tristeza virus based on ELISA tests. Sweet orange manifested main symptoms like bud union crease, pin holing, bark cracking, incompatibility, yellowing and dropping of leaves. Pin holing was particularly prevalent in Sweet orange (Citrus sinensis Osbeck), Kinnow (Citrus reticulata Blanco) and Grapefruit (Citrus paradisii Mcaf.), while incompatibility was only in Sweet orange and Grapefruit.