Reports from all over the world show that several destructive isolates of CTV, not dependent on sensitive rootstocks, may suddenly appear as a result of rearrangements or mutations of the genome. Also, it appears that bio-indexing on indicator plants or other official equivalent methods cannot help to limit the introduction of exotic strains, given that biological indexing is time consuming and molecular methods have a limited range of discrimination. With this in view, and thanks to the recent progress in CTV genome sequencing, we developed a fast diagnostic assay in which multiplex RT-PCR combined with a sequential hybridization step on the InCheck Platform allows the genotyping of CTV isolates. The 44 probes were designed on the complete genome of 38 CTV representative isolates of six phylogenetic clades . Tests carried out with isolates from different countries were used to validate the diagnostic procedure: single or mixed MMM genotypes inducing seedling yellows and stem pitting on sour orange and grapefruit; VT+T3 and VT genotypes inducing SY/SP on sweet orange; and mild isolates, with T30+T3 or T30 genotypes. Quadruplex primers Qua1 and Qua2 targeting eight genes, and, coupled with the panel of specific probes, after the hybridization step,mobile grow system resulted in signals of VT-like group probes for SY isolates and RB probes for isolates inducing SY and SP.
T30-like isolates from asymptomatic as well as combined infections were also detected. According to the results the detection and hybridization process is easy, rapid and accurate and can also be run by someone with no background in biology. With such potential it could dramatically increase the capability of diagnostic laboratories and contribute to minimizing the impact of new emerging CTV strains.Unlike most citrus infecting viruses, Citrus tristeza viruspossesses a number of distinct strains that produce a range of disease syndromes on different host species. In addition, these strains are frequently found in mixed populations within a single host. While we have some knowledge of the symptoms produced by a single strain, there is little understanding of how combinations of strains affect symptom expression and disease severity. To test the effect of population structure on symptom expression, we inoculated Citrus paradisi and C. aurantii with a range of single strains and mixed field populations of CTV. Seedling yellows symptom development was assessed six months post-inoculation. Real-time qRT-PCR was used to assess relative population titre, and these data were compared with symptom severity. We found that there was no obvious correlation between the dominance or presence of specific isolates in the population and the incidence or severity of seedling yellows; there was also no correlation between total viral load within infected tissues and symptom severity. These data further suggest that symptom severity is not due to quantitative differences between strains in a population, but indicates that qualitative differences within and between each population influence symptom development.
Citrus tristeza virusis the cause of an important and devastating citrus disease with great economic loss to some citrus species in certain regions of the world. In Chile, the disease has spread throughout the entire national citrus zone. Tthe major damage has been caused in the northern region where isolates capable of causing severe stem pitting have been detected. The CTV genome is close to 20.000 bp and encodes three suppressors that block the natural defence strategy of the plant, the “silencing mechanism”. This research work proposes a strategy to generate transgenic rootstocks resistant or tolerant to the disease through posttranscriptional gene silencing in which the silencing signals will move from rootstock to scion preventing future infections. We have developed two hybrid genetic-constructs CTV-p25-p20 and CTV-p20-p23 based in the T36-strain. These transgenes were synthesized and integrated in hairpin mode in the pHellgate12 plasmid. These constructs were used in transformation of Citrus macrophylla in order to obtain rootstocks resistant or tolerant to CTV. The methodology employed was the transformation of epicotyl tissues; the transformation was mediated by Agrobacterium tumefaciens, strain EHA105. The transformed explants were incubated at 25ºC in co-cultivation medium in the dark for 72 hrs and then the explants were regenerated in a medium with kanamycin selection. We have obtained transgenic shoots that show transgene integration into the plant-DNA. In the near future these shoots will be grafted with a CTV infected Lime scion to challenge the transgenic rootstocks and evaluate the silencing process in the scion.
Twenty four samples from non-symptomatic Pummelo trees as well as trees expressing virus-like symptoms such as inverted leaf cupping and/or stem pitting were collected randomly across citrus-growing regions in the Philippines between 2012 and 2013. Samples were propagated on calamandarin rootstock and maintained under standard greenhouse conditions at the Bureau of Plant Industry in Davao City, Philippines. Pummelo budwood obtained from the greenhouse propagations was shipped to the University of California, Riverside under permit. Total RNA was extracted and analyzed by reverse transcription polymerase chain reaction using primers targeting the major coat protein gene of the Citrus tristeza virus . All samples from Luzon Island tested negative for CTV while eleven samples from Mindanao and Visayas Island were CTV positive. The CTV genotype of the positive samples was determined using multiple molecular markers targeting different CTV genome regions. The predominant CTV genotype identified was VT in single or mixed infections with T30. A single sample contained the T36 CTV genotype. Preliminary phylogenetic analysis based on the full length sequence of the CP gene indicated that ten Philippines CTV isolates with VT and VT+T30 genotypes clusThered in one clade closely related to VT isolates from China and Japan . The single Philippines isolate with a T36 genotype clusThered with CTV isolates from India , Hawaii , and Taiwan . To minimize losses in the local citrus industry, the South African Citrus Improvement Scheme implemented cross-protection using mild CTV sources to reduce the effect of challenges by endemic severe CTV strains. This management strategy also referred to as “pre-immunisation” or “mild strain protection” was implemented by the CIS at its initiation. The use of cross-protection in South Africa has been mostly successful,mobile vertical rack but cases of cross-protection breakdown have been experienced and a change in the pre-immunising source for grapefruit crossprotection was made to address this. A number of countries apply cross-protection and report diminished expression of disease and improved production including Australia, Japan, Brazil, Argentina, Peru and South Africa. All pre-immunisation sources used for cross-protection, except the South African sources, are single variant sources. CTV is a complex of strains. This insight and the subsequent development of diagnostics for genotyping enabled the analysis of mixed populations. We have expanded on a published CTV genotype testing system and have tested various maintenance sources of the GFMS12 and GFMS35 pre-immunisation sources at 3 different institutions including grapefruit mother trees maintained at the Citrus Foundation Block. Also segregation of genotypes is noted in different multiplications of the two sources and this is in all probability a contributing factor to cross-protection breakdown in the field.Bio-indexing and characterization by CE-SSCP, MMM and phylogenetic analysis of representative samples collected in a highly infected Citrus tristeza virus area of Sicily reveals that two main groups are present: one includes isolates inducing severe seedling yellows and a second is asymptomatic on sour orange. Severe isolates cluster in a single subclade within the group of seedling yellows and stem pitting isolates ; mild ones are similar to T30-like isolates. In order to investigate the phylogenetic relationship of the CTV population with the isolates of respective clades we undertook the complete genome analysis of two of them, namely SG29 and Bau282. SG29 is a severe isolate, from Sanguinello sweet orange, inducing seedling yellows on sour orange and, rarely, stem pitting on Duncan grapefruit, but not on sweet orange. Bau282 is from sweet orange TDV and host susceptibility showed it is asymptomatic on sour orange and Duncan grapefruit. The genomes were obtained after sequencing of the small RNAs and assembly of overlapping sequences by reference alignment from libraries sequenced by Next-generation platforms. The viral small interfering RNAs found were in the predominant 22 and 21 nucleotide-size classes.
The complete SG29 and Bau282 genome in length are 19,259 bp and 19,250 bp, respectively, with 12 open reading frames , structurally identical to the other known CTV isolates. Phylogenetic analysis based on 31 full CTV genomes showed that SG29 clustered with the “Asian” VT-lineage in which T318A , AT- 1 , Nuaga and CT11A isolates segregate and has the highest homology identity with T318A and AT-1 . Bau282 clustered within the mild isolates T30 and T385 and BLAST analysis showed a very high identity equal to 99%. Since its appearance two centuries ago, Tristeza has been classified as the most devastating viral disease which affects citrus worldwide. Its causative agent, Citrus tristeza virus , is transmitted by infected grafts or by insect vectors such as the aphid Toxoptera citricida. In all affected citrus growing areas, the existence of genetic variants of the virus with different degrees of severity has been reported. Characterization of CTV isolates can provide epidemiological information and can be useful for disease control. The presence of CTV and its efficient vector has been known in Uruguay since the 1940s. However, there is no data based on molecular biology reflecting genetic variants circulating in the country. In the present study, using RT-PCR amplification of three regions of the CTV genome , we established phylogenetic relationships of the strains in the country at the present time and 20 years ago. The samples used were collected in 1990 and maintained in planta in vector free greenhouses, and also were compared with current field samples. This valuable historical collection provides a sample of past CTV occurrence in Uruguay. The results show that circulating strains in the country are severe and unsurprisingly resemble strains reported in Argentina. However, some of the strains under study are similar to reference strains from Israel or Hawaii. This may reflect the introduction of infected buds or trees in the past.Citrus Sudden Death remains a challenge for citrus production in nonirrigated areas in the North and Northwest regions of Sao Paulo State, Brazil. Its incidence has increasingly affecting the drought-tolerant but CSD-susceptible ‘Rangpur lime’ rootstock. So far, since the first report of the disease in the 2000s its etiology remains uncertain, but vectored-viruses have been considered as the main hypothesis . Here we checked the occurrence of these viruses in a deep sequencing dataset of total RNA by Illumina platform run for transcriptomic studies of sweet orange. Pools of reads of Valencia graphed on Rangpur lime and Sunki mandarin, both CSDsymptomatic and also from its non-symptomatic pairwise trees growing side-by-side were analyzed. Around 74 million reads for each experimental condition were assembled using the CLCbio platform de novo assembly algorithm using a CTV reference genome . For the NS-CSD dataset only 0.005% of reads matched with CTV virus sequences, whereas for the S-CSD dataset 0.014% of reads come from the CTV virus. However, almost three times more reads of CSDdisease trees matched with CTV. A careful comparison of CTV reads from S-CSD with NS-CSD allowed us to identify some sets of reads more frequently occurring in diseased trees, as also some CTV reads specifically occurring in the CSD diseased plants. Those genetic differences which make the CTV-reads specific for the CSD symptomatic plants were mapped through the whole CTV genome reference.On the other hand, using the same assembly strategy we observed only few reads matching the Tymovirus genome in the analyzed dataset, independent of whether they come from diseased or non-diseased trees. Despite being poorly represented, the Tymovirus’s reads were shown to be genetically diverse, which was confirmed by Sanger sequencing of amplicons from five different Tymovirus’ genomic regions amplified by specific primers. Citrus tristeza virusis the causal agent of the most important citrus disease and exists as numerous strains which may cause different symptoms. Due to the wide biological diversity of the virus, identification of the actual genotypes present in an area is useful in adopting adequate control strategies. In recent years a plethora of methods for molecular characterization of the virus have been developed. Among others RT-PCR, Real-Time PCR and CE-SSCP have been adapted for discrimination of severe and mild CTV strains. In this work, based on the fully sequenced genomes of the virus available on GenBank, we have developed two quadruplex primer sets, one for simultaneously obtaining amplicons from the 5’UTR, ORF1a, RdRp and p27 regions , the other for p20, p23 and p33, and the Citrus elongation factor EF 1 alpha .