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 tissue 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.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, but cases of cross-protection breakdown have been experienced and a change in the pre-immunising source for grapefruit cross protection 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,hydroponic indoor growing system 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 non irrigated 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’ root stock. 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 CSD symptomatic 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,macetas 1 litro whereas for the S-CSD dataset 0.014% of reads come from the CTV virus. However, almost three times more reads of CSD disease 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 . One VerechipTM, an electrically active system that integrates a PCR module, consisting of two independent PCR reactors, and a hybridization chamber, is used for both quadruplex reactions. RNA and RT-PCR master mix are combined and loaded to the chip, which is placed in a thermal reactor for rapid end point PCR. Hybridisation mix is then added and the RT-PCR products, with sizes between 73 and 239 bp, flow into the microarray portion of the chip, which is returned to the thermal reactor. Hybridisation complete, the array is then read. The array was represented by 44 probes designed for the phylogenetic groups with type strains T36, T3, T30, VT, NZ-RB, B165 and other emerging clusters such as HA16-5. The distribution of the target genes throughout the genome and the specific probes designed on the nucleotide sequences, enables the identification of any known strain even if recombinant or in mixed infections, and is likely to distinguish new groups.

Historically, decline has been the most devastating disease caused by Citrus tristeza virus , although stem pitting greatly limits production in many citrus industries around the world. Decline has been the major problem caused by CTV in Florida because fortunately severe stem-pitting isolates have been kept out so far. Decline is a man-made disease based on propagation of sweet orange, grapefruit, and mandarins on the sour orange root stock. Although this disease can be controlled by using alternative root stocks, there are soils in which all other root stock choices are less desirable in terms of fruit quality and yield. One of our major goals has been to develop a way to allow growers to use the sour orange root stock in the presence of CTV. Florida has two predominant strains of CTV, a decline strain and a non-decline strain . A first goal has been to map the viral determinant that induces decline. This was done by creating hybrids by substituting T30 sequences into T36. A parallel goal was to use a non-decline T36 hybrid to cross protect trees on the sour orange root stock from decline. This project was delayed considerably because we were not able to definitively assay decline in the greenhouse because we were unable to distinguish decline symptoms from seedling yellows symptoms with these hybrids. In order to examine decline in field trees, we had to obtain permission to do a field test of recombinant DNA produced virus hybrids from the USDA Biotechnology Regulatory Service. Valencia sweet orange on sour orange root stocks were inoculated with T30/T36 hybrids in the greenhouse and transferred to the field in 2010. The test will end August 2013. Results of this field test will be presented showing that the p23 and 3’ non-translated sequences of T36 contain the determinant of decline.A high-throughput sequencing pipeline to characterize Citrus tristeza virus isolates was developed. Three alternative viral templates combined with random RT-PCR amplification were first tested on a single, previously characterized GFMS12 sub-isolate for their enrichment qualities and subjected to Illumina paired-end sequencing. Comparisons between the sequencing data obtained and gene-specific phylogenies were also made in order to test their validity. Double-stranded RNA was found to be most enriching and was utilized for further characterization of additional glasshouse-kept isolates. A novel T68-1-like South African genotype, named CT-ZA3 was assembled de novo and shown to be the dominant component in all GFMS12 sub-isolates tested. This study underlined the effectiveness of next-generation sequencing for genotype discovery as well as whole-genome characterization of CTV isolates to a level of detail not previously attainable with classical methods such as single stranded conformational polymorphism and Sanger sequencing of multiple clones, which have been shown previously to yield incongruences in genotype identification.Next-generation sequencing has been established as a reliable approach to study metagenomic samples. In plant pathology, the application of NGS has not been limited to sequencing for pathogen discovery, but has also been used for applications such as the study of plant-pathogen interactions. In this study, we used metagenomic NGS to establish the virome of citrus samples. Double stranded RNA was extracted from the cambium tissue of virus diseased citrus trees with a cellulose extraction protocol and sequenced in an unbiased manner using Illumina sequencing-by-synthesis technology on a HiScanSQ. Two samples were analyzed; one displayed atypical “psorosis” symptoms and the other was a source plant containing Apple stem grooving virusand Citrus tristeza virus .