Studies On The Citrus Tristeza Virus And Citrus Viroids In Pakistan

Citrus tristeza virus (CTV) is distributed worldwide, and is the causal agent of one of the most economically important and destructive diseases of citrus. CTV, a member of the genus Closterovirus within the family Closteroviridae, is phloem-limited and is dispersed to new areas by grafting of buds infected and then locally spread by aphids in a semi-persistent manner. CTV virion are flexuous filamentous of around 11×2,000nm, composed of a single-stranded, positive-sense genomic RNA(gRNA) molecule of approximately 19,300nt, the largest known for plant RNA viruses. The gRNA is organized in 12 open reading frames (ORFs), potentially encoding at least 19 protein products, and un-translated regions (UTRs) of 107 and 275 nucleotides at the 5'and 3' termini. Complicated diversity of CTV strains or isolates has occurred, which cause such a range of symptoms as decline and death of sweet orange and grapefruit on sour orange rootstock, stem-pitting, seedling yellows, etc. Stem-pitting tristeza has been become a severe threat to some susceptible cultivars such as sweet orange and grapefruit in the Punjab province of Pakistan. So far, according to the experience obtained from other countries, mild strain cross protection (MSCP) has been proven to be the most effective way of preventing sensitive citrus varieties from CTV damage where both severe CTV strains and brown citrus aphids [Toxoptera citricida (Kirkaldy)] co-exist. The application of MSCP bases are a priority to investigation, isolation and identification of the severe and mild CTV strains from infected citrus.Direct tissue blot immuno-assay (DTBIA) and reverse transcription polymerase chain reaction (RT-PCR) were used to pre-select CTV isolates from the sample of citrus plants collected from the Sargodha, Bhalwal, Sahiwal, Faisalabad and Toba Tek Singh districts of Punjab, Pakistan in this study. CTV-infected samples were then preserved in the sweet orange by graft-inoculation and maintained in a green-house. The target sequences of the major coat protection gene (p25 gene) were amplified from the CTV-infected samples using RT-PCR, and the p25 genes of these isolates were analyzed by restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) for investigating the p25/HinfⅠRFLP groups and molecular characteristics of CTV isolates found in Pakistani isolates. Further assessment of the genetic differences and genetic relationships and the sequences of 4 genomic regions p23, p20,p18, and p25 of the 21 CTV isolates from different cultivars were amplified and sequenced using RT-PCR and with a sequencer. Their sequences were subject to sequence analysis and compared with those of foreign isolates from GenBank and with each other by BioEdit and MEGA softwares. Using sequence analysis, the differences were therefore estimated among the CTV isolates from cultivated citrus, in order to provide theoretical data for controlling tristeza by MSCP. Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd) were detected by one-step multiplex RT-PCR assay simultaneously.The main experimental results are as follows1 Collection of Citrus Samples and Detection of CTVIn 2008,230 samples collected from five districts of Punjab, Pakistan were tested by DTBIA to determine the CTV infection which was also confirmed by RT-PCR.85 samples were found to be infected with CTV by DTBIA and RT-PCR. In 2009,260 samples collected from five districts (Sargodha, Bhalwal, Sahiwal, Faisalabad and Toba Tek Singh of Punjab, Pakistan) were tested by DTBIA and RT-PCR.104 samples were found to be infected with CTV by DTBIA and RT-PCR.2 RFLP and SSCP Analysis of the CTV Isolates2.1 RFLP analysis of the samples collected in 2008 and 2009p25/HinfⅠRFLP groupⅢwas the main epidemic isolate of the CTV isolates from Pakistan, followed by groupⅠwith the infection (single and mixed) rate 78.57%, 28.57% respectively. p25/Hinf I RFLP group VII was not found in samples from Pakistan.The ratio of mixed infection and single infection from all the citrus was 35.6% and 64.2% respectively. The ratio of mixed infection in mandarin field tree was 57% and the ratio of single infection was 43%. The ratio of mixed and single infection in sweet orange was 35.7% and 64.28%, respectively.2.2 SSCP analysis of samples collected in 2008 and 2009SSCP analysis of within-isolate haplotype population and profiles from different clones of each isolate showed that most isolates contained only one predominant sequence variant.3. Variability and Sequence Identity Analysis3.1 Sequence identity analysisNucleotide and deduced amino acid sequences of four genomic regions (p23, p20, p18, and p25) of different clones from 21 different CTV isolates were determined. Ten of the 21 isolates were collected from sweet orange, one was collected from sweet lime, and 10 were collected from mandarin cultivars. The reason for selecting different clones for different genes was that some clones could not be sequenced for all four genes. The nucleotide and deduced amino acid sequences of these clones showed an identity range of 93.1% to 100% and 89.8% to 100%, respectively, within the population of CTV isolates and the identity with reference isolates were 69.6% to 99.4%(nt) and 88.6% to 98.2%(aa). These results showed that the identity within the Punjab, Pakistan CTV population was somehow conserved and that there was high divergence with the isolates from other countries (completely or partially described). The degree of identity is similar to the earlier reports on p25 sequences of various CTV.3.2 Phylogenetic analysisPhylogenetic trees of nucleotide sequences for each gene were inferred by neighbor-joining method (NJ). Phylogenetic trees from amino acid deduced sequences showed the same grouping when compared with the trees from nucleotide deduced sequences by using the above mentioned methods, and moreover, the trees from protein deduced sequences also had the same results. Nucleotide changes among sequences are represented as branch lengths in the trees. Most of the isolates from Punjab, Pakistan grouped in clusters regardless of their host varieties and geographical differences. The trees from p18 and p25 showed more groups while these isolates in p20 and p23 phylogenetic trees were clustered. In the p20 phylogenetic tree, most of the isolates grouped in one cluster except the isolate 109 which was more distantly closer to NuagA (seedling yellows isolate) from Japan and SY568 (quick decline and stem-pitting isolate) from California, and isolate 188 and 215 were closely related to VT (Decline and grapefruit stem-pitting isolate) from Israel, while in the p23 gene tree clones 180,231 and 215 from Punjab, Pakistan were closely related to VT, B165, SY568 and NuagA, and clone 157 isolate from Punjab, Pakistan from a symptomless sweet lime tree was most distant from all other groups and sub-groups. The p18 and p25 gene trees showed more groups. The p18 gene tree isolate 242 from Punjab, Pakistan was closely associated with CT-9 (mild isolate) from China, while isolate 109 and 180 were closely associated with T36 and Mexico-ctv (both are decline isolates). The p25 gene tree isolate 142 from Punjab, Pakistan Punjab, Pakistan showed absolute divergence and was distantly far from all other groups, while isolate 146 was somewhow associated to isolate VT from Israel. Isolate T30, T385, T36 and Mexico-ctv joined were closely associted, while CT-9, NuagA, SY 568 and B165 were closely associated, respectively.3.3 Alignment of deduced amino acid sequences analysisThe alignment of p23 sequences showed that amino acids of isolate 157 are similar to the mild isolates CT-9 from China, T385 from Spain and T30 from Florida. Their amino acid positions are already represented in our results chapter. The polymorphism of a specific region of p23 allows the discrimination between mild and severe isolates. Our results for the discrimination of mild and severe isolates by p23 gene are supported by Iglesias et al., (2008). 4 Biological indexingEleven CTV isolates from citrus areas of Punjab, Pakistan were used for biological indexing. Most of the 11 CTV isolates tested did not show prominent symptoms in all indicator plants except Mexico lime (10 showed mild to severe vein-clearing and stem-pitting) and Duncan grapefruit (some showed mild to severe stem-pitting), proving that most of the CTV isolates in Pakistan ranged from mild to moderate except two isolates tested might be the severe isolates, which caused moderate stem-pitting symptom in sweet orange. Two isolates caused moderate seedling-yellows reaction on sour orange seedlings and Duncan grapefruit.5 First Report of Citrus bent leaf viroid and Citrus dwarfing viroid from Citrus in Punjab, PakistanSamples collected in 2008 from Punjab, Pakistan showed the presence and distribution of citrus viroids in this area. On the basis of multiplex RT-PCR assay CEV), CBLV), HSVd, CDVd, and CBCVd were detected simultaneously. With the amplification of the appropriately sized DNA, four kind of viroids (CEVd, CBLVd, HSVd, and CDVd) were detected in 12,8,31, and 17 samples, respectively, whereas CBCVd was not detected in these samples collected from Punjab, Pakistan. BLAST analysis showed that these nucleotide sequences had greater than 97% nucleotide identity to the most similar genome sequences in GenBank.6 ConclusionIn summary, there existed CTV infections of citrus. RFLP and SSCP analyses on the p25 gene demonstrated that most of CTV-infected citrus carried single CTV isolates, and the dominate p25/Hinf I RFLP groups in citrus were groupⅢand groupⅠin the citrus cultivated areas of Punjab, Pakistan Sequence identity, deduced amino acid sequence, genetic evolution and phylogenetic analyses of the CTV isolates collected from Punjab, Pakistan in comparison to those overseas showed that of the four genomic regions of CTV isolates analyzed, the CTV isolates clustered in a unique grouping and shared higher homology, while some isolates showed relationships with CTV isolates from different geographical origins, isolates with similar biological characteristics usually dropped into the same clusters. It is suggested that there exists complicated genetic relationships among the CTV isolates, and that pathogenicity is critical to evaluation. This Biological indexing experiment showed some mild isolates which can be used as MSCP in citrus. CBLVd and CDVd are reported for the first time from citrus in Punjab, Pakistan.