Biological indexing and genetic analysis of Citrus tristeza virus in Pakistan

Citrus tristeza virus (CTV) is one of the major threats to production and fruit quality of citrus worldwide. In Pakistan, more than 95% of the citrus trees are grown on sour orange rootstock, which is highly susceptible to CTV. We studied the genetic variability of four genomic regions (p18, p20, p23 and p25) of CTV isolates collected from the citrus orchards. High divergence was revealed among the isolates from Pakistan and also with reference isolates. An inter-isolate identity range of 92.1-99.4% at the nucleotide level and 92.3-98.8% at the amino acid level was found. Phylogenetic analysis of the predominant sequence variants of each isolate revealed almost similar grouping of isolates for each gene. The groups revealed by phylogenetic trees include sequences from isolates that cause severe quick decline, seedling yellows and stem pitting (SP) and also from mild isolates. The high percentage of mixed infections is alarming because of the threat of further diversification and spread of severe variants into additional citrus-growing areas of Pakistan and neighboring countries. Eleven CTV isolates from Pakistan were biologically indexed, and most induced mild or mild to moderate reactions on all biological indicators. Four genomic regions of isolate 21C from the biological indexing experiment were sequenced and phylogenetically analysed. These results provide the basis for mild strain cross protection (MSCP) in Pakistan and in neighbouring countries in the near future.