First Report of Citrus tristeza virus in National Germplasm of Citrus in Afghanistan.

Rejuvenation of the horticulture industry is a government priority in Afghanistan. With that purpose, European Commission-supported programs specifically focus on greater access to improved and appropriate planting materials to increase the quantity and quality of more competitive horticultural products. Establishment of a biotechnology laboratory was considered essential support to horticulture sector development. This laboratory has begun screening the health status of the Afghan Germplasm National Collection to ensure multiplication of not only the best selected varieties or ecotypes but also to avoid reproduction and distribution of virus-infected fruit trees. Symptom inspection and sample collection for viral diseases was carried out in the citrus orchard during survey activity at the National Collection Experimental Farm in Jalalabad (Nangarhar Province). Ninety-nine variety plots (one row of five plants) were inspected visually and samples from two plants for each plot were collected and analyzed by double-antibody sandwich (DAS)-ELISA. Plants showing vein flecking, yellowing, and plant decline symptoms were observed in several plots. Four accessions were found to be infected by Citrus tristeza virus (CTV): kumquat cv. Margarita (isolates J4 and J8), orange cv. Mahali (J61), mandarin group cv. Fruter (J76), and rough lemon cv. Mahali (J101). Identified isolates have been characterized molecularly. A 655-nt fragment, corresponding to the major coat protein gene, has been amplified from all ELISA-positive samples by reverse transcription (RT)-PCR using CTVF (5'-TAATGGACGACGAACAAAGA-3') and CTVR (5'-CCAAGCTGCCTGACATTAGT-3') primers. Sequence analysis revealed high similarity, ranging from 91.1 to 99.8%, within CTV isolates detected in Jalalabad. In accordance with the phylogenetic groups previously defined (page 8 in: Proceedings of the 15th Conference of the International Organization of Citrus Virologists, 2002), nucleotide sequences of Afghan CTV isolates investigated in the current work cluster in Group 1 (J4 and J8), Group 4 (J61 and J76), and Group 5 (J101). In particular, J4 and J8 isolates show, respectively, identity of 99.4 and 99.2% with reference isolate T36 (GenBank Accession No. M76485) from the United States (Florida). Moreover, in Group 4, isolate J61 and J76 were more similar to ANO-1 isolate (GenBank Accession No. DQ211658) from Egypt (identity of 98.5 and 98.0%, respectively) than to isolate 443-4 (GenBank Accession No. AY791844) from Croatia (97.4 and 97.5%, respectively). Finally, isolate J101 in Group 5, shows identity of 95.6% with isolates C268-2 (GenBank Accession No. AY750770) and C269-6 (GenBank Accession No. AY750775) from Argentina. To our knowledge, our results identified for the first time CTV-infected plants in Afghanistan. The presence of CTV in four accessions of the national citrus collection is of concern for Afghan horticulture. Implementation of the certification schemes is therefore necessary to guarantee the production and the employment of virus-free propagating material.