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 the same purpose the EC supported programs that specifically focus on increase access to improved and appropriate planting materials in order to increase quantity and quality of more competitive horticultural products. Establishment of a biotechnology laboratory and its services in quality control and management of the horticulture input chains were considerate an essential support to horticulture sector development. This laboratory started screening the health status of the Afghan Germplasm National Collection in order to ensure multiplication of not only the best selected varieties or ecotypes, but also to avoid production and distribution of virus-infected fruit trees. Viral symptoms inspection and samples collection has been performed on the citrus orchard during survey activity on National collection experimental farm in Jalalabad (Nangarhar province). Ninety nine variety plots (1 row of 5 plants) are been visually inspected and samples from 2 plants for each plot were collected and analyzed by DAS-ELISA test. Plants showing vein flecking, yellowing and plant decline symptoms are been observed in several plots. Four accessions resulted 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). In order to preserve citrus national collection from CTV infection, identified isolates has been molecularly characterized. A 655 nucleotides long fragment, corresponding to the major coat protein gene, has been amplified from all ELISA-positive samples by 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 previous defined (1), nucleotide sequences of Afghan CTV isolates investigated in the present 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% against reference isolate T36 (M76485) from USA (Florida) . Moreover, in the Group 4, isolate J61 and J76 resulted more similar to ANO-1 isolate (DQ211658) from Egypt (identity of 98.5 and 98.0% respectively) than to isolate 443-4 (AY791844) from Croatia (97.4 and 97.5% respectively). Finally isolate J101, in the Group 5, shows identity of 95.6% with isolates C268-2 (AY750770) and C269-6 (AY750775) from Argentina. Our results identified, for the first time, CTV infected plants in Afghanistan. Presence of CTV in four accession of the citrus National collection field is a worrying aspect of the Afghan horticulture. An implementation of the certification schemes is therefore necessary in Afghanistan in order to guarantee the production and the employment of virus-free propagating material.