PRELIMINARY BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF MILD PPV ISOLATES NATURALLY INDUCED IN N. BENTHAMIANA PLANTS

Natural induction of variation of Plum pox virus (PPV-D-GR) in Nicotiana benthamiana plants was achieved by keeping plants at 15°C for at least 15 days. A total of 802 single lesion isolates were screened for mildness and 20 mild ones (2.5% variation) were assayed for cross protection efficiency towards the severe PPV-M isolates occurring in Greece. Two isolates, PPV-B2 and PPV-B18, were selected as they appeared to be the most stable and mild, giving at the same time 100% protection. Their HC-Pro, P3-6K1 and coat protein genes were sequenced and compared with the homologue sequences of the wild type PPV-D-GR. In the case of PPV-B2 two substitutions in the HC-pro (Thr85Ile and Thr400Ala) and two in the P3 (Thr123Ser and Asp176Glu) protein were determined, while in the case of PPV-B18 one substitution in the HC-pro (Lys369Arg), two in P3 (Phe112Leu and Phe118Ile) and one in CP (Ala64Val) were found, the role of which in virus pathogenicity has to be further examined. Both isolates retained intact the “FRNK” domain of their HC-Pro, responsible for aggressiveness in other potyviruses, indicating that this domain is not the only one associated with their symptomatology. In addition, both isolates carried the “DAG” motif in the CP and the “PTK” one in the HC-pro. Since PPV-B2 was not aphid transmissible, other molecular determinants have to be implicated in this loss of transmissibility.