IDENTIFICATION OF A SECOND MEMBER OF THE FAMILY CLOSTEROVIRIDAE IN MOSAICDISEASED FIGS
T. Elbeaino, M. Digiaro, A. De Stradis, G.P. Martelli
A double-stranded RNA (dsRNA) ca. 19 kb in size was recovered from a fig tree of Algerian origin (F3) showing chlorotic mottling of the leaves and clearing of the second and third veins. Filamentous closteroviruslike particles up to 2100 nm in length and with distinct cross banding were observed in leaf dips. Cytopathology of thin sectioned phloem cells was similar to that elicited by members of the family Closteroviridae. RTPCR assays with primers specific to the recently described Fig leaf mottle-associated virus (FLMaV) were negative. No virus was recovered by mechanical inoculation to herbaceous hosts. Using denatured F3 dsRNA preparations as template, a 625 bp cDNA fragment was amplified by RT-PCR using degenerate primers designed on the phosphate motifs 1 and 2 of the heat shock-protein 70 homologue (HSP70) of members of the family Closteroviridae. Sequence analysis demonstrated that the sequenced 625 bp fragment was part of a closteroviral HSP70 gene, with sequence identity to FLMaV of ca. 45% and 28% at the nucleotide and amino acid level, respectively. Comparison with the HSP70 amino acid sequences of other closteroviral species showed homologies of 48%, 37% or 36% with Little cherry virus 2 (LChV-2,) Grapevine leafroll-associated virus 1 (GLRaV-1) and Pineapple mealybug wilt-associated virus 2 (PMWaV-2), respectively. In a phylogenetic tree constructed with the amino acid sequences of the HSP70 genes of several members of the family Closteroviridae, isolate F3 clustered with members of the genus Ampelovirus. In RT-PCR assays with specific primers designed to match the F3 HSP70 sequence, 4 of 57 field-grown Italian figs trees yielded amplicons of the expected size, while Multiplex-PCR proved effective for the simultaneous detection of double infections by FLMaV and isolate F3. The different sizes of dsRNA and virus particle lengths of F3 and FLMaV, the low sequence homology of the F3 HSP70 gene with that of FLMaV, and the different phylogenetic affiliation constitute evidence that isolate F3 belongs to a virus species different from FLMaV, for which the name of Fig leaf mottle-associated virus 2 (FLMaV-2) is proposed.