First Report of Fig Leaf Mottle-Associated Virus 1 Infecting Common Fig Tree in Greece

Abstract

In Greece, fig tree (Ficus carica L.) is commercially cultivated mainly in Evia, Attiki and Peloponnese regions and constantly expanding towards the northern parts of the country, due to the climate change. Common fig is naturally infected by several viruses and viroids (Alkowni et al., 2015). Most fig trees with viral infection display symptoms such as chlorotic mottling and blotching, mosaic, vein banding, chlorotic-necrotic ring spots and line patterns, all associated with the fig mosaic disease (FMD), one of the most important fig infectious disorder (Elbeaino et al., 2009). Surveys were conducted in three fig orchards during autumns of 2018 in Chrisopigi district, and 2019 in Agrokipio and Nerokourou districts of Chania province (Crete island). Leaf samples were collected from a total of 32 different trees, for which 22 were from trees with mosaic, vein clearing and mottling symptoms, whereas the remaining 10 samples were from asymptomatic trees. All samples were tested by RT-PCR for the presence of the four viruses reported to be associated with the FMD, i.e. fig leaf mottle-associated virus 1 (FLMaV-1), fig leaf mottle-associated virus 2 (FLMaV-2), fig mild mottle-associated virus (FMMaV) and fig mosaic virus (FMV), as described by Aldhebiani et al. (2015). RT-PCR results showed the presence of FMV in 50% of the 32 tested samples, FLMaV-1 in 31% and FMMaV in 22%, whereas the FLMaV-2 was not detected. All viral infections were found in the symptomatic trees. The symptomatic trees showed also mixed infections with different viruses’ combinations, i.e. FMV and FLMaV-1 (4 out of 22), FMV and FMMaV (2 out of 22) and FLMaV-1 with FMMaV (5 out of 22); whereas 10 trees were single-infected with FMV. From Nerokourou district the only one symptomatic tree was found single-infected with FLMaV-1. Sanger sequencing of the purified PCR products (one virus isolate from each district) reconfirmed the presence of the viruses found in the analyzed samples. The seven reconstructed sequences of the Greek isolates from FMV (Fc-FM-2 and Fc-FM-11 from Chrisopigi and Agrokipio districts), FLMaV-1 (Fc-3Br, Fc-FLa1-6 and Fc-Fla1-10 from Nerokourou, Crisopigi and Agrokipio districts) and FMMaV (Fc-FMa-8 and Fc-FMa-15 from Chrisopigi and Agrokipio districts) were deposited in the GenBank (MN400047, MT122753-MT122756, MT122759, MT122760). BLAST analysis of nucleotide sequences showed that isolates of FLMaV-1, FMV and FMMaV from Greece share 92-94%, 92-95% and 95% identity, respectively; with deposited homologue sequences in the NCBI database. The presence of FLMaV-1 in the 10 symptomatic trees was reconfirmed by an additional RT-PCR which targets the helicase domain of FLMaV-1, using newly designed specific primers (LM1-Hel-F: 5΄-AAAGATGAGCGGGGAAACTT-3΄, LM1-Hel-R: 5΄-ACAACATTGTGCCCAGTTGA-3΄). Sequences of the 308 bp PCR amplicons (same isolates as above) showed to share 82-83% nucleotide identity (MN400048, MT122757, MT122758) with the unique sequence available from the polyprotein gene of FLMaV-1 in the database. Our study, together with that of Dimitriadou et al., (2016) which reports the molecular detection of different fig viruses, constitute the only information available on fig viruses in Greece. To our knowledge, this is the first official report of FLMaV-1 infecting common fig in Greece with exclusive data on partial sequences of Greek isolates. Future studies with larger survey and more viruses, investigated in the main producing areas will allow a better evaluation of the sanitary status of fig viruses in the country.