Mixed infection by Eggplant Mottled Dwarf Virus and an Ophiovirus species in Japanese Pittosporum

In spring of 2014, an oustanding vein yellowing sometimes expanding to the interveinal tissues, was observed on the foliage of a Japanese pittosporum (Pittosporum tobira) plant growing in a private garden at Bari (Apulia, Italy). These symptoms resembled very much those induced by Eggplant mottled dwarf virus (EMDV), a nucleorhabdovirus endemic in the Mediteranean region that affects also P. tobira (Martelli et al., 2011). Electron microscope observations of ultrathin sections from symptomatic pittosporum leaves showed the consistent presence of enveloped bacilliform or bullet-shaped particles accumulating in dilations of the nuclear envelope, whereas leaf dips revealed the presence of coiled filaments ca. 3 nm in diameter resembling ophioviruslike virions. A next generation sequencing analysis of small RNA libraries from the symptomatic pittosporum plant yielded a number of contigs matching the genome of both EMDV and of the ophiovirus species Ranunculus white mottle virus (RWMV) genome (Vaira et al., 1997). Two primer sets were therefore designed on the assembled contigs and used for RT-PCR amplification. Products of the expected size (426 and 400 bp for EMDV and RWMV, respectively) were amplified and sequenced. BLAST analyses showed that the cloned sequences, deposited in GenBank under the accession Nos. KT211397 and KT211398, shared ca. 96% identity at the nucleotide level with the sequence of an EMDV isolate from Agapanthus sp. (KJ082087) and of 91% with that of a RWMV isolate (AY542957). To our knowledge, this seems to be the first record of a mixed infection by a rhabdoand an ophiovirus in P. tobira.