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First Report of Plum pox virus Strain M Isolates in Apricot in Sicily, Italy

Sharka or plum pox disease is one of the most economically important virus diseases of stone fruits. Plum pox virus (PPV), the causal agent, is a member of the genus Potyvirus of the family Potyviridae transmitted by aphids in a non-persistent manner and by grafting. To date, nine PPV strains have been described on the basis of their biological, serological, and molecular properties: M and D are the most widespread and economically important strains, PPV-Rec and PPV-C have been reported mainly in Europe, PPV-EA confined to Egypt, PPV-T to Turkey, PPV-W from Canada, Ukraine, Latvia, and Russia, PPV-CR detected in Russia, and finally a putative PPV strain infecting plum in Albania described as the ancestor of the M. PPV-M is responsible for major epidemics in many Italian regions and despite phytosanitary measures, the infection rate increases each year. The D and Rec isolates are sporadically reported while PPV-C, once signaled in Apulia, has been successfully eradicated. Except for a report from the 1980s, which is no longer traceable, Sicily was considered free from the virus (2). In 2012, two new foci of sharka in a coastal area of Catania in Sicily were first reported by the national plant protection service to the European Commission (DG-SANCO). In spring 2013, plants of different varieties of apricot (Prunus armeniaca) and peach (P. persica) showing typical symptoms of flower color break, yellowing and leaf deformation, chlorotic spots or rings, and malformation on fruits were tested positive to PPV by DAS-ELISA using polyclonal antibodies. In order to characterize two isolates from apricot varieties (Carmen Top and Ninfa), total RNAs, extracted using the RNeasy Plant Mini Kit (Qiagen) from ELISA-positive samples, were analyzed by RT-PCR with primers P1/P2, targeting the 3′-terminal region of the coat protein (CP) gene (5) followed by RFLP analysis after digestion with Rsa1. Subsequently total RNAs were analyzed with the type-specific primers P1/PM and P1/PD (3), P3M/P4b and P3D/P4b amplifying the N-terminal region of the CP gene (1) and, finally, with primers mD5/mD3, mM5/mM3, and mD5/mM3, amplifying the region 3′NIb-5′CP, including the recombination site of Rec isolates (4). Only primer pairs P1/P2, P1/PM, P3M/P4b, and mM5/mM3 produced amplicons of the expected size (243, 198, 466, and 459 bp, respectively). The RFLP assay confirmed both isolates belonging to the M strain. Moreover, no reaction was obtained with primer pair mD5/mM3, excluding isolates belonging to Rec-type. Isolate characterization was completed by direct sequencing in both directions of the of P1/P2 and P3M/P4b amplicons obtained from apricot samples L9-1 (Carmen Top isolate) and 9-335 (Ninfa isolate). The P1/P2 sequences (KJ994235, KJ994237) showed 98% similarity with PPV-M or PPV-Rec isolates. The P3M/P4b sequences (KJ994236, KJ994238) confirmed that Sicilian isolates belong to the PPV-M strain showing 99% similarity with those already present in GenBank, thus ruling out the possibility of an infection with a PPV-Rec isolate. This outbreak of the Marcus strain of PPV in Sicily represents a high risk for the expanding production of stone fruit in southern Italy. An eradication plan was quickly activated by the regional phytosanitary service.

References: (1) T. Candresse et al. Phytopathology 101:611, 2011. (2) EPPO. PQR-EPPO database on quarantine pests (available online). http://www.eppo.int, 2014. (3) A. Olmos et al. J. Virol. Methods 68:127, 1997. (4) Z. Subr et al. Acta Virol. 48:173, 2004. (5) T. Wetzel et al. J. Virol. Methods 33:355, 1991.