First report of ‘ <i>Candidatus</i> Phytoplasma mali’ in <i>Prunus avium</i> , <i>P. armeniaca</i> and <i>P. domestica</i>

Abstract

Apple proliferation (AP) phytoplasma (‘Candidatus Phytoplasma mali’; Seemüller & Schneider, 2004) is the causal agent of a serious proliferation disease of apple (Malus domestica). It has also been found in hazelnut (Corylus spp.) (Marcone et al., 1996), pear (Pyrus communis) and Japanese plum (Prunus salicina). In this report, AP phytoplasma was found in cherry (Prunus avium), apricot (P. armeniaca) and plum (P. domestica). In cherry, wilting, dying, floral and phloem necrosis were first observed in 2004 in south western Slovenia. No phytoplasmas could be isolated from the leaves or phloem from 40 trees, but were detected in roots of three trees with, and one without 3 symptoms using DAPI staining and electron microscopy. These were subsequently identified as AP by PCR, nested PCR, RFLP and sequencing. AP phytoplasma was also identified in 2 out of 29 apricot trees expressing stem necrosis and leaf wilting, and in 1 out of 34 plum trees expressing late blooming. Total DNA was extracted from the roots (cherry) or roots and shoots (apricot and plum) using a modified CTAB method or the Plant DNeasy mini kit (Qiagen GmbH). PCR was performed using universal phytoplasma rDNA primers P1/P7 (Seemüller et al., 1998), followed by a nested PCR using the AP group specific primers f01/r01 (Lorenz et al., 1995). Controls included DNA extracts from healthy plants and AP-infected Catharanthus roseus and apple. RFLP analysis of nested PCR products was performed using restriction enzymes BsaAI and SspI. All positive samples produced a restriction profile identical to the AP-infected apple. The f01/r01 PCR products were cloned into pGEM-T vector and sequenced (GenBank Accession No. EF025917-20). BLAST analysis of 1018 bp sequences showed the highest identity (greater than 99·6%) with the ‘Ca. Phytoplasma mali’ (GenBank Accession No. AF248958.1). This is the first report of AP phytoplasma infecting cherry, apricot or plum (P. domestica). Although the AP-infected cherries in the present study tested negative for other pathogenic bacteria, further experiments are needed to verify whether the AP phytoplasma caused the observed symptoms. We thank M. Tušek Žnidarič (NIB, Slovenia) and P. Ermacora (Univ. of Udine, Italy) for helping with the phytoplasma identification.