Accepted for publication 07/07/05

Symptoms observed on Acer negundo (ashleaf maple; box elder) at several sites in Poland, included reduced apical growth, severe leaf malformation and necrosis (Fig. 1), witches’ broom and dieback of twigs and branches. Leaves were sparse or in tufts at the tips of the shoots (Fig. 2) and trees died within a few years of the onset of symptoms. Young (<3 years old) trees of cv. ‘Flamingo’, growing in two nurseries showed less severe shoot proliferation, leaf chlorosis or reddening, malformation of newly expanding leaves and stunting.

Figure 1: Abnormalities in the growth of shoots at the base of phytoplasma-affected Acer negundo	Figure 2: Leaves showing severe leaf malformation and necrosis from affected ashleaf maple	Figure 3: Periwinkle (Catharanthus roseus) experimentally infected by chip bud grafting from diseased A. negundo

Chip-grafting from affected A. negundo hybrids to 15 healthy Catharanthus roseus resulted in 2 plants with very narrow, etiolated branches (Fig. 3); whose symptoms developed slowly over 13 months. The remaining 13 periwinkles developed small, chlorotic leaves. Nucleic acids were extracted from sprouts and leaves by the method of Ahrens & Seemüller (1992). A nested PCR was done using the universal phytoplasma rDNA primer pair P1/P7, followed by primers fA/rA, R16F2n/R16R2 or the phytoplasma rRNA 16SrI group-specific primers R16(I)F1/R16(R1)R1. RFLP analysis of R16F2n/R16R2 primed PCR products was done using AluI, MseI and RsaI endonucleases. Phytoplasma rDNA was amplified from five A. negundo hybrids, two cv ‘Flamingo’, two apparently healthy 2 year old seedlings collected from a park, two C. roseus experimentally inoculated by grafting with etiolated symptoms and two C. roseus with small chlorotic leaves (Fig. 4). 

Figure 4 (top): Nested polymerase chain reaction (PCR) amplification of ribosomal DNA fragments obtained with DNA from C. roseus plants experimentally infected by grafting (C.ros), affected maple hybrids with disease symptoms (A1) and symptomless (A2) and maple ‘Flamingo’ (A3) using the following primer pair combinations: (a) P1/P7 followed by R16F2n/R16R2; (b) P1/P7 followed by R16(I)F1/R16(I)R1. M – 1 kb DNA molecular marker (Sigma–Aldrich). The phytoplasma reference strains were: AP = Ca.P. mali (16SrX-A) group, CX = Ca. P. pruni (16III-A) group, ULW = Ca. P. ulmi (16SrV) group and AY1 = Ca. P. asteris (16SrI-B) group. Figure 5 (bottom): RFLP profiles after AluI, RsaI and MseI digestion of the nested PCR products (primed by R16F2n/R16R2 primer pair) from C. roseus experimentally infected by grafting (C.ros), affected maple hybrids with (A1) and without (A2) disease symptoms, and maple ‘Flamingo’ (A3). M - molecular weight marker PhiX 174 DNA/HinfI (Promega Symbios). The phytoplasma reference strains were as Fig. 4.

No bands were amplified from three healthy ashleaf maple seedlings maintained in the glasshouse or healthy C. roseus. RFLP patterns of 16S rDNA indicated that the bands were amplified from phytoplasmas belonging to the 16SrI group - subgroup B (Fig. 5); now re-classified as the species ‘Candidatus Phytoplasma asteris’. In Poland, 16SrI-B subgroup strains (Ca. P. asteris) affect several ornamental crops including magnolia and rose (Kaminska et al., 2001; Kaminska et al., 2003). This is the first report of a phytoplasma-associated disease of Acer negundo.

Ahrens U, Seemüller E, 1992. Detection of plant pathogenic mycoplasmalike organisms by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82, 828-832.

Kamińska M, Śliwa H, Rudzińska-Langwald A, 2001. The association of phytoplasma with stunting, leaf necrosis and witches’ broom symptoms in magnolia plants. Journal of Phytopathology 149, 719-724.

Kamińska M, Śliwa H, Malinowski T, Skrzypczak Cz, 2003. The association of aster yellows phytoplasma with rose dieback disease in Poland. Journal of Phytopathology 151, 469-476.

The British Society for Plant Pathology