First Report of Witch's Broom Phytoplasma (16SrII-D Group) in Purple Coneflower in Australia
Pearce, T and Scott, JB and Pethybridge, SJ, First Report of Witch's Broom Phytoplasma (16SrII-D Group) in Purple Coneflower in Australia, Plant Disease: An International Journal of Applied Plant Pathology, 95, (6) pp. 773. ISSN 0191-2917 (2011) [Letter or Note in Journal]
Pale purple coneflower, Echinacea pallida (Nutt.) Nutt., is a herbaceous perennial, cultivated for its ornamental and medicinal properties. In 2005, phytoplasma-like symptoms, including virescence, phyllody and chlorotic leaves were first observed in coneflower fields in northern Tasmania, Australia. During the 2010/11 growing season, the incidence of affected plants was estimated to be 12% within a single field. Total DNA was extracted from symptomatic plants using a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer’s instructions. DNA was also extracted, as described above, from asymptomatic coneflower seedlings obtained by germinating surface-sterilized seed on water agar. DNA was amplified via a nested polymerase chain reaction (PCR) using universal primer pairs; P1/P7 followed by R16F2n/R16R2 to detect putative phytoplasmas (2).
Amplifications yielded expected products of 1.8 kb and 1.2 kb, respectively, only from symptomatic samples. Subsequently, PCR products from six arbitrarily selected samples were used for sequencing (Genome Lab Dye Terminator Cycling Sequence with Quick Start chemistry) and read in a CEQ8000 sequencer (Beckman Coulter Inc., Brea, CA). Sequence homology indicated 100% similarity between isolates designated EWB1 to EWB4 (GenBank accessions JF340075, and JF340077 to JF340079) and between EWB5 and EWB6 (JF340076 and JF40080). Sequence homology between the two observed groups was 99.7%, resulting from a 4-bp difference in the R16F2n primer region. Blast search revealed isolates EWB1 to EWB4 were 100% homologous with Catharanthus roseus phytoplasma (EU096500.1),Tomato big bud phytoplasma (EF193359.1), Scaevola witches’-broom phytoplasma (AB257291.1) and Mollicutes sp. (Y10097.1 and Y10096.1). Moreover, isolates EWB5 and EWB6 shared 99% sequence identity with the above isolates. iPhyClassifier (4) was used to
perform sequence similarity and generate virtual restriction fragment length polymorphism (RFLP) profiles. The 16S rDNA sequence of isolates EWB1 to EWB4, and EWB5 to EWB6, shared 100% and 99.7% similarity respectively, to he ‘Candidatus Phytoplasma australasiae’reference strain (Y10097). RFLP profiles from all isolates suggested they belonged to the 16SrII-D group. To our knowledge, this is the first report of a 16SrII-D group phytoplasma
infecting E. pallida in Australia. Aster yellow phytoplasmas (16SrI-C group), infections of E. purpurea have been recorded in Slovenia (3) and southern Bohemia (1).