First report of ‘Candidatus Phytoplasma asteris’ (16SrI-B) causing witches’-broom disease of Boehmeria pannosa Nakai & Satake in Korea

Eggplant (Solanum melongena L.) belongs to the family Solanaceae and is an important vegetable cash crop grown in most parts of Oman. In February 2010, plants showing phyllody symptoms and proliferation of shoots resembling those caused by phytoplasma infection were observed at Khasab, 500 km north of Muscat. Total genomic DNA was extracted from healthy and two symptomatic plants with a modified (CTAB) buffer method (2) and analyzed by direct and nested PCR with universal phytoplasma 16S rDNA primers P1/P7 and R16F2n/ R16R2, respectively. PCR amplifications from all infected plants yielded an expected product of 1.8 kb with P1/P7 primers and a 1.2-kb fragment with nested PCR, while no products were evident with DNA from healthy plants. Restriction fragment length polymorphism (RFLP) profiles of the 1.2-kb nested PCR products of two eggplant phyllody phytoplasma and five phytoplasma control strains belonging to different groups used as positive control were generated with the restriction endonucleases RsaI, AluI, Tru9I, T-HB8I, and HpaII. The eggplant phytoplasma DNA yielded patterns similar to alfalfa witches'-broom phytoplasma (GenBank Accession No. AF438413) belonging to subgroup 16SrII-D, which has been recorded in Oman (1). The DNA sequence of the 1.8-kb direct PCR product was deposited in GenBank (Accession No. HQ423156). Sequence homology results using BLAST revealed that the eggplant phyllody phytoplasma shared >99% sequence identity with Scaevola witches'-broom phytoplasma (Accession No. AB257291.1), eggplant phyllody phytoplasma (Accession No. FN257482.1), and alfalfa witches'-broom phytoplasma (Accession No. AY169323). The RFLP and BLAST results of 16S rRNA gene sequences confirm that eggplant phyllody phytoplasma is similar to the alfalfa phytoplasma belonging to subgroup 16SrII-D. To our knowledge, this is the first report of a phytoplasma of the 16SrII-D group causing witches'-broom disease on eggplant in Oman. References: (1) A. J. Khan et al. Phytopathology 92:1038, 2002. (2) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA, 81:8014, 1984.

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First Report of a 16SrII-D Subgroup Phytoplasma Associated with Pale Purple Coneflower Witches'-Broom Disease in Australia

Plant Disease ◽

10.1094/pdis-03-11-0155 ◽

2011 ◽

Vol 95 (6) ◽

pp. 773-773 ◽

Cited By ~ 7

Author(s):

T. L. Pearce ◽

J. B. Scott ◽

S. J. Pethybridge

Keyword(s):

Sequence Homology ◽

Sequence Similarity ◽

Universal Primer ◽

First Report ◽

Purple Coneflower ◽

Echinacea Pallida ◽

Pcr Products ◽

Broom Disease ◽

Plant Pathol ◽

Single Field

Pale purple coneflower, Echinacea pallida (Nutt.) Nutt., is an 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 2010–2011 growing season, the incidence of affected plants was estimated to be 12% within a single field. Total DNA was extracted from symptomatic plants with 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 PCR using universal primer pairs P1/P7 followed by R16F2n/R16R2 to detect putative phytoplasmas (2). Amplifications yielded expected products of 1.8 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 Accession Nos. 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 the ‘Candidatus Phytoplasma australasiae’ reference strain (Y10097). RFLP profiles from all isolates suggested that they belonged to the 16SrII-D subgroup. To our knowledge, this is the first report of a 16SrII-D subgroup phytoplasma infecting E. pallida in Australia. Aster yellow phytoplasmas (16SrI-C subgroup) infections of E. purpurea have been recorded in Slovenia (3) and southern Bohemia (1). References: (1) J. Franova et al. Eur. J. Plant Pathol. 123:85, 2009. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) S. Radišek et al. Plant Pathol. 58:392, 2009. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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