First Report of ‘Candidatus Phytoplasma asteris’ Associated with Oilseed Rape Phyllody in Poland

Abstract

Winter oilseed rape (Brassica napus L.) is widely grown in Poland to produce vegetable oil for industrial processing of human and animal feed. In recent years, according to European Union directives on the use of biofuels (Directive 2003/30/EC), the area under oilseed rape cultivation in Poland has dramatically increased to 810,000 ha in 2009 and is still increasing. Morphological deformations of winter oilseed rape indicative of phytoplasma infection have been observed sporadically in Poland since 2000 (3). Plants exhibiting floral virescence, phyllody, as well as auxiliary bud proliferation, reduced leaves, and malformation of siliques were identified during surveys of research fields in Wielkopolska during May and June of 2009 and 2010. To confirm phytoplasma infection of these plants, inflorescence and leaf tissues were collected from nine diseased and three symptomless plants from three different field locations with 1 to 16% disease incidence. Total DNA was extracted from each plant tissue sample with a modified cetyltrimethylammoniumbromide method (2). Samples were analyzed for phytoplasma DNA with a nested PCR assay employing phytoplasma universal rRNA operon primer pair P1/P7 followed by R16F2n/R16R2, using previously described conditions (1). PCR products of 1.8 and 1.2 kb were obtained from all diseased plants only following PCRs with P1/P7 and nested primer pair R16F2n/R16R2, respectively. PCR products were not obtained from symptomless plants. Eight 1.2-kb amplicons were sequenced (GenBank Accession Nos. JN193475 to JN193482). Comparative analysis of the R16F2n/R16R2 rDNA sequences confirmed the phytoplasma origin of the rDNA sequences that shared 100 to 99% identity with Maize bushy stunt phytoplasma (GenBank Accession No. HQ530152), Alfalfa stunt phytoplasma (GenBank Accession No. GU289675), Primula green yellows phytoplasma (GenBank Accession No. HM590623), and other aster yellows group phytoplasmas. A 1.8-kb amplicon of isolate designated RzW14 was sequenced (GenBank Accession No. HM561990) and had 99% identity with Aster yellow group phytoplasmas from Lithuania (GenBank Accession Nos.GU223208 and AY744071). A virtual restriction fragment length polymorphism analysis of the 16S rDNA sequences from the R16F2n/R16R2 amplicons was performed with iPhyClassifier (4). Restriction profile comparisons identified all aster yellows group phytoplasmas as subgroup 16SrI-B strains. To our knowledge, this is the first report of a ‘Candidatus Phytoplasma asteris’-related strain infecting oilseed rape in Poland. References: (1) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) A. C. Padovan et al. Aust. J. Grape Wine Res. 1:25, 1995. (3) M. Starzycki and E. Starzycka. Oilseed Crops 21:399, 2000. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.