Detection and molecular characterization of ‘Candidatus phytoplasma asteris’ related phytoplasmas infecting canola in North Dakota

Abstract

Canola (Brassica napus) plants with Aster yellows-like symptoms were collected in 2011 and 2012 from canola fields in North Dakota, USA. The presence of phytoplasmas in the symptomatic plants was confirmed by a nested PCR assay using universal primer pairs P1/P7 and R16F2n/R2. Phytoplasmas were identified as ‘Candidatus phytoplasma asteris’ related strains with >99% nucleotide identity to the reference strain M30790 based on in silico analysis of the F2nR2 fragments using the iPhyClassifier online tool. Based on sequence homology searches, RFLP and phylogenetic analyses of 16S rDNA gene, phytoplasmas belonging to two distinct lineages, 16SrI-A and 16SrI-B subgroups, were identified infecting canola in North Dakota. Further characterization of the phytoplasmas based on ribosomal protein operon and secY genes also supported the presence of two distinct lineages. Sequence comparison and RFLP analyses based on the three regions confirmed the presence of mixed infection by phytoplasmas belonging to subgroups 16SrI-A and 16Sr1-B in two samples, one each in 2011 and 2012. To our knowledge, this is the first report of 16SrI-A subgroup phytoplasma and mixed infection by 16SrI-A and 16SrI-B phytoplasmas infecting canola in the USA.