Target gene selection for primers and probes design

To select the target genes, the four representative genomes of the three genetic populations RT-I, RT-II and RT-III were aligned using Mauve²¹ and BRIG (BLAST Ring Image Generator)²². The complete genome of R. toxicus strain WAC3373 (Western Australia; NCBI GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP013292.1","term_id":"961558191","term_text":"CP013292.1"}}CP013292.1; size 2.346 MB) was used as a reference genome to align the genome assemblies (Fig. 1). Locally Collinear Blocks (LCBs) sequences generated by Mauve were annotated for target selection using Geneious version 9 (Biomatters, Inc. Newark, NJ). The Average Nucleotide Identity (ANI) of complete genomes was calculated using OrthoANI²³. Endpoint PCR and qPCR assays for the detection of all R. toxicus isolates were designed using the highly conserved rpoD gene. Rathayibacter toxicus population-specific primers were designed based on genomic regions unique to each population (Fig. 1; Table ​Table2).2). The primers and probes were designed using the online software Primer3²⁴ following the parameters described by Arif and Ochoa-Corona (2013). Primer3 and mFold²⁵ were used to predict the internal structure and the potential for self-dimer formation for each primer and probe. Details for all primers and probes are provided (Tables ​(Tables22 and ​and3).3). Primers and probes were synthesized by IDT (Integrated DNA Technologies, Inc., Coralville, IA). Genome locations for primer and probe sites are provided (Supplement Fig. ³).