Comparative Genomic Analysis Confirms Five Genetic Populations of the Select Agent, Rathayibacter toxicus

Abstract

Rathayibacter toxicus is a Gram-positive, nematode-vectored bacterium that infects several grass species in the family Poaceae. Unique in its genus, R. toxicus has the smallest genome, possesses a complete CRISPR-Cas system, a vancomycin-resistance cassette, produces tunicamycin, a corynetoxin responsible for livestock deaths in Australia, and is designated a Select Agent in the United States. In-depth, genome-wide analyses performed in this study support the previously designated five genetic populations, with a core genome comprising approximately 80% of the genome for all populations. Results varied as a function of the type of analysis and when using different bioinformatics tools for the same analysis; e.g., some programs failed to identify specific genomic regions that were actually present. The software variance highlights the need to verify bioinformatics results by additional methods; e.g., PCR, mapping genes to genomes, use of multiple algorithms). These analyses suggest the following relationships among populations: RT-IV ↔ RT-I ↔ RT-II ↔ RT-III ↔ RT-V, with RT-IV and RT-V being the most unrelated. This is the most comprehensive analysis of R. toxicus that included populations RT-I and RT-V. Future studies require underrepresented populations and more recent isolates from varied hosts and geographic locations.

Loop-mediated Isothermal Amplification (LAMP) assay for reliable detection ofXanthomonas axonopodispv.vasculorum

“…Despite the threat posed by gumming disease in sugarcane, no such assay is currently available for Xav. The field-deployable LAMP is a widely used and well-established method for pathogen detection and diagnosis due to its cost-effectiveness, high sensitivity, and ease of use 16,29,33 . Consequently, we have developed a robust LAMP assay for the specific detection of the sugarcane systemic pathogen Xav.…”

Field-Deployable Recombinase Polymerase Amplification Assay for Specific, Sensitive and Rapid Detection of the US Select Agent and Toxigenic Bacterium, Rathayibacter toxicus

“…Comparative genomics analysis to identify distinctive genomic regions of high diagnostic value has now become a common approach to support the design of robust and highly specific assays for genus-, species-, and strain-level discrimination [14,24,26,31,36,37]. Designing primers and probes from unique genomic regions enhances assay robustness and reduces the probability of non-specific amplification.…”

“…Whole-genome sequence analysis was performed to identify unique gene regions of R. toxicus to specifically detect this pathogen [31]. Six genomes of R. toxicus representing the known genetic populations RT-I to RT-V (respectively, RT-I: SA03-04, RT-II: SAC7056, RT-III: WAC3373 and WA40-23C, RT-IV: CS36, and RT-V: CS39) along with the genomes of six other Rathayibacter species, including the R. tritici strain NCPPB1953 (GenBank: CP015515), R. rathayi strain DSM7485 (GenBank: CP028129), R. iranicus strain NCCPB2253 (GenBank: CP028130.1), R. caricis strain DSM15933 (GenBank: GCF_003044275.1), R. festucae strain DSM15932 (GenBank: CP028137), and R. oskolensis strain VKM Ac-2121 (GenBank: GCF_900177245.1); the genomes of other Rathayibacter species were retrieved from the NCBI GenBank database (reference numbers provided).…”

“…Representative genomes of all five genetic populations of R. toxicus (SA03-04, SAC7056, WAC3373, WA40-23C, CS36, and CS39) along with other Rathayibacter species, including R. tritici, R. rathayi, R. iranicus, R. caricis, R. festucae, R. oskolensis, and R. tanaceti, were evaluated to identify taxon-specific diagnostic markers in R. toxicus (Figure 1). Based on the genomes of different populations of R. toxicus [11,30,31] and other Rathayibacter species, galE was selected for primers and probe design. The BLASTn outcomes showed 100% query coverage and 100% identity only with R. toxicus genomes when the primers and probe were evaluated using NCBI GenBank database; no 100% matching with any other species was observed.…”

“…The BLASTn outcomes showed 100% query coverage and 100% identity only with R. toxicus genomes when the primers and probe were evaluated using NCBI GenBank database; no 100% matching with any other species was observed. The primers/probes were also evaluated with 18 R. toxicus genomes present in our in-house database [31]-the selected signature region was highly conserved among all the genomes and showed 100% identity with all R. toxicus-specific primers and probe. Primers were thermodynamically competent to obtain the highest sensitivity.…”