Emergence of a New Population of Rathayibacter toxicus: An Ecologically Complex, Geographically Isolated Bacterium

Citation: Arif, M., Busot, G. Y., Mann, R., Rodoni, B., Liu, S. Z., & Stack, J. P. (2016). Emergence of a New Population of Rathayibacter toxicus: An Ecologically Complex, Geographically Isolated Bacterium. Plos One, 11(5), 20. https://doi.org/10.1371/journal.pone.0156182Rathayibacter toxicus is a gram-positive bacterium that infects the floral parts of several Poaceae species in Australia. Bacterial ooze is often produced on the surface of infected plants and bacterial galls are produced in place of seed. R. toxicus is a regulated plant pathogen in the U.S. yet reliable detection and diagnostic tools are lacking. To better understand this geographically-isolated plant pathogen, genetic variation as a function of geographic location, host species, and date of isolation was determined for isolates collected over a forty-year period. Discriminant analyses of recently collected and archived isolates using Multi-Locus Sequence Typing (MLST) and Inter-Simple Sequence Repeats (ISSR) identified three populations of R. toxicus; RT-I and RT-II from South Australia and RT-III from Western Australia. Population RT-I, detected in 2013 and 2014 from the Yorke Peninsula in South Australia, is a newly emerged population of R. toxicus not previously reported. Commonly used housekeeping genes failed to discriminate among the R. toxicus isolates. However, strategically selected and genome-dispersed MLST genes representing an array of cellular functions from chromosome replication, antibiotic resistance and biosynthetic pathways to bacterial acquired immunity were discriminative. Genetic variation among isolates within the RT-I population was less than the within-population variation for the previously reported RT-II and RT-III populations. The lower relative genetic variation within the RT-I population and its absence from sampling over the past 40 years suggest its recent emergence. RT-I was the dominant population on the Yorke Peninsula during the 2013-2014 sampling period perhaps indicating a competitive advantage over the previously detected RT-II population. The potential for introduction of this bacterial plant pathogen into new geographic areas provide a rationale for understanding the ecological and evolutionary trajectories of R. toxicus

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

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

An ISSR phylogenetic tree of 54 isolates of <i>Rathayibacter toxicus</i> achieved using the neighbor-joining method.

<p>Isolates were grouped into three clusters named as population RT-I, RT-II and RT-III. The scale bar at the bottom indicates the dissimilarity among the isolates. Details for all isolates are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156182#pone.0156182.t001" target="_blank">Table 1</a>. This consensus tree was generated using bootstrap resampling method in Resample module of NTSYSpc with 1000 replicates.</p

A phylogenetic tree was generated using consensus partial 16S ribosomal RNA gene sequence (about 1015 bp) of <i>Rathayibacter toxicus</i>, <i>R</i>. <i>tritici</i>, <i>R</i>. <i>agropyri</i>, <i>R</i>. <i>rathayi</i>, <i>R</i>. <i>iranicus</i>, <i>R</i>. <i>caricis</i> and <i>R</i>. <i>festucae</i>.

<p><b><i>Clavibacter michiganensis</i> subsp. <i>nebraskensis</i> was included as an outgroup.</b> The tree was constructed using neighbor-joining method and the Tamura-Nei genetic distance model. Detail of isolates and accession numbers of submitted sequences are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156182#pone.0156182.t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156182#pone.0156182.s007" target="_blank">S1 Table</a>, respectively. A consensus tree was generated through bootstrap analysis using Geneious Tree Builder program with 1000 cycles; the obtained values labeled at the forks indicate the confidence limits for the grouping. The scale bar at the bottom indicates the substitution rate.</p

A phylogenetic tree of 54 isolates of <i>Rathayibacter toxicus</i> was generated using concatenated consensus partial gene sequences six genes.

<p>A total of 5,182 nucleotides from vancomycin resistant protein <i>vanA</i>, CRISPR-associated protein <i>cse4</i>, <i>secA</i> ATPase, chromosome partition protein SMC, tRNA dihydrouridine synthase, and cysteine desulfurase genes, were analyzed to generate this tree. Three distinct groups RT-I, RT-II and RT-II were formed. The tree was constructed using neighbor-joining and Tamura-Nei genetic distance model. A consensus tree was generated through bootstrap analysis using Geneious Tree Builder program with 1000 cycles; the obtained values labeled at the forks indicate the confidence limits for the grouping. The scale bar at the bottom indicates the substitution rate. Detail for all isolates and gene accession numbers submitted to NCBI GenBank are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156182#pone.0156182.t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156182#pone.0156182.s007" target="_blank">S1 Table</a>.</p

Primer sequences and results for ISSR analysis of 54 isolates of <i>Rathayibacter toxicus</i> collected from South and Western Australia.

<p>Primer sequences and results for ISSR analysis of 54 isolates of <i>Rathayibacter toxicus</i> collected from South and Western Australia.</p

ISSR loci data for the three <i>Rathayibacter toxicus</i> populations: RT-I, RT-II and RT-III.

<p>ISSR loci data for the three <i>Rathayibacter toxicus</i> populations: RT-I, RT-II and RT-III.</p

Isolates recently collected in 2013–14 from South Australia and old isolates received from different culture collections, universities and institutes were used to determine the population structure of <i>Rathayibacter toxicus</i>.

<p>Isolates recently collected in 2013–14 from South Australia and old isolates received from different culture collections, universities and institutes were used to determine the population structure of <i>Rathayibacter toxicus</i>.</p

The ISSR profiles of 54 isolates of <i>Rathayibacter toxicus</i>.

<p>(A) Primer P15 produced an unique locus of 1.2 kb to differentiate population RT-I (locus absent) from RT-II and RT-III populations (locus present); (B) primer P16 produced an unique locus of 1.7 kb only with population RT-II; (C) primer UBC 810 produced an unique locus of 3.5 kb only with population RT-III of <i>R</i>. <i>toxicus</i>. The numbers above the gel images correspond to the individual isolates listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156182#pone.0156182.t001" target="_blank">Table 1</a>.</p

Nucleotide differences in the partial gene sequences used for multi-locus sequence typing (MLST) analyses of <i>Rathayibacter toxicus</i> populations RT-I, RT-II, and RT-III.

<p>Nucleotide differences in the partial gene sequences used for multi-locus sequence typing (MLST) analyses of <i>Rathayibacter toxicus</i> populations RT-I, RT-II, and RT-III.</p