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Multiple internal controls enhance reliability for PCR and real time PCR detection of Rathayibacter toxicus.

ABSTRACT: Rathayibacter toxicus is a toxigenic bacterial plant pathogen indigenous to Australia and South Africa. A threat to livestock industries globally, the bacterium was designated a U.S. Select Agent. Biosecurity and phytosanitary concerns arise due to the international trade of seed and hay that harbor the bacterium. Accurate diagnostic protocols to support phytosanitary decisions, delineate areas of freedom, and to support research are required to address those concerns. Whole genomes of three genetic populations of R. toxicus were sequenced (Illumina MiSeq platforms), assembled and genomic regions unique to each population identified. Highly sensitive and specific TaqMan qPCR and multiplex endpoint PCR assays were developed for the detection and identification of R. toxicus to the population level of discrimination. Specificity was confirmed with appropriate inclusivity and exclusivity panels; no cross reactivity was observed. The endpoint multiplex PCR and TaqMan qPCR assays detected 10 fg and 1 fg of genomic DNA, respectively. To enhance reliability and increase confidence in results, three types of internal controls with no or one extra primer were developed and incorporated into each assay to detect both plant and artificial internal controls. Assays were validated by blind ring tests with multiple operators in three international laboratories.

SUBMITTER: Arif M

PROVIDER: S-EPMC8052354 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Rathayibacter toxicus is a species of Gram-positive, corynetoxin-producing bacteria that causes annual ryegrass toxicity, a disease often fatal to grazing animals. A phylogenomic approach was employed to model the evolution of R. toxicus to explain the low genetic diversity observed among isolates collected during a 30-year period of sampling in three regions of Australia, gain insight into the taxonomy of Rathayibacter, and provide a framework for studying these bacteria. Analyses of a data set of more than 100 sequenced Rathayibacter genomes indicated that Rathayibacter forms nine species-level groups. R. toxicus is the most genetically distant, and evidence suggested that this species experienced a dramatic event in its evolution. Its genome is significantly reduced in size but is colinear to those of sister species. Moreover, R. toxicus has low intergroup genomic diversity and almost no intragroup genomic diversity between ecologically separated isolates. R. toxicus is the only species of the genus that encodes a clustered regularly interspaced short palindromic repeat (CRISPR) locus and that is known to host a bacteriophage parasite. The spacers, which represent a chronological history of infections, were characterized for information on past events. We propose a three-stage process that emphasizes the importance of the bacteriophage and CRISPR in the genome reduction and low genetic diversity of the R. toxicus species.IMPORTANCERathayibacter toxicus is a toxin-producing species found in Australia and is often fatal to grazing animals. The threat of introduction of the species into the United States led to its inclusion in the Federal Select Agent Program, which makes R. toxicus a highly regulated species. This work provides novel insights into the evolution of R. toxicusR. toxicus is the only species in the genus to have acquired a CRISPR adaptive immune system to protect against bacteriophages. Results suggest that coexistence with the bacteriophage NCPPB3778 led to the massive shrinkage of the R. toxicus genome, species divergence, and the maintenance of low genetic diversity in extant bacterial groups. This work contributes to an understanding of the evolution and ecology of an agriculturally important species of bacteria.

Project description:Rathayibacter 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.

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Multiple internal controls enhance reliability for PCR and real time PCR detection of Rathayibacter toxicus.

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