Evaluating the impacts of stressors of Pseudomonas syringae pathovar tomato on the effectiveness of multi-locus variable number tandem repeat analysis and multi-locus sequence typing in microbial forensic investigations

BACKGROUND: Crops in the USA are vulnerable to natural and criminal threats because of their widespread cultivation and lack of surveillance, and because of implementation of growing practices such as monoculture. To prepare for investigation and attribution of such events, forensic assays, including determination of molecular profiles, are being adapted for use with plant pathogens. The use of multi-locus variable number tandem repeat (VNTR) analysis (MLVA) and multi-locus sequence typing (MLST) in investigations involving plant pathogens may be problematic because the long lag periods between pathogen introduction and discovery of associated disease may provide enough time for evolution to occur in the regions of the genome employed in each assay. Thus, more information on the stability of the loci employed in these methods is needed. RESULTS: The MLVA fingerprints and MLST profiles were consistent throughout the experiment, indicating that, using a specific set of primers and conditions, MLVA and MLST typing systems reliably identify P.s. tomato DC3000. This information is essential to forensic investigators in interpreting comparisons between MLVA and MLST typing profiles observed in P.s. tomato isolates. CONCLUSIONS: Our results indicate that MLVA and MLST typing systems, utilizing the specified primers and conditions, could be employed successfully in forensics investigations involving P.s. tomato. Similar experiments should be conducted in the field and with other high-consequence plant pathogens to ensure that the assays are reliable for pathogens infecting plants in their natural environment and for organisms that may display faster rates of mutation.