Abstract
BackgroundCrops 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.ResultsThe 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.ConclusionsOur 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.
Highlights
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
We examined the ability of MLVA and multi-locus sequence typing (MLST) typing methods to type P.s. tomato that was subjected to various treatments, in order to evaluate the appropriateness of their use in microbial forensic investigations involving plant pathogens
Multi-locus variable number tandem repeat analysis The MLVA typing system employed in this project utilized primers specific for five known VNTR loci within the P.s. tomato genome (Table 1)
Summary
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. Methods used commonly to fingerprint pathogens, differentiate between microbial strains, and determine microbial relatedness include multi-locus variable number tandem repeat (VNTR) analysis (MLVA) and multi-locus sequence typing (MLST) [4]. Variation in the number of repeats at a particular locus results in the production of amplicons of different sizes, creating a VNTR fingerprint for the bacterial strain of interest [7,4]. Hypervariability at a given VNTR locus, which can be used to indicate that different bacterial isolates originated from a common source, may be especially useful for the purposes of attribution [5,8]
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