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Preliminary application of DNA barcoding toward the detection of viable plant propagules at an initial, international point-of-entry in Georgia, USA

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Abstract

Over 90% of global commercial trade occurs between seaports, which are initial points-of-entry for nonnative, potentially invasive propagules. As such, there is a need to develop means to both rapidly intercept and identify propagules as they arrive. Here, we focus on plant propagules that are assumed to be non-native, in seed form. Because standard morphological techniques alone are laborious and require taxonomic expertise, we sought to address if identification through barcoding of the plastid DNA (rbcL + matK genes) of plant seeds could improve current processes in the early detection and rapid response to prevent entry/establishment of nonnative plant species. This research conducted a preliminary foray to evaluate the utility of widely accepted plant plastid DNA barcodes to identify plant propagules (seeds, hereafter) collected from the air-intake grilles of refrigerated shipping containers of a single agricultural commodity arriving at the Port of Savannah, Georgia, USA. We ask four questions: (1) Can DNA barcoding be used to detect seeds collected from shipping containers at the port? (2) What is the genetic composition of propagules entering the port? (3) How do morphological identifications compare to those based on genetic analysis? (4) Are nonnative invasive plant species present on shipping containers entering the Port of Savannah? This research collected 11,044 seeds from 628 refrigerated shipping containers between 2015 and 2017. Seeds were then morphologically sorted into Seed Types. Barcoding of the matK and rbcL gene regions of the plastid genomes directly isolated from seeds resulted in poor amplification. This is likely due to a host of potential confounding factors. Therefore, we germinated seeds and utilized leaf-tissues for sequencing of these two gene regions. From BLASTn analyses, results returned top hits for a variety of species, with up to 22 possible nonnative plant species and one definite Federal Noxious Weed. This work investigates the interception application of DNA barcoding to improve agro- and bio-security issues posed by nonnative and invasive species. Though this study required the germination of the seeds to obtain leaf-tissue suitable for our DNA barcoding method, we effectively demonstrated seed viability. Our seed identification process was lengthy and understandably not feasible for real-time application. Therefore, we seek to improve our methods for future applications by testing other approaches that may better complement morphological identification. Next reasonable steps include improved extraction protocols, metabarcoding to generate DNA barcode sequences directly from groups of seeds harvested from shipping containers and implementing other next-generation sequencing techniques.

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Acknowledgements

Our work would have not been possible without the assistance, cooperation, and significant support provided by the US Customs and Border Protection (MA King, RA Rhinehart, LB Brown), USDA APHIS-PPQ (W Kauffman), Georgia Ports Authority and Police (G Hammer, B Bolt, S King, G Buck), and the Georgia Forestry Commission (CS Bates, C Barnes, B Brown). We also thank our colleague, Jarron K. Gravesande, for his significant technical contributions to the project. We acknowledge the Arkansas State University Herbarium (STAR), where vouchers of fruits, seeds, and seedlings have been deposited. We would like to acknowledge several sources of funding from Columbus State University: The Department of Biology, Flora M. Clark Foundation Research Scholarship, and the Department of Academic Affairs for numerous Student Research and Creative Endeavors (SRACE) Grants for providing financial assistance with research and travel. This research was supported in part by the USDA Forest Service, Southern Research Station (15JV11330129031) to KSB.

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  1. L. E. Whitehurst

    Present address: Department of Biology, University of Florida, Gainesville, FL, 32611, USA

Authors and Affiliations

  1. Department of Biology, Columbus State University, Columbus, GA, 31907, USA

    L. E. Whitehurst, S. J. Worthy & K. S. Burgess

  2. Department of Biological Sciences, Arkansas State University, State University, AR, 72467, USA

    C. E. Cunard, J. N. Reed & T. D. Marsico

  3. United States Department of Agriculture, US Forest Service, Southern Research Station, Athens, GA, 30602, USA

    C. E. Cunard & R. D. Lucardi

  4. University of Maryland, 4094 Campus Drive, College Park, MD, 20742, USA

    S. J. Worthy

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Whitehurst, L.E., Cunard, C.E., Reed, J.N. et al. Preliminary application of DNA barcoding toward the detection of viable plant propagules at an initial, international point-of-entry in Georgia, USA. Biol Invasions 22, 1585–1606 (2020). https://doi.org/10.1007/s10530-020-02204-w

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