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Browsing by Author "Miller, Sally A."

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    Metagenomic Sequencing for Identification of Xylella fastidiosa from Leaf Samples
    Roman-Reyna, Veronica; Dupas, Enora; Cesbron, Sophie; Marchi, Guido; Campigli, Sara; Hansen, Mary Ann; Bush, Elizabeth; Prarat, Melanie; Shiplett, Katherine; Ivey, Melanie L. Lewis; Pierzynski, Joy; Miller, Sally A.; Hand, Francesca Peduto; Jacques, Marie-Agnes; Jacobs, Jonathan M. (2021-09)
    Xylella fastidiosa (Xf) is a globally distributed plant-pathogenic bacterium. The primary control strategy for Xf diseases is eradicating infected plants; therefore, timely and accurate detection is necessary to prevent crop losses and further pathogen dispersal. Conventional Xf diagnostics primarily relies on quantitative PCR (qPCR) assays. However, these methods do not consider new or emerging variants due to pathogen genetic recombination and sensitivity limitations. We developed and tested a metage-nomics pipeline using in-house short-read sequencing as a complementary approach for affordable, fast, and highly accurate Xf detection. We used metagenomics to identify Xf to the strain level in single-and mixed-infected plant samples at concentrations as low as 1 pg of bacterial DNA per gram of tissue. We also tested naturally infected samples from various plant species originating from Europe and the United States. We identified Xf sub-species in samples previously considered inconclusive with real-time PCR (quantification cycle [C-q], > 35). Overall, we showed the versatility of the pipeline by using different plant hosts and DNA extraction methods. Our pipeline provides taxonomic and functional infor-mation for Xf diagnostics without extensive knowledge of the disease. This pipeline demonstrates that metagenomics can be used for early detection of Xf and incorporated as a tool to inform disease management strategies. IMPORTANCE Destructive Xylella fastidiosa (Xf) outbreaks in Europe highlight this pathogen's capacity to expand its host range and geographical distribution. The current disease diag-nostic approaches are limited by a multiple-step process, biases to known sequences, and detection limits. We developed a low-cost, user-friendly metagenomic sequencing tool for Xf detection. In less than 3 days, we were able to identify Xf subspecies and strains in field-collected samples. Overall, our pipeline is a diagnostics tool that could be easily extended to other plant-pathogen interactions and implemented for emerging plant threat surveillance.
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    Phylogenomics of Xanthomonas field strains infecting pepper and tomato reveals diversity in effector repertoires and identifies determinants of host specificity
    Schwartz, Allison R.; Potnist, Neha; Milsina, Sujan; Wilson, Mark; Patane, Jose; Martins, Joaquim, Jr.; Minsavage, Gerald V.; Dahlbeck, Douglas; Akhunova, Alina; Almeida, Nalvo F.; Vallad, Gary E.; Barak, Jeri D.; White, Frank F.; Miller, Sally A.; Ritchie, David; Goss, Erica; Bart, Rebecca S.; Setubal, Joao C.; Jones, Jeffrey B.; Staskawicz, Brian J. (Frontiers, 2015-06-03)
    Bacterial spot disease of pepper and tomato is caused by four distinct Xanthomonas species and is a severely limiting factor on fruit yield in these crops. The genetic diversity and the type Ill effector repertoires of a large sampling of field strains for this disease have yet to be explored on a genomic scale, limiting our understanding of pathogen evolution in an agricultural setting. Genomes of 67 Xanthomonas euvesicatoria (Xe), Xanthomonas perforans (Xp), and Xanthomonas gardneri (Kg) strains isolated from diseased pepper and tomato fields in the southeastern and midwestern United States were sequenced in order to determine the genetic diversity in field strains. Type Ill effector repertoires were computationally predicted for each strain, and multiple methods of constructing phylogenies were employed to understand better the genetic relationship of strains in the collection. A division in the Xp population was detected based on core genome phylogeny, supporting a model whereby the host-range expansion of Xp field strains on pepper is due, in part, to a loss of the effector AvrBsT. Xp-host compatibility was further studied with the observation that a double deletion of AvrBsT and XopQ allows a host range expansion for Nicotiana benthamiana. Extensive sampling of field strains and an improved understanding of effector content will aid in efforts to design disease resistance strategies targeted against highly conserved core effectors.