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POSTERS: Pathogen detection, quantification and diagnosis

Protecting the U.S. pome fruit industry from foreign pathogens at the USDA APHIS using high-throughput sequencing technologies
Oscar Hurtado-Gonzales - USDA-APHIS Plant Germplasm Quarantine Program. Bishwo Adhikari- USDA-APHIS Plant Germplasm Quarantine Program, Joseph Foster- USDA-APHIS-PPQ-FO-Plant Germplasm Quarantine Program, Clint McFarland- USDA-APHIS-PPQ-Field Operations, Leticia Hendrickson- USDA-APHIS Plant Germplasm Quarantine Program, Mart

The pome fruit industry is a multibillion dollar business with apples as the most consumed fruit in the United States. Improved varietal development depends on access to valuable imported pome germplasm, mainly introduced into the U.S. through the National Clean Plant Network in Washington and the USDA-APHIS Plant Germplasm Quarantine Program (PGQP) in Maryland. The PGQP Pomes Program screens imported germplasm for a wide variety of well-characterized high-risk pathogens including viruses, viroids and phytoplasmas using PCR techniques, bioassays, and field indexing. If pathogen(s) are detected, germplasm undergoes therapy to generate pathogen-free trees before their released. Heightened demand for imported germplasm and limitations with conventional pathogen detection increases the risk of undetected pathogens during the screening process. To reduce this risk, the PGQP Pomes Program is combining high-throughput sequencing (HTS) technologies and improved bioinformatic pipelines to rapidly detect quarantine pathogens and uncharacterized disease agents. Ten unique apple and pear fruit trees routinely used as positive controls were subjected to HTS as a proof-of-concept. Illumina TruSeq cDNA libraries were constructed from ribosomal RNA-depleted total RNA extracts. Approximately 50 million reads (1x75 bp) were obtained per sample. Bioinformatic analyses identified most expected plant pathogens as well as unreported agents. A summary of our findings will be presented.