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  • Writer's pictureDebbie Woodbury

High Throughput Sequencing: A New Diagnostic Protocol for Quarantine Release

One major advancement to reach the FPS rose program in recent years is the adoption of high throughput sequencing (HTS) for disease testing. HTS is an alternative to traditional diagnostic methods for the detection and identification of organisms in a sample by using DNA sequencing to provide a comprehensive picture of the entire microbial profile. Biological indicators are among the earliest of traditional diagnostic methods. Indicators are specific plant species or varieties that are known to display symptoms when exposed to certain disease agents. Herbaceous indicators are used by spreading sap of the plant being tested across the leaves of the indicator plant. Woody indicators are used by grafting material of the subject plant to a variety known to display symptoms if infected. After the grafting is complete, disease agents present in the subject plant are expected to infect the indicator, which would display symptoms. Indicators are only known to display symptoms for certain agents; it is possible that an indicator may show no symptoms, but a disease agent is still present. It can take many years for woody indicators to establish and show symptoms. From indicators, diagnostic technology evolved to ELISA and PCR molecular methods, often in conjunction with indicator plants. ELISA and PCR are advancements over biological indexing because they allow determination of specific disease agents, and typically require less time to complete testing, but they require knowledge of the agent to test for its presence. FPS scientists conducted side-by-side studies comparing the efficacy of woody and herbaceous indexing to PCR and HTS testing in rose, grapevine, and Prunus diagnostics. The results of these studies indicate that biological indicators give false negative results a significant percentage of the time and do not provide sufficient sensitivity in detecting target viruses or unknown viral pathogens.

FPS has integrated this research knowledge into an improved protocol that adopts the most reliable and efficient techniques using HTS and real-time quantitative RT-qPCR/qPCR, which are known to be highly sensitive tests. For roses, the adoption of the revised protocol has led to the elimination of woody indexing to multiflora and shirofugen. The revised protocol (see testing flowchart) uses informative HTS testing of source material to provide customers with feedback on the phytosanitary status of the plant material and determines if virus elimination therapy is necessary. Once plants are propagated and have developed appropriate tissue to test, initial HTS/PCR testing provides information on viruses that may be present in the plant material in the summer-to-fall timeframe. Final HTS/PCR testing after a six-month minimum period of dormancy provides a third and final test, testing for the presence of viruses in spring to summer and verifying earlier test results or detecting pathogens previously undetected due to low titer. The entire process captures pathogens that may be present in plant source material by testing upon arrival, then in two different seasons separated by a period of dormancy to account for fluctuations in virus titer. Although there are slight variations between the grapevine, Prunus, and rose protocols per the horticultural needs of each crop, the length of time from introduction to release of material using the revised protocol is approximately 18-24 months.

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