Comparison of High Throughput Sequencing to Standard Protocols for Virus Detection in Berry Crops

Ioannis E. Tzanetakis

Professor of Plant Virology and Director of the Arkansas Clean Plant Center for Berries

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The Problem

​Virus testing ensures that clean plants are distributed by nurseries to producers and home gardeners. Viruses can lead to reduced plant vigor, lower fruit quality and reduced yields, costing producers significant profit loss. Because of this, all foundation (or generation 1) plant materials are tested by USDA-funded National Clean Plant Network laboratories for the presence of systemic pathogens including viruses. Standard protocols include either biological indexing or RT-PCR. Biological indexing inoculates susceptible plants with samples from plants suspected of being infected. The inoculated plants are grown in a nursery to see if severe symptoms appear. RT-PCR is a genetic, pathogen-specific, detection test. Research at the Arkansas Agricultural Experiment Station, the research arm of the University of Arkansas System Division of Agriculture, has shown that more than 50 percent of viruses were going undetected by standard test protocols.

The Work

Ioannis Tzanetakis, virologist and professor of plant pathology, and research associate Dan Villamor conducted a comparison study of the standard detection protocols against HTS, also known as next-generation sequencing, a genetic test that can examine many samples at once for all viruses present — something that RT-PCR cannot do. They used 30 varieties of blackberries, raspberries, blueberries and strawberries with known virus profiles provided by the USDA National Clonal Germplasm Repository (NCGR). The study examined temporal virus detection at four sampling times encompassing two growing seasons.  They discovered that both RT-PCR and HTS outperformed biological indexing in virus detection. RT-PCR and HTS had nearly identical results for detecting known viruses. However, HTS is able to detect viruses not known to be present but also variants undetectable by RT-PCR. More importantly, 11 viruses known to be present in 16 berry samples went undetected in some time points by both RT-PCR and HTS.

The Results

Tzanetakis and Villamor concluded that more than 50 percent of viruses were going undetected using standard NCPN protocols that call for two tests of plant material. Based on these data they proposed amending NCPN requirements to include four sampling times over two years for berry and potentially other crops to ensure that no virus remains undetected independent of titer, distribution or other virus/virus or virus/host interactions. Tzanetakis also recommends that HTS replace RT-PCR as the sole testing platform. Not only can it detect variants that escape RT-PCR but it can also identify novel viruses. HTS is also much faster than RT-PCR because it can analyze a large number of samples simultaneously. Also, RT-PCR requires that testers know what they are looking for. During the course of the research, using HTS, the team identified six new viruses that had remained undetected in NCGR plants that had been studied for years.

The Value

The United States ranks second worldwide in berry fruit production, according to FAOSTAT, a collection of databases covering international agricultural statistics for the Food and Agriculture Organization of the United Nations. According to USDA’s National Agricultural Statistics Service, berry production provides nearly $4 billion to the U.S. economy. Accurate testing to detect viruses, therefore, is essential to making sure that berry producers can maintain quality, high-yield crops from plants that continue to produce for as many years as possible.

Fundings

Primary – USDA-APHIS through National Clean Plant Network
Secondary — Arkansas Agricultural Experiment Station

About the Researcher

Ioannis E. Tzanetakis

Professor of Plant Virology and Director of the Arkansas Clean Plant Center for Berries

• Ph.D. in Molecular Biology, Oregon State University, Corvallis

Research focuses on plant virus epidemiology, virus-vector interactions and next generation diagnostics based on crop viromes. He also is director of the Arkansas Clean Plant Center for Berries at the Arkansas Agricultural Experiment Station.