Issue link: http://winesandvines.uberflip.com/i/513570
VIEWPOINT June 2015 WINES&VINES 31 Viewpoint tion has provided virologists with powerful new tools that have potential to overcome the prime limitation of labora- tory tests. Although they are powerful and accurate, they are often overly specific. For example, the causal agents of grapevine leafroll disease (collectively referred to as GLRaVs) include five viruses and many different strains, each of which requires a different ELISA reagent and/or DNA-based test for its detection. There may be strains or new viruses that are undetectable with current laboratory tests but might be revealed in a woody indexing bioassay. Enter "deep sequencing," also known informally as "next-generation sequencing." Modern molecular and com- putation techniques have reduced the cost of sequencing the entire genome of any organism 100,000-fold since 2001, to the extent that it is now practical to simply sequence the entire DNA (or millions of markers) in a sample, subtract the bits that are known to be the grape genome and identify viral DNA amongst the "leftover" DNA. With the proper protocols, this should allow virologists to iden- tify not only pathogenic viruses in the tissue but also other, perhaps unknown, viruses. Laboratory tests based on sequencing are rapidly dropping in cost, to the extent that plant breeders are able to use a technique called "genotyping by sequence" to provide detailed genetic information about hundreds of selections at an affordable cost (less than $100 per sample for 95-sample minimum from the Cornell Institute of Biotechnology, for example). Scientists have ac- cess to computational re- sources and databases that G rapevine certification programs are based on the premise that the foundation planting stocks propagated by nursery growers have been tested and shown to be free of pathogens of concern to the industry. Pathogen testing saves growers millions of dollars by providing a good (though not perfect) level of protection from pathogens that shorten the productive life of vineyards and reduce productivity and grape quality. There are three basic methods of verifying that grape- vines are free of harmful viral pathogens: 1) inoculating indicator plants (mostly herba- ceous ones) with tissue from the grapevine being tested; 2) a process called "woody index- ing," which involves grafting buds on to a grapevine variety that expresses symptoms; and 3) laboratory testing. For many viruses, woody indexing is the reference method (and final arbiter) of the phytosanitary status on the vine. Yet this step takes two to four years, delaying introduction and adding enormously to the expense of in- troducing germplasm from abroad and produc- ing certified propagation material. What if we could cut two to four years off the time needed to introduce new cultivars or clones to the marketplace? Chip grafting on woody indicator vines is the earliest testing method, and it has endured for a good reason. Historically it has been capable of casting a broader net, capturing a wider range of pathogens than laboratory tests. And its long duration and biologi- cal basis neatly sidestepped sampling issues with viruses whose titer varies widely over a growing season (when do I sample?) or is unevenly distrib- uted in the vine (where do I sam- ple?). It has provided protection against false-negatives in laboratory tests and an extra level of insurance as a final demonstration of pathogen elimi- nation. Because of these attributes, woody indexing is written into both state and na- tional regulations as the "gold standard." Meanwhile, the molec- ular genetics revolu- n TIM MARTINSON It's Time to Replace Woody Indexing with DNA Testing A non-technical argument for remov- ing a time-consuming and costly step from the process of introducing new grape varieties and clones to the United States.