Development of a PCR-Based Protocol for In Planta Detection of Pathogens In Asymptomatic Horseradish (Armoracia rusticana) Tissues

Horseradish (Armoracia rusticana P. Gaertn., B. Mey. & Scherb.) is grown for its white, fleshy, and pungent roots.  Approximately half of the total commercial production of horseradish in the United States occurs in Illinois.  However, this situation is currently unstable due to yield declines.  These declines are due to a disease complex, and the primary causal agents are thought to be Verticillium dahliae, V. longisporum, and the Potyvirus, Turnip mosaic virus (TuMV).  Each plays a role in horseradish decline: Verticillium is related to internal root tissue discoloration and TuMV is related to yield decreases.  In this study, four formerly productive horseradish cultivars were freed of pathogens via meristem culture in vitro.   To make and maintain pathogen-free (PF) plants in vitro for the development of a certification program, a sensitive pathogen screening method was required.  To test for the presence or absence of TuMV, leaf samples were harvested from putative PF plantlets growing in vitro.  Half of each leaf sample from an individual plant was subjected to the enzyme-linked immunosorbant assay (ELISA).  Total RNA, including viral RNA, was extracted from the other half of each leaf and was evaluated via the reverse transcriptase and polymerase chain reaction (RT-PCR).  The degenerate primers used were designed to amplify DNA fragments specific to highly conserved areas from potyvirus coat protein genes.   To test for the presence or absence of Verticillium spp. fungi in planta, a streamlined PCR-based testing system capable of species differentiation was developed.  Root samples were collected from symptomatic plants, and the segments were placed inside a resealable plastic sandwich bag with a piece of moistened, sterile paper towel to encourage fungal growth.  Bags were incubated at 22^oC, DNA was extracted at 0, 3, and 7 days, and subjected to PCR testing.  For TuMV detection, RT-PCR was found to be 100-1000 times more sensitive than ELISA and should be used to screen certified propagation materials in vitro.  For Verticillium detection, the streamlined testing protocol is superior to traditional plating techniques because it does not require the use of a laminar flow hood, surface disinfestation of roots (even from field samples), or a detailed knowledge of fungal morphology.  In addition, PCR-based techniques can differentiate between infected, but symptomless, plants and non-infected plants.  This information is critical for the development of a production protocol for rapid and cost effective propagation of PF horseradish sets.