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Enzyme-Linked Immunosorbent Assay Testing of Shoots Grown In Vitro and the Use of Immunocapture-Reverse Transcription-Polymerase Chain Reaction Improve the Detection of Prunus necrotic ringspot virus in Rose

May 2000 , Volume 90 , Number  5
Pages  522 - 528

Benoît Moury , Loïc Cardin , Jean-Paul Onesto , Thierry Candresse , and Alain Poupet

First, second, third, and fifth authors: INRA, Unité de recherche Santé végétale et environnement, Villa Thuret, 62 bd du Cap, BP 2078, F-06606 Antibes cedex, France; and fourth author: INRA, IBVM, Virologie végétale, BP 81, F-33883 Villenave d'Ornon cedex, France

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Accepted for publication 24 January 2000.

We developed and evaluated two different methods to improve the detection of the most prevalent virus of rose in Europe, Prunus necrotic ring-spot virus (PNRSV). Immunocapture-reverse transcription-polymerase chain reaction was estimated to be about 100 times more sensitive than double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and showed an equivalent specificity. Based on the observation that PNRSV multiplies actively in young growing tissues (axillary shoots and cuttings), an in vitro culture method allowing rapid (about 15 days) and homogeneous development of dormant axillary buds with high virus titers was standardized. ELISA tests of these young shoots showed, in some cases, a 104 to 105 increase in sensitivity in comparison to adjacent leaf tissues from the rose mother plants. Between 21 and 98% (depending on the season) more samples were identified as positive by using ELISA on samples from shoot tips grown in vitro rather than on leaves collected directly from the PNRSV-infected mother plants. This simple method of growing shoot tips in vitro improved the confidence in the detection of PNRSV and eliminated problems in sampling appropriate tissues.

Additional keywords: season effect, woody plant.

© 2000 The American Phytopathological Society