Improved PCR detection of potyviruses in Allium species Pablo Lunello 1 , Daniel Ducasse 2 and Vilma Conci 2, * 1 Department of Biotechnology, INIA, Autopista A-6, km 7. 28040, Madrid, Spain; 2 Instituto de Fitopatologı´a y Fisiologı´a Vegetal, Instituto Nacional de Tecnologı´a Agropecuaria (IFFIVE-INTA), Camino 60 cuadras km 5½ (5119), Co ´rdoba, Argentina; *Author for correspondence (Phone: +54 (0351) 4973636; Fax: +54 (0351) 4974343; E-mail: vconci@correo.inta.gov.ar) Accepted 25 April 2005 Key words: Alliaceae, garlic, indexing, Leek yellow stripe virus, molecular tests, Onion yellow dwarf virus, RT-PCR, Shallot yellow stripe virus, Turnip mosaic virus Abstract Protocols for producing virus-free Allium plants require an indexing system that is more sensitive than DAS-ELISA and can detect low virus concentrations in infected plants. In the present work, degenerate primers were designed and a one-step IC-RT-PCR protocol was developed to differentiate between Leek yellow stripe virus (LYSV) and Onion yellow dwarf virus (OYDV) in single and mixed infections in several Allium spp. A 566-bp band was observed for LYSV, a 489-bp band for OYDV in single infections, and two bands of the same sizes in mixed infections in different species of Alliaceae. A 508-bp band of Shallot yellow stripe virus and a 594-bp band of Turnip mosaic virus were also amplified with the same primers. RT- nested-PCR was also conducted directly in microtitre plate wells after negative or questionable reactions were produced in an ELISA experiment. The detection limit of the DAS-ELISA for LYSV was 16.5– 27.3 ng ml )1 . The RT-nested-PCR done after DAS-ELISA was 10 2 times more sensitive than the DAS- ELISA alone. In parallel, an IC-RT-nested-PCR in microcentrifuge tubes was 10 4 times more sensitive than the DAS-ELISA. The DAS-ELISA-RT-nested-PCR enables the initial screening of samples by DAS- ELISA to eliminate a high percentage of virus-positive plants, considerably reducing the number of plants to analyze further by RT-PCR. Introduction Viruses infecting Alliaceae are widespread throughout the world and affect a great number of Allium species, causing significant yield losses. Some of the crops affected are Allium sativum var. sativum (garlic), A. cepa (onion), A. ampeloprasum var. porrum (leek), A. ampeloprasum var. ampelo- prasum (‘gigantajo’ or great-headed garlic) and A. sativum var. ophioscorodon (‘castan˜o’ garlic). Among the potyviruses affecting Allium spp., Onion yellow dwarf virus (OYDV) and Leek yel- low stripe virus (LYSV) have been detected world- wide (Conci et al., 1992; Van Dijk, 1993; Barg et al., 1997; Tsuneyoshi et al., 1998b; Chen et al., 2001). These are the most important viruses in terms of the damage, with garlic bulb weight re- duced between 24 and 60% for OYDV and be- tween 17 and 54% for LYSV (Canavelli et al., 1998; Lot et al., 1998). Shallot yellow stripe virus (SYSV) and Turnip mosaic virus (TuMV) have less frequently been reported in Allium spp. (Van Dijk, 1993; Gera et al., 1997; Van der Vlugt et al., 1999). Because international health standards are steadily becoming stricter and product health is so closely linked to quality and thus marketing, the production of virus-free plants is of vital impor- European Journal of Plant Pathology (2005) 112:371–378 Ó Springer 2005 DOI 10.1007/s10658-005-6232-3