*Corresponding Author E-mail: katis@agro.auth.gr © 2002 Association of Applied Biologists Ann. appl. Biol. (2002), 140:21-28 Printed in Great Britain 21 Fluctuations in concentration of two potyviruses in garlic during the growing period and sampling conditions for reliable detection by ELISA By C I DOVAS 1 , A P MAMOLOS 2 and N I KATIS 1 * 1 Plant Pathology Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, 540 06, Greece 2 Ecology & Environmental Protection Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, 540 06, Greece (Accepted 26 September 2001; Received 23 November 2000) Summary To optimise sampling conditions for the detection by ELISA of Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV), the most important viral pathogens of garlic worldwide, relative virus concentrations were determined during the growing period and in different leaf parts by DAS- ELISA. Both viruses were found to have uneven distributions in garlic plants, with the tips of the two latest fully developed leaves showing the highest concentrations and the oldest leaves the lowest concentrations. The tips of the youngest leaves were found to have higher virus concentrations than their middle and basal sections. In the older leaves, viruses were distributed more uniformly in the three leaf sections. In the oldest leaves virus levels in the leaf tips were significantly decreased. The concentrations of OYDV and LYSV increased until March, whereas later on they decreased. During storage of leaf samples at 6ºC for 15 days, a loss was found of both virus antigens of more than 80%, and during 109 days of storage at -30ºC a loss of more than 90% was found. Key words: Onion yellow dwarf virus, Leek yellow stripe virus, garlic, ELISA, sampling conditions, virus concentration Introduction Garlic is usually infected by a large number of different viruses, of which the most widespread are: i) two aphid-transmitted potyviruses, Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV), causing mosaic symptoms and significant yield reduction (Van Dijk, 1993a), ii) two aphid- transmitted carlaviruses Shallot latent virus, and Garlic common latent virus, causing latent infections (Van Dijk, 1993b) and iii) several Allexivirus species transmitted by the eriophyid mite Aceria tulipae, and inducing faint short stripes, mild mosaic or no symptoms in Allium spp. (Van Dijk et al., 1991). OYDV and LYSV are the most important viral pathogens of garlic, in which they cause significant yield reduction. Recently, the effects of OYDV- and LYSV-infections on the yields of different garlic cultivars were studied and a reduction of bulb weight was reported to range from 39% to 60%, and 17% to 54%, respectively (Lot et al., 1998). For epidemiological studies and for production of certified planting material, reliable detection methods are essential. Our preliminary experiments to detect LYSV by ELISA revealed problems when elderly garlic leaves were used. In addition, ELISA results were negative when leaf samples infected by OYDV and LYSV were tested after 6 months of storage at -30 o C (no problems were observed with allexiviruses even after 9 months of storage at the same temperature). We have, therefore, investigated more systematically the distribution and concentration of the two viruses in garlic plants during the growing season, and also the effect of temperature on virus concentration in leaf samples stored for testing. Materials and Methods Infected plants and sampling procedures Three garlic clones from three different parts of Greece were planted at the farm of the Aristotle University of Thessaloniki, 15 km north-east of Thessaloniki in northern Greece (40°54N, 23°E) on 28 October 1998 and 1999. All plants were naturally infected with both OYDV and LYSV. Maximum and minimum temperatures for the region during the experimental period are shown in Fig. 1. The three garlic clones were planted in plots of 4 m ´ 6 m, and arranged randomly in each of four blocks. Samples were taken during the period of major plant development, from January to May (sampling dates 30 Jan, 18 Feb, 12 Mar, 7 Apr, 6 May, 20 May in 1998 and 11 Feb, 15 Mar, 14 Apr, 17 May in 1999). At each sampling date one plant was randomly collected from each plot and from