1.8.55
USE OF MONOCLONAL ANTIBODIES TO INVESTIGATE THE PRE-INVASIVE PROCESSES INVOLVED IN INFECTIONS OF WHEAT BY STAGONOSPORA NODORUM

E ZELINGER1, FM DEWEY2, SJ GURR2 and CR HAWES1

1Research School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford OX3 0BP, UK; 2Department of Plant Sciences, University of Oxford, Oxford OXI 3RB, UK

Background and objectives
Stagonospora nodorum (Septoria nodorum) is an airborne foliar pathogen. It is responsible for the leaf and glume-blotch disease in wheat (Triticum aestivum) and other cereals, and has become a significant problem in recent years [1, 2]. To date most of the work has concentrated on the post-invasive course of action. Little is known about the early stages occurring between S. nodorum and wheat on the leaf surface prior to penetration. The aim of this study was to investigate these stages focusing on three particular processes: adhesion of pycnidiospores and germ tubes to the leaf surface, germination, and penetration. This was achieved by raising specific monoclonal antibodies (MAbs) for use as tools for immunolabelling the pathogen.

Materials and methods
A large panel of MAbs were raised against (i) a suspension of pycnidiospores; (ii) pycnidiospores germinated on agar; and (iii) extracts from infected wheat leaves. The MAbs were screened by immunofluorescence against spores germinated in 96-well microtitre plates to select the best ones to distinguish between pycnidiospores, mycelium and extracellular matrices (Ecm). For detailed in vitro studies the spores were germinated on multi-well glass slides, 1x105/ml suspension per well, at 25C for 7-8 h in high relative humidity. The germinated spores were labelled with the MAbs and viewed by epi-fluorescence microscopy. For in vivo studies wheat seedlings were grown in a growth cabinet at 17C with 16 h daylight for 9 days. Seedlings were inoculated by spraying a volume of 1x106/ml spore suspension until run-off, then covered with a polyethylene bag and put back in the phytotron. Leaves were sprayed with distilled water before inoculation. Samples were taken after 1, 2, 4, 8 and 12 days. Leaf segments of 2 cm were immunolabelled and viewed as before. A variety of light, epi-fluorescence, laser scanning and electron microscopy methods were employed to visualize the immunolabelled material.

Results and conclusions
Three MAbs were finally selected: (i) SN.MG11-EF7 that recognized only spores in vitro and spores plus surrounding Ecm in vivo; (ii) WI.CB2-DD2 which recognized only mycelium; and (iii) SN.CH9-E68 which recognized Ecm at the germ-tube periphery in vitro and secretion areas of different sizes along the germ tubes in vivo. We propose that Ecm covering the spores may have a role in adhesion of spores to the leaf surface. The Ecm along the germ tubes may also have a role in adhesion particularly around penetration sites. Experiments to determine the role of Ecm are ongoing.

References
1. Shaner G, Buechley G, 1995. Plant Disease 79, 928-938.
2. Leath S, Scharen AL, Lund RE, Dietz-Holmes ME, 1993. Plant Disease 77, 1266-1270.