1.9.28
CONTROL OF ERYSIPHE GRAMINIS IN BARLEY BY FUNGAL CELL WALL PREPARATIONS

H HAUGAARD, HJL JORGENSEN, DB COLLINGE AND V SMEDEGAARD-PETERSEN

Royal Veterinary and Agricultural University, Department of Plant Biology, 40 Thorvaidsensvej, DK-1871 Frederiksberg C, Denmark

Background and objectives
Preliminary experiments showed that fungal cell wall preparations from ascomycetes, oomycetes and zygomycetes were able to reduce infection of powdery mildew (Erysiphe graminis f.sp. hordei) in barley (Hordeum vulgare cv. Pailas) almost completely. Northern blot analysis showed induction of defence related transcripts eg peroxidase and a pathogenesis related protein. The objective of this study is to estimate the disease controlling ability of fungal cell wall preparations in the barley -powdery mildew system and to investigate the plant-pathogen interactions following treatment of barley with cell wall preparations from Bipolaris oryzae, Pythium ultimum, and Rhizopus stolonifer by using histological and molecular methods.

Materials and methods
Fungal cell wall preparations: The fungi are grown in potato dextrose broth (PDB, Difco) in flasks on an orbital shaker at 125 r.p.m., 22-25C in light for 5 days. Mycelia are harvested through cheese cloth and rinsed with sterile H2O. The mycelia are frozen, thawed and homogenized with an Ultraturrax and then treated with 2 g Novozym/ml over night on a magnetic stirrer. Again the cell wall preparations are filtrated through cheese cloth, then centrifuged, sterile filtrated (0.45 pm, Sartorius, Minisart) and supplied with 0.1% Tween 20. The preparations are stored at -20C until use.

In the bioassay the primary leaves of 11-day-old barley plants are fixed horizontally, abaxial side up, on bent plexiglass plates covered with nylon net. The plants are treated with cell wall preparations applied with a cotton stick. Controls are treated with sterile water. The plants are inoculated in a fall tower with virulent races of E. graminis (A6 or C15) 24 h after treatment. The inoculation density is approximately 5-10 spores/m2. The plants are incubated in a growth chamber at 20C, 16 h light and 8 h darkness. The plants are scored after 7 days, by counting the colonies and assessing them according to infection type. Each experiment consists of three replicates per treatment.

Northern blots are made in accordance with [1] and [2]. Histological observations are made on epidermis strips from the abaxial side of barley leaves 8, 24 and 48 h after inoculation.

Results and conclusions
The bioassay shows a significant decrease in disease incidence by all cell wall preparations compared to water-treated plants. The control of E. graminis is primarily seen as a reduction in the number of colonies, but a shift in infection type from a compatible to an incompatible interaction, visualized as a reduction in size and sporulation ability, is observed too. Depending on the assessment method, the protection ranges from approximately 70-100%. Northern blots have shown induction of a chitinase as early as 1 h after treatment with fungal cell wall preparations from Bipolaris oryzae and Pythium ultimum. Histological observations compare the development of powdery mildew (papillae, haloes, hypersensitive response, appressoria, haustoria and elongating secondary hyphae) with and without treatment with fungal cell wall preparations in order to determine when and how the fungus is arrested. We infer that induced resistance is involved in the suppression of E. graminis following treatment with fungal cell wall preparations.

References
1. Thordal-Christensen et al., 1992. Physiological and Molecular Plant Pathology 40, 395-409.
2. Bryngeisson et al., 1994. Molecular Plant-Microbe Interactions 7, 267-275.