1.3.1
ULTRASTRUCTURAL COMPARISON OF THE DEVELOPMENT OF NET AND SPOT LESIONS ON BARLEY BY DRECHSLERA

MG CROMEY

New Zealand Institute for Crop & Food Research Ltd, Private Bag 4704, Christchurch, New Zealand

Background and objectives
Net blotch of barley is caused by the fungus Drechslera teres. The two forms, D. teres f. sp. teres (Dtt) and D. teres f. sp. maculata (Dtm), cause net and spot lesions, respectively [1]. Net lesions have reticulate patterns of transverse and longitudinal brown necrotic flecks, while spot lesions have circular to oval brown necrotic areas. Mature lesions of both types are surrounded by cholorotic zones. This study aims to characterize the developmental differences in the host/pathogen interactions between barley and the two forms of D. teres, leading to the two lesion types.

Materials and methods
Barley plants were inoculated with spore suspensions of Dtt or Dtm. Leaf pieces were sampled at intervals and prepared for transmission electron microscopy using standard techniques for fixing, embedding, sectioning and staining.

Results and conclusions
Both net and spot lesions were first observed as water-soaked spots 2 days after inoculation. These spots became necrotic 1 day later. Characteristic net or spot lesions developed over the following 4 days. Chlorosis developed around lesions 8-10 days after inoculation.

Both Dtt and Dtm directly penetrated barley epidermal cells by means of appressoria, usually formed over periclinal host walls. Infection hyphae developed in penetrated epidermal cells prior to exit of the fungus into host mesophyll, after which only intercellular hyphae were observed. Death of penetrated epidermal cells and a few adjacent mesophyll cells occurred. These dead mesophyll cells were often filled with lightly stained granular material, which corresponded to the water-soaked spots visible within 2 days of inoculation.

Hyphae began to grow away from initial points of infection within 2 days of inoculation, when differences were first observed between the two pathogen forms. Hyphae of Dtt grew longitudinally or transversely through leaves, branching to form reticulate patterns. While some hyphae of Dtm grew longitudinally or transversely, many hyphae grew at oblique angles. In both cases, hyphae were sparse in lesions in the first 3 days after inoculation.

Hyphae beyond initial infection zones were at first associated with living host cells, although papillae (granular deposits just inside host walls) were soon formed in host cells where host and pathogen came into close contact. Death of host cells occurred behind the zones of hyphal spread. Dead host cells in contact with hyphae were often electron-dense and frequently compressed. Nearby cells showed senescence, losing membrane integrity and accumulating granular material in vacuoles. Necrosis was therefore closely associated with the presence of hyphae, which led to the development of characteristic net or spot lesions. Hyphae proliferated in the vicinity of penetration only after the net or spot lesions became visible. Many hyphae of Dtm were much smaller in diameter that those of Dtt.

Chlorosis developed once lesion shape was fully formed, and was accompanied by host degeneration similar to normal leaf senescence, with loss of membrane integrity, and breakdown of chloroplasts and other organelles. There was little obvious host defence reaction in response to Dtt, although papillae were sometimes formed in degenerating host cells in contact with hyphae of Dtm. Hyphae of Dtm proliferated in the chlorotic zones around necrotic lesions, while hyphae in necrotic lesions were often moribund. As reported in an earlier study [2], hyphae of Dtt continued to be most common in necrotic lesions, and contained large numbers of mitochondria.

References
1. Smedegaard-Peterson V, 1971. Yearbook of the Royal Veterinary and Agricultural University (Copenhagen), 124-144.
2. Keon JPR, Hargreaves JA, 1983. Physiological Plant Pathology 22, 321-329.