University College Dublin, Belfield, Dublin 4, Ireland

Background and objectives
Microdochium nivale (teleomorph Monographella nivalis) occurs on cereal hosts in Ireland. Infections of seedlings can result in pre- or post-emergence death or may be limited to lesions on the leaf sheaths or the leaf axils of surviving plants [1]. Disease can progress to the ear and may spread to adjacent spikelets, causing severe grain shrivelling and latent infection of the grain. Foliar infections have attracted more attention in recent years since it is now thought that leaf lesions might be important for conidial spread to the ears [2]; however, such a dispersal mechanism for M. nivale conidia is as yet unproven. The aim of this work was to investigate the infection process, host penetration, colonization and spore production by M. nivale on cereal leaves using cryoscanning electron microscopy (cryo-SEM).

Materials and methods
Seeds of wheat (cv. Avalon), oats (cv. Barra) and barley (cv. Pastoral) were sown in John Innes Compost No. 2 in 10-cm pots in an unheated isolation plant propagator (IPP) unit with a 16-h daylength. At 42 days, the seedlings were harvested and leaf segments 5 cm in length were cut from the second seedling leaf, and placed on the surface of 0.5% water agar containing 10 mg/l kinetin as a senescence retarder. Leaf segments were inoculated with a 10 l droplet at the centre of the adaxial surface and incubated at 20C under continuous light. Using own-host treatments, detached leaves were inoculated with isolates of M. nivale var. nivale and var. majus from wheat, barley and oats. Spore concentration used was low (103 spores/ml) to prevent conidial overcrowding on leaf surfaces. Control leaves were inoculated with sterile distilled water. For SEM studies, segments of leaf material (wheat, oats and barley) were examined after 1, 3, 5, 9, 11, 15 and 17 days from the time of inoculation using a Jeol 5410 scanning electron microscope. Segments of the inoculated leaflets (5x5 mm) were mounted on aluminium stubs using 'Tissue-Tek' cryo-glue. Specimens were immediately frozen by immersion in sub-cooled nitrogen (slush nitrogen) at -200C for 20 s. This cryofixes the material in its fully hydrated and chemically unmodified state. The frozen specimens were transferred to the viewing stage of the microscope which was then heated to (-90C) to allow for the sublimation of surface ice for 2 min. Specimens were then transferred back to the cryo-chamber, coated with gold for 2 min and then viewed under the scanning electron microscope.

Results and conclusions
Conidia germinated on the leaf surfaces 24-48 h after inoculation, and hyphae were observed running adjacent to epidermal cell walls in the leaf. Stomatal colonization and penetration were seen 4-5 days after infection. Sporodochia and annellidic (percurrent) conidiogenesis were observed after 15-17 days; the annellate conidiogenesis cells were clearly visible. It is difficult from this work to identify clear differences in spore germination, host penetration and spore production of the two fungal varieties on the three hosts. It would appear that M. nivale enters the host by stomatal colonization and penetration. The morphology of sporodochia produced on wheat and barley by M. nivale var. nivale appears to be different from that of sporodochia produced on oats by the same pathogen. In this study it was not possible to identify differences in incubation and latent periods for different host-isolate combinations.

1. Diamond H, Cooke BM, Dunne B, 1995. Plant Varieties and Seeds 8, 171-174.
2. Jenkinson P, Parry DW, 1994. Mycological Research 98, 506-510.