1.3.31
DIRECTIONAL GROWTH OF DOWNY MILDEW HYPHAE WITHIN INFECTED LEAVES

A OTT, P WHITEHEAD and PTN SPENCER-PHILLIPS

Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK

Background and objectives
It has been suggested that the growth of endophytic hyphae of biotrophic pathogens, such as downy mildew and rust fungi, may respond to the relative availability of photo-assimilates in the apoplast (e.g. cell walls) within infected tissues [1] . Directional growth towards vascular tissue, and frequent insertion of haustoria into mesophyll cells, might be expected in order to intercept nutrients in host species with type 1 veins. In these, mainstream products of photosynthesis remain symplastic (i.e. in the cytoplasm) between mesophyll cells and the vascular tissue, and perhaps hyphae would grow towards the relatively high concentration of sugars in veins. A similar effect might occur with infections of hosts with type 2 veins, which have an apoplastic step in the phloem-loading pathway. This would depend, however, on whether photo-assimilates are released to the apoplast either from each mesophyll cell, or only in close proximity to the phloem. We have initiated a survey of downy mildew infections in order to evaluate this transfer intercept hypothesis. The methods to be used have been applied to Peronospora viciae infections of Pisum sativum.

Materials and methods
The directional growth of downy mildew hyphae immediately after penetration (first directional growth), and at 48 h and 5 days post-inoculation, have been measured within leaves of the susceptible pea cv. Krupp Pelushka. Data for first directional growth and hyphae at 48 h were obtained using localized inoculation droplets as in previous work [1] , whilst for 5-day infections whole plants were sprayed with a sporangiospore suspension. Discs of leaf tissue were cleared overnight in a 1:1:1 mixture of ethanol, lactic acid and chloroform, and then for 1.5 h in 2% NaOH at 60C. They were stained in Trypan Blue in ethanolic lactophenol and mounted in lactophenol for microscopic examination. First directional growth and orientation of hyphal tips at 48 h were determined using a method developed for rust infection structures [2] . Directional growth after 5 days was determined using an eyepiece graticule with a grid of intersecting lines. The grid was positioned against veins located at random, and the number of intersections made by hyphae with lines orientated in parallel versus perpendicular to the veins was compared.

Results and conclusions
The first intercellular hyphae formed after penetration of the upper epidemis showed directional growth (P<0.001), with 38% of hyphae growing into the quarter sector towards the nearest vein and 18% into the opposite quarter sector. This was also the case for vein endings (P<0.01; 44 and 17% towards and away from the vein, respectively). Hyphal tips at 48 h, however, were not oriented towards veins to a statistically significant degree, although 31% were located in the sector towards veins, as opposed to 23% in the opposite sector. In contrast, first directional growth towards vein endings was highly significant (P<0.001) when data for nearest and second nearest veins were pooled; 48 h hyphal tips also appeared to be oriented similarly (P<0.05) when pooled data were analysed. Hyphae at 5 days after infection did not seem to show directional growth in relation to veins. Thus it would appear that, whilst the first hyphae formed after penetration respond positively to a factor (e.g. nutrient concentration) related to the vascular tissue, this influence reduces with time as colonization proceeds. Similar data for downy mildew infections of other hosts will help elucidate the validity of the transfer intercept hypothesis.

References
1. Spencer-Phillips PTN, 1997. Advances in Botanical Research (incorporating Advances in Plant Pathology) 24, 309-333.
2. Jacobs TH, 1990. Mycological Research 94, 122-124.