1.10.2
EXTRACELLULAR MATRIX OF ERYSIPHE GRAMINIS: INFLUENCE OF SUBSTRATUM ON ACCUMULATION

TLW CARVER1, H KUNOH2, RL NICHOLSON3 and BJ THOMAS1

1IGER, Aberystwyth, Dyfed SY23 3EB, UK; 2Laboratory of Plant Pathology, Mie University, Tsu 514, Japan; 3Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA

Background and objectives
We followed extracellular matrix (ECM) accumulation associated with ungerminated and germinated conidia of Erysiphe graminis incubated on plant and artificial surfaces, to determine whether substratum influences time, location or quantity of accumulation. Penetration of the leaf epidermal cell by E. graminis occurs after differentiation of the appressorium, about 16 h post-inoculation. However, ECM appears in advance of attempted penetration, even before germination. Many plant pathogenic fungi produce an ECM, but the stimuli for its release are poorly understood. ECM may be involved in adhesion, preparation of the infection court and morphogenesis.

Within 30 min of contacting a cellulose membrane, E. graminis conidia release ECM containing cutinase that can degrade the plant cuticle [1]. However on leaves, ECM is rarely seen in association with ungerminated conidia but is seen beneath germ tubes and appressoria [2]. The substratum greatly influences E. graminis morphogenesis [2]. For example, germlings develop normally on leaves and on different hydrophobic substrata, whereas on most hydrophilic substrata germling development is typically abnormal [2]. Our goal is to develop an axenic system where morphogenesis and development is comparable to that on leaves.

Materials and methods
Conidia of E. graminis f. sp. hordei were incubated at 100% RH on natural and artificial substrata of different hydrophobicities (assessed by water drop contact angle, CA). These were barley leaf (i) with epicuticular wax (CA=134), and (ii) without epicuticular wax (CA=102); (iii) glass (CA=38); (iv) silanized plastic (CA=80); (v) cellulose membrane (CA=27). Observation was by low-temperature scanning electron microscopy (LTSEM) using low angle stubs, and by light microscopy of living specimens.

Results and conclusions
LTSEM showed that on leaves, with or without epicuticular wax, no ECM was visible beneath conidia, although localized ECM could be seen beneath primary (PGT) and appressorial (AGT) germ tubes (confirming [2]). On glass (hydrophilic surface), ECM was absent beneath ungerminated conidia but was seen beneath germ tubes. By contrast, on cellulose membrane (hydrophilic surface), ECM was seen beneath conidia 15 min after contact, correlating to the time when cutinase is released on the membrane [1]. ECM increased in abundance to ca 1 h and remained beneath conidia until 12 h when it was also seen beneath germ tubes. Abundant ECM was seen on silanized plastic. Here, ECM accumulated beneath conidia within 5 min and continued to accumulate for 30-60 min when it extended well beyond the area of direct conidial contact. As PGTs and AGTs emerged (45 min and 6 h), the ECM often formed a continuous layer beneath conidia, PGTs and AGTs. By 12 h, the layer of ECM beneath conidia had decreased considerably in thickness. We speculate that ECM component(s) may have been absorbed back into conidia.

Light microscopy revealed ECM beneath conidia on silanized plastic. ECM appeared within 2 min and accumulated up to 30 min, when it covered the entire area subtended by conidia. By 60 min the ECM was less obvious. When conidia were moved by microneedle, the ECM remained at the conidium/substratum interface, indicating that it was a mobile liquid. If conidia were deposited at an angle, ECM accumulated at the substratum interface and the PGT emerged there. On glass, light microscopy was unable to reveal ECM up to 3 h (PGTs emerging), confirming LTSEM observations.

While germling morphogenesis on some artificial substrata appears normal, ECM accumulation beneath conidia is not apparent on a host leaf.

References
1. Nicholson RL, Kunoh H, 1996. Canadian Journal of Botany 73 (suppl. 1), S609-S615.
2. Carver TLW, Ingerson SM, Thomas BJ, 1996. In Kerstiens, G., ed. Plant Cuticles - An Integrated Functional Approach. Bios Scientific Publishers, Oxford, pp. 255-266.