5.5.2S
MICROSCOPY: OPENING A WINDOW ON FUNGICIDE MODE OF ACTION

R CARZANIGA

Istituto di Patologia Vegetale, Universita degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy and IACR-Long Ashton Research Station, University of Bristol, Long Ashton, Bristol BS41 9AF, UK

The elucidation of fungicide mode of action remains a high priority in the identification of novel target sites for intervention and the development of more effective compounds and formulations. Microscopy can provide important clues and insights into mode of action by revealing changes in the morphology and organisation of the treated cells. It can also indicate which developmental stage of the target fungus is affected by preventive or curative treatments.
Transmission electron microscopy (TEM) of thin sections can provide information on how fungicide treatment affects the ultrastructure of both the pathogen and host. Cryo-fixation by rapid freezing followed by freeze-substitution is now the preferred preparation method for TEM, The technique avoids many of the artefacts caused by chemical fixation, preserves labile organelles and water-soluble material such as mucilage, and retains better antigenicity for immunolabelling. Low temperature preparation is also the method of choice for scanning electron microscopy (SEM), where direct examination of frozen-hydrated specimens at low temperature gives optimum preservation of external morphology.
Immunocytochemistry takes microscopy beyond mere description of structure by allowing high resolution localisation of cell components in situ through the specific binding of affinity probes such as antibodies, lectins and enzymes. This precise spatial information generally cannot be obtained by conventional biochemical analyses of fractionated cells and tissues. A vast array of polyclonal and monocional antibodies are available for labelling specific fungal and plant antigens, the distribution of which may be affected by fungicide treatment. Other affinity probes include lectins and enzymes for the localisation of specific sugar residues and substrates, respectively. Labelling is typically visualised using fluorescent markers for light microscopy and electron-opaque colloidal gold particles for electron microscopy.
Recent advances in optical microscopy and computer image analysis, combined with the availability of an expanding range of fluorescent probes, have provided powerful new tools for dissecting cell structure and function. In particular, the advent of confocal laser scanning microscopy (CLSM) has greatly enhanced our ability to visualise what is happening inside living cell and tissues. In conventional fluorescence microscopy (especially of thick specimens), out-offocus fluorescence seriously degrades the image. In CLSM, a pin-hole is placed so that light from above or below the plane of focus is rejected, resulting in greatly improved image contrast and resolution. In addition, the non-invasive collection of serial optical sections allows reconstruction of blur-free 3-D images of intact cells and tissues, without the need to embed and cut sections mechanically. Using fluorescent vital dyes, time-resolved imaging of dynamic processes in living cells is now possible, and double- or triple- labelling with different fluorochromes can be viewed simultaneously. Perturbation of physiological parameters such as calcium, pH and membrane potentiais can be monitored in situ using a variety of fluorescent indicators. Other probes are available to visualise the effects of fungicides on nuclei, endoplasmic reticulum, Golgi, mitochondria, vacuoles, the cytoskeleton, the cell wall and other cell components.
Green fluorescent protein (GFP), from the jellyfish Aequorea victoria, is a valuable new marker for monitoring gene expression and protein localisation in living cells. Expression of GFP under the control of a constitutive promoter in the target fungus can be used to observe the effects of fungicide treatment on mycelial expansion within host tissues. Under the control of appropriate metabolic promoters, GFP can also be used for sensing the microenvironment of the fungus in planta to help optimise fungicide activity. Each of the above imaging techniques offer a different view of cell structure and organisation and how these are affected by fungicide treatment. Although microscopy cannot reveal the biochemical site of action, it can inform and guide biochemical and molecular studies.