School of Biological Sciences, University of Birmingham, Birmingham B15 2TT, UK

Background and objectives
To be effective, antimicrobial defence in the long-lived secondary tissues of woody plants must be unusually durable, as individual tree-pathogen interactions often continue for years. Fungal lesions in the living sapwood of trees are generally bounded by dark or coloured reaction zones (column boundary layers; CBL reaction zones). Although the precise mechanism by which they may confer durable protection in living trees has remained obscure, reaction zones are commonly assumed to have a defensive function. Cell wall alterations and the accumulation of phytoalexin-like compounds have been reported in such zones. In the present study the nature, development and function of reaction zones in the sapwood of sycamore, Acer pseudoplatanus, have been investigated using a wide range of methodologies.

Materials and methods
The structure and composition of reaction zones, formed in response to natural infections or experimentally induced in juvenile pot-grown trees by wound inoculation with wood-inhabiting fungi (principally Ustulina deusta and Chondrostereum purpureum), was determined using light microscopy and a range of chemical and biochemical approaches. Dynamic studies of lesion development and host response were made possible by non-invasive NMR imaging, which also allowed the analysis of tissue water content [1]. The elemental composition of lesion tissues was determined and mapped using proton-induced X-ray emission (PIXE) microanalytic methods [2], and free radicals were detected using electron spin resonance (ESR) spectroscopy [3].

Results and conclusions
Scopoletin, an antifungal coumarin, started to accumulate in sycamore xylem within 24 h of wounding and inoculation, and the coumarin glucoside fraxin after about 3 days. Other phytoalexin-like compounds, some of which have been identified as coumarinolignans, accumulated subsequently. At about this time, when green polymeric deposits were being laid down in the wood, NMR imaging revealed that the water content of the reaction-zone tissues increased. Gas normally present in the lumen of xylem fibres was replaced by water containing the coumarins in solution. An accumulation of ions, especially potassium, in this region was shown by PIXE. The consequent increased osmotic pressure may effect the movement of water to the developing reaction zone, in opposition to the xylem tension forces that would otherwise promote drying in functionally compromised xylem. Water saturation of the xylem tissues could itself create an inimical microenvironment for fungal growth, or it could enable phytoalexin-like compounds produced by the xylem parenchyma to infuse both living and non-living xylem cells. This would permit the formation of a continuous reaction-zone barrier within the differentiated wood, despite the structural heterogeneity of secondary xylem. High concentrations (up to ca 2.5 mM) of long-lived, immobilized, organic free radicals were detected in reaction zones using ESR spectroscopy. These radicals were associated with the coloured, insoluble materials that appear to be formed by the oxidative polymerization of the induced coumarins, and may have a key role in defence, protecting wood from decay by acting as scavengers for the degradative free radicals produced during fungal depolymerization of wood cell walls.

This multi-component response could allow the formation of relatively durable barriers at the margins of fungal lesions in sapwood, and confer the long-term protection required for the defence of long-lived plant organs such as the sapwood of trees.

1. Pearce RB, Fisher BJ, Carpenter TA, Hall LD, 1997. New Phytologist 135, 675-688.
2. Grime GW, Pearce RB, 1995. Nuclear Instruments and Methods in Physics Research B 104, 299-305.
3. Pearce RB, Edwards PP, Green TL et al., 1997. Physiological and Molecular Plant Pathology 50, 371-390.