The view that nutrient acquisition by most plants growing in natural ecosystems is mediated by mycorrhiza-forming symbiotic fungi is now largely accepted. With this perception comes the need to learn more about the identity of the organisms responsible for these key processes. Herein lies a challenge taken up by Vrålstad et al. on pp. 549–563 in this issue.

Patterns of molecular genetic variation were examined in seed collections of Plantago major and Plantago intermedia, used previously to investigate the variations in ozone (O3) resistance of these species across Europe. Total genomic DNA was amplified with random primers (random amplied polymorphic DNA (RAPD) and inter- single sequence repeats (SSR)) to produce 73 genetic markers. In addition, allozyme and chloroplast variations were surveyed. Genetic markers were examined for association with O3 resistance in 18 British populations of P. major as well as 27 continental European populations of P. major and P. intermedia. Two populations that exhibited increased resistance to O3 following several years' exposure to high O3 concentrations in the field showed decreased genetic variation over time. In addition, their genetic composition showed no drastic change, which suggests that the change in resistance to O3 was probably the result of selection on genotypes already present in local populations (selection in situ). It appears that selection for O3 resistance may occur in independent populations, and also may involve a number of genetically determined traits. Consequently the finding that plants with similar degrees of O3 resistance are not closely related was not unexpected. However, the finding of an association of several genetic markers with O3 resistance merits further investigation.

Individual arbuscular mycorrhizal fungi can differ markedly in their ability to improve the phosphate nutrition and growth of host plants. In particular, Scutellospora calospora is relatively ineffective with some hosts and a growth depression is often observed. We have examined the abilities of S. calospora and Glomus sp. ‘City Beach’ strain WUM 16, grown in soils which promote extensive mycorrhizal colonization, to transfer phosphate (P) to Allium porrum. Phosphate uptake from the low-P soils (P0) was compared with uptake from soils amended with extra P (P1). In order to relate P transfer to physiological characteristics of the two fungi, inflow of P via fungus to the plant was combined with the surface areas of intercellular hyphae and arbuscules (symbiotic interfaces) to calculate the amount of P transferred per unit area of interface (P fluxes). ‘Hyphal inflows’ and ‘hyphal fluxes’ were also calculated on the assumption that P uptake by the plant was the same in mycorrhizal and equivalent non-mycorrhizal (NM) plants (the validity of this assumption is discussed). With both soil P treatments, P was taken up by plants colonized by both mycorrhizal fungi to a greater extent than by the equivalent NM controls. Hyphal inflows to plants in P0 soil that were colonized by Glomus sp. ‘City Beach’ were greatest from 14–21 d and decreased at later harvests. With P1 soil the inflow via Glomus sp. ‘City Beach’ peaked at a very high value at 21–28 d. Hyphal inflows into plants in P0 soil that were colonized by S. calospora increased throughout the experiment, and with P1 soil they remained steady at high values. With both fungi, the surface areas of the symbiotic interfaces increased greatly after 14 d, and generally there was little effect of higher soil P. With P0 soil, Glomus sp. ‘City Beach’ showed no significant differences in the ratio of surface areas of the two interfaces over the course of the experiment. Scutellospora calospora tended to produce a lower percentage of interfacial area contributed by arbuscules. With the mycorrhizal plants growing in their respective soils, there appeared no consistent differences between the two fungi with respect to fluxes of P across the interfaces. With P0 soil, fluxes via Glomus sp. ‘City Beach’ were initially higher than those via S. calospora, but later they were higher with S. calospora. With P1 soil the only difference was the 28–42-d period, when Glomus sp. ‘City Beach’ produced the higher flux (reflecting the low surface area at the time). The results show that relative inefficiency of S. calospora in its ability to transfer P, as reported by others, may result from different environmental conditions, use of different hosts, or even of different fungal isolates (strains).

Using field plots, three species (Mercurialis perennis L., Rubus fruticosus L., and Trientalis europaea L.) were tested for their potential to emit gaseous ammonia to the atmosphere. Canopies were misted with 5 mM methionine sulphoximine (MSO) to inhibit glutamine synthetase (GS), the enzyme of ammonium assimilation. Leaf tissue NH4+ concentration of control plants was 0·03–0·1 μmol g−1 f. wt. Although NH4+ accumulated in the leaf tissue of MSO-treated plants of all three species to similar concentrations (6-10 μmol g−1 f. wt after 4 d), emissions were only detected from the leaves of M. perennis, with potential rates of 2·5 nmol m−2 leaf s−1. Experiments carried out in a controlled environment confirmed this rate of emission over 9 d, during which time leaf tissue ammonium increased to 66 μmol g−1 f. wt. Comparisons with Hordeum vulgare grown under the same conditions showed that tissue NH4+ concentration reached a plateau of about 40 μmol g −1f. wt after 2 d. Emissions of NH3 during the 5 d of treatment reached a maximum rate of 10 nmol m−2 s−1 by the third day.

Apoplastic pH of the plants was determined, and it is suggested that this is an important factor explaining the differences in NH3 emission between species. The higher the apoplastic pH, the greater the likelihood of loss of NH3 from sub-stomatal spaces to the atmosphere. T. europaea (non-emitter) had an apoplastic pH of 5, R. fruticosus (non-emitter) a pH of c. 5·6, whereas that of M. perennis (emitter) was c. pH 6·3. The apoplastic pH is thought to be dictated in part by the N nutrition of a species, nitrophilous species tending to have high pH. Without NO3− fertilization, H. vulgare had an apoplastic pH of 6·8 but this increased to 7·3, 3 d after feeding with NO3−.

Short-term fumigation (2 h) of shoots of H. vulgare with 60 μg (≈32 mg NH3 m−3) of labelled gaseous 15N-NH3 (in the absence of MSO) showed that a substantial proportion (60%) of the applied label was found in the leaves, as well as in stems and roots (3%). There was also a change in amino acid pools, with an increase in shoot amino acids and a decrease in those in the root, while tissue NH4+ was very low in both shoots and roots. This provided indirect evidence that some of the applied label had been incorporated into an organic form. Following the fumigation treatment, emissions of NH3 were collected for 3 h, then c. 6·5 μg of N was recovered, of which c. 17% was 15N-labelled. Some of this label could have resulted from desorption of NH3 from leaf surfaces, but it was more likely that the remaining 14N isotope was from sub-stomatal emissions of NH3.

It is argued that non-nitrophilous plants tend to rely on mixed sources of N (NO3−, NH4+ or organic-N) and are more likely to favour root rather than shoot assimilation. Under these circumstances, their apoplastic pH is relatively low (compared with that of nitrophiles, which tend to assimilate NO3− mainly in their shoots), and at atmospheric concentrations most wild species are likely to be net assimilators, rather than emitters, of atmospheric ammonia.

Segmentation of the aeration system and its anatomy was studied in rhizomes of common reed (Phragmites australis). Segmentation is achieved by nodal diaphragms which allow the passage of pressurised gas-flow but which also form effective barriers against flooding of the internal space in case of local injury. The pressures required to force water through the diaphragms were measured and compared with the anatomical structure of the diaphragm. The fine hydrophilic stellate parenchyma of the diaphragms was shown to act as a matrix supporting menisci of air–water interfaces and therefore preventing water movement until the ‘limit’ pressure (measured values ranged from 18 to >40 kPa) was overcome. Although the surfaces of the large stellate parenchyma and sclerenchyma strands covering the diaphragm are hydrophobic these components do little to prevent the ingress of water.

Microseris douglasii (DC.) Sch.-Bip. and M. bigelovii (Gray) Sch.-Bip. are two small annual autogamous species of Compositae with nearly non-overlapping distribution ranges in Western North America. Specifically, M. bigelovii occurs directly along the Pacific coast, whilst M. douglasii has an inland distribution including patches of serpentine soil. Both species are variable, and artificial hybrids between them vary widely in fertility depending on the individual parents. Segregating offspring of one hybrid (strain H27) is being used to analyse the genetic basis of characters differentiating the two species by QTL mapping with RAPDS and ALFPs as molecular markers. Technical problems with mapping dominant markers in a wide cross will be briefly listed and QTL analysis will be discussed. For the genetic analysis of physiological characters, the precise definition of the characters is crucial and the methods of scoring or measuring phenotypes in different environments eventually require more time and effort than the molecular characterization. We are establishing recombinant inbred lines to provide material for more complex physiological analyses requiring several plants per genotype. An increasing number of characters is being studied in this cross, and the possibility of shared pleiotropic QTLs is high. The potential number of QTL genotypes by far exceeds the number of actual genotypes in these lines. We are characterizing the gene interactions as closely as possible and making quantitative genetic models to predict the genotypes corresponding to all possible genotypes. These predictions are converted via computer modelling into an increasingly realistic three-dimensional representation of the growing plant useful for a simulation of plant evolution.

Plants of Molinia caerulea were grown in sand culture for two seasons. All nitrogen (N) supplied to the plants was enriched with 15N throughout the first season, and at natural isotopic abundance throughout the second season. A series of destructive harvests was taken during the second season. At each harvest, the N mobilized from roots and swollen basal internodes and the protease activity (ability to degrade azocasein) of these storage tissues were measured. The pH response curves of protease activity of both basal internodes and roots exhibited optima at pH 5 throughout the season. The protease activity of roots and basal internodes increased in spring, both on a unit fresh weight and unit protein basis, concomitant with mobilization of N from both these tissues to new shoot growth. In absolute terms more N was mobilized from roots than from basal internodes. However, basal internodes which, compared with roots, showed the greatest protease activity (on a f. wt or unit protein basis) were also the tissues that mobilized a greater proportion of N present in the tissue over winter to new shoot material. Individual clones of M. caerulea varied both in the amount of N mobilized from roots and in root protease activity (per plant). Individuals with a higher protease activity mobilized more N compared with individuals of lower protease activity. Therefore in M. caerulea, relationships between N mobilization and protease activity exist at several levels: (1) between different tissues; (2) temporally throughout the season; and (3) between individual clones.

The influence of variation in the concentration of dissolved inorganic carbon (DIC) in the form of CO2 and HCO3− in the root media on the C and N metabolism of Lycopersicon esculentum cv. F144 was investigated under both saline and non-saline conditions. Tomato seedlings were grown in hydroponic culture (pH 6.5) with or without NaCl, and the root solution was aerated with either ambient CO2 (360 μmol mol−1) or CO2-enriched air (5000 μmol mol−1). Nitrate uptake and root tissue NO3− concentrations were increased slightly by elevated rhizosphere DIC concentrations in both control and salinity-treated plants. This was associated with 46% higher nitrate reductase activity in the roots of control plants supplied with elevated DIC than in those supplied with ambient DIC. The activity of phosphoenolpyruvate carboxylase (PEPc) in vitro in control and salinity-treated plants was unaffected by the supply of elevated rhizosphere DIC concentrations. However, PEPc activity in vitro was considerably higher than the rates of PEPc activity in vivo reported previously, indicating that PEPc activity was not in itself a limitation on the provision of anaplerotic C. Therefore elevated DIC concentration in the rhizosphere stimulated the uptake of NO3− and provided alternative C skeletons for the assimilation of the NH4+ resulting from NO3− reduction into amino acids within the roots. Salinity stimulated root glutamine synthetase (GS) activity up to double that in control plants. Furthermore, elevated DIC caused an increase in leaf and root GS activity of control plants while inhibiting GS activity in the roots of salinity-treated plants. Glutamine[ratio ]2- oxoglutarate aminotransferase (GOGAT) activity of salinity-treated plants was doubled by elevated rhizosphere DIC concentrations. These changes in GS and GOGAT activity must reflect changes in amino acid synthesis. Under saline conditions the xylem transport of NO3− is partly blocked and a larger root assimilation develops, requiring not only the transamination of 2-oxoglutarate to glutamate but also that of oxaloacetate to aspartate and the transamidation of aspartate to asparagine.

Eupatorium makinoi plants with or without geminivirus infection were grown in shading frames with 70, 15 and 5.5% sunlight. Growth characteristics of these plants in the early vegetative phase were compared by means of growth analysis. We also measured leaf photosynthetic gas exchange rates and examined relationships between leaf photosynthesis and whole-plant growth. Relative growth rate (RGR=(1/W)×(dW/dt), where W is plant dry mass) of virus-infected plants was lower than that of uninfected plants under all three light conditions. The reduction of RGR by infection was increased with irradiance. The net assimilation rate (NAR=(1/A)×(dW/dt), where A is total leaf area of the plant) was also reduced both by infection and shading. NARs that were estimated from light-response curves of leaf photosynthesis, in situ measurements of irradiance, and respiration rates of leaves, stems and below-ground parts, agreed very well with the values obtained by conventional growth analysis techniques. Decreases in the estimated NAR value from infection and shading were mostly explained by the decreases in leaf photosynthesis. These results clearly showed that lowered RGR in virus-infected plants was attributed mainly to impaired photosynthesis in virus-infected leaves.

Extracellular matrices associated with conidia and germ tubes of Botrytis fabae (Sard.) sporelings grown on Vicia faba L. leaves were clearly visualized by epi-fluorescence microscopy following immunolabelling with the monoclonal antibodies, BC-KH4 and BC-FD7-G9. These antibodies were raised against surface washings of B. cinerea, are directed against B. cinerea and B. fabae, and are known to recognize carbohydrate epitopes on a glycoprotein. Both BC-KH4 and BC-FD7-G9 also labelled matrix material located at the surface of penetration and infection hyphae inside the leaf tissue by epi-fluorescence microscopy. Such matrix material was not visible by DIC microscopy.

Immunoelectron microscopy of B. fabae-infected leaf tissue, prepared by progressive low-temperature dehydration and embedding in acrylic resin, allowed further investigation of the spatial distribution of the antibody-binding sites. An abundance of BC-KH4 and BC-FD7-G9 antigenic sites were observed throughout the fibrillar-like matrix material surrounding the germ tubes on the leaf surface and the infection hyphae inside the host cells. However, close examination of the V. faba–B. fabae interface inside the host tissue showed that this fibrillar material extended some distance from the surface of the infection hyphae and through the swollen epidermal and mesophyll cell walls. Such fibrillar matrix material is thought to be of fungal origin. The possible role(s) of this matrix material in the infection process are discussed.

Double-immunolabelling studies using the BC-KH4 MAb and a polyclonal antiserum directed against oligosaccharides containing β-(1→3)-glucose were carried out in order to localize and distinguish between the fungal extracellular matrix material and translucent cell wall respectively. This technique allowed a closer examination of the interactions of the fungal matrix components with the host walls and degenerate host cytoplasm. Finally, inward curling of the leaf cuticle suggested that mechanical pressure is involved in the penetration process.

To study the role of fructan in leaf development of barley seedlings, fructan contents and the activities of enzymes involved in fructan metabolism were measured in segments of the third leaf at the stage of maximal growth. Fructans amounted to more than 10% of the d. wt in the growth zone at the base of the developing leaf and decreased sharply in the zone above. The specific activities of sucrose[ratio ]sucrose 1-fructosyltransferase (1-SST) and sucrose[ratio ]fructan 6-fructosyltransferase (6-SFT), two key enzymes in fructan synthesis, also peaked in the growth zone but decreased more slowly in the zone above. By contrast, the specific activities of invertase and fructan exohydrolase (FEH) were low in the growth zone at the leaf base and had their peak just above it. The specific activity of fructan[ratio ]fructan 1-fructosyltransferase (1-FFT) was approx. constant over the whole leaf. These results indicate that transitory accumulation of fructans in the growth zone plays a key role in the development of barley leaves.

This study compares the effects of the fungicide Aliette® (fosetyl-Al) on non-mycorrhizal and mycorrhizal onion (Allium cepa L.) with effects of the degradation products, aluminium (applied as aluminium chloride) and phosphonate (applied as dimethyl phosphonate). We sought to determine the extent to which the plants absorb and accumulate phosphonate and ascertain why, as shown previously, toxic effects of fosetyl-Al on mycorrhizal plants are less severe than effects on equivalent non-mycorrhizal plants.

Fosetyl-Al markedly reduced growth, especially of roots, and also inhibited mycorrhizal colonization. Dimethyl phosphonate caused smaller effects on growth and did not decrease colonization. Aluminium chloride did not affect growth of non-mycorrhizal or mycorrhizal plants, or mycorrhizal colonization. In all cases, mycorrhizal plants grew better than equivalent non-mycorrhizal plants (with no added soil phosphate), and non-mycorrhizal plants given supplementary soil phosphate grew best.

Concentrations and contents of total P in shoots were increased by dimethyl phosphonate and more so by fosetyl-Al. Concentrations of P were also increased in roots. 31P nuclear magnetic resonance (NMR) spectroscopy was used to determine relative concentrations of phosphonate and phosphate. Application of fosetyl-Al led to accumulation of more phosphonate than did application of dimethyl phosphonate, and non-mycorrhizal plants treated with fosetyl-Al accumulated much more phosphonate than did equivalent mycorrhizal plants (both with no added soil phosphate). Internal phosphate concentrations increased, especially in mycorrhizal plants, following application of both dimethyl phosphonate and fosetyl-Al. Non-mycorrhizal plants given supplementary soil phosphate also showed restricted growth when treated with fosetyl-Al even though they accumulated phosphate in relatively high amounts and had lower ratios of phosphonate to phosphate, particularly in shoots. In general, high internal concentrations of phosphonate were correlated with large reductions in plant growth among treatments.

Possible causes of these effects include conversion of phosphonate to phosphate in the tissues (especially in mycorrhizal plants), enhanced uptake of phosphate via mycorrhizal fungi and additional accumulation of P in plants with restricted growth. Competition for uptake between phosphate and phosphonate is also discussed.

Modern yew (Taxus baccata) is demonstrated to have considerable potential for dendrochronological use. A replicated 303 yr chronology has been formed from Hampton Court Palace yew and cross-matches well with a number of different tree species, including oak. Growth is positively correlated with rainfall but inversely related to late summer temperature. Range limits and present and prehistoric sources of yew are also briefly discussed, highlighting the future potential of yew to become the third major tree species for British dendrochronological study.

Responses of gas exchange and photosynthesis to changes in CO2 concentration and PPFD were examined in well watered plants of Delosperma tradescantioides Bgr. to establish the relative importance of these environmental changes on the photosynthetic machinery in this CAM-cycling species which grows naturally in both exposed and partly shaded environments. Plants were grown at two PPFDs (220 [LL] and 550 [HL] μmol m−2 s−1). HL plants had larger leaves with higher specific weight, water content and diurnal malic acid fluctuation. Photosynthetic PPFD responses were typically those of sun and shade species for HL and LL plants, both under 21% O2 and non-photorespiratory (2% O2) conditions. The CO2 compensation point in the absence of non-photorespirational CO2 evolution in the light (Γ∗) was c. 30 μmol mol−1. Irradiation reduced mitochondrial respiration by >50%. Comparison of the PPFD responses of linear electron flow rates derived from gas exchange measurements and from fluorescence analysis ([Jscr ]F) indicated effective photosynthetic control. [Jscr ]F was always larger than electron flow rates calculated from gas exchange, indicating that processes other than carboxylation and oxygenation were consistently important in energy consumption under all sampled environmental conditions. Regardless of PPFD during growth, electron flow to carboxylation and [Jscr ]F were linearly correlated, demonstrating that the photosynthetic apparatus was well adapted to PPFD during growth. In HL plants, non-photochemical quenching increased, and photochemical quenching and the quantum yield of linear electron transport through PS II decreased more slowly with increasing PPFD than in LL plants. In plants of both treatments non-photochemical energy dissipation seemed to be exhausted when the proportion of photons not utilizable by photochemistry exceeded 0·7. Results illustrate a pronounced ability of D. tradescantioides to acclimate to a 100% change in the prevailing PPFD and lend support to the hypothesis that CAM cycling might act as a photoprotective process.

Many human activities, such as ore mining and smeltering, sewage sludge treatment and fossil fuel consumption, result in toxic soil concentrations of ‘heavy metals’ (Al, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Ti, Zn and others) (Gadd, 1993). There are also natural soils, such as serpentine, with levels of heavy metals that inhibit or preclude the growth of many plants and soil micro-organisms. However, certain plants and microorganisms do grow in these metalliferous sites. Understanding the physiology, ecology and evolution of tolerance to elevated soil metal concentrations is important in an applied setting, and is also of interest in theoretical biology. Applied importance relates to the improvement of forest health in areas subject to increasing pollution, rehabilitation of severely polluted sites by phytostabilization of metals, and metal removal using hyperaccumulating plants (Krämer, 2000; Ernst, 2000). Areas of theoretical interest include the evolution of local adaptation (Sork et al., 1993) and how it is shaped by the combined influences of natural selection, gene flow and genetic architecture, as well as metal influences on various species interactions (Pollard, 2000). A paper appears on pages 367–379 in this issue by Jan Colpaert and coworkers which adroitly combines the disparate fields of physiology, genetics and ecology to answer several outstanding questions concerning heavy metal tolerance in mycorrhizal fungi.

Mycorrhizal fungi, which interact mutualistically with the majority of plant species, are well known for improving the P status of their hosts (Smith & Read, 1997). Some mycorrhizal fungi are also able to mobilize N and P from organic substrates and to provide plants with improved micronutrient and water acquisition, pathogen resistance, and a variety of other benefits (Smith & Read, 1997). One of these additional benefits is the amelioration of toxicity in metalliferous soils.

The photobiont ultrastructure of the epiphytic lichens Bryoria fuscescens and Bryoria fremontii was studied along the pollution gradient from two Cu-Ni smelters in Nikel and Monchegorsk in northern Finland and north-western Russia. The relationship between ultrastructural characteristics of B. fuscescens and environmental factors (i.e. climate, atmospheric SO2 and bark element concentrations) was studied by using a principal component analysis (PCA) aiming to assess the air quality in a northern environment. Based on PCA, increased plasmolysis and mitochondrial changes in the Trebouxia photobiont were significantly correlated with elevated pollutant concentrations. Degenerated cells, showing altered chloroplasts and electron-translucent pyrenoglobuli, occurred in lichens growing 35–50 km from the Monchegorsk smelter. Cell wall and cytoplasmic lipid volumes, and size of pyrenoglobuli, positively correlated with the distance from the Monchegorsk smelter. Vacuoles and electron-opaque vacuolar deposits were significantly increased at the Finnish site in the vicinity of a pulp mill. Swelling of mitochondrial cristae and thylakoids showed little correlation with environmental factors, but indicated of initial stage of injuries and were observed at several slightly polluted sites in northern Finland and north-western Russia. The results suggest that the severe photobiont injuries of lichens are strongly associated with poor air quality.

Canopies of heterophyllous trees expand by production of long shoots. We have previously shown in mountain birch (Betula pubescens ssp. czerepanovii) that damage to internode leaves within long shoots does not impede shoot growth, indicating that long-shoot elongation occurs by means of external resources. To study to what extent leaves other than true long-shoot leaves are necessary for the normal growth of mountain birch long shoots, we simulated herbivore damage to the two basal leaves of shoots (which flush simultaneously with short-shoot leaves) and the short-shoot leaves nearest to the long shoot within the branch. Damage to the two basal long-shoot leaves significantly reduced long-shoot growth. Additional damage to short-shoot leaves, situated proximally to the long shoot, did not retard long-shoot growth any more than damage to basal leaves alone. To determine the extent to which short-shoot leaves within a large branch are responsible for the pooled long-shoot production of the branch, we clipped differing proportions of short-shoot leaves from such branches. We found small but significant reduction in the pooled length of the long shoots of the branch, presumably indicating a limited role in long-shoot elongation of current photosynthates within the branch. Our experiments indicate that long shoots are not independent modular units in their carbon economy.

Wheat stems serve as a store for fructans to buffer the plant against nutritional and environmental influences. It has been suggested that fructan storage influences yield stability and tolerance of environmental factors. Near infra-red spectroscopy (NIR) analysis provides a rapid and accurate assessment of the fructan content of the wheat stem, as well as allowing detection of growth-limiting nutrient stresses, and so is proving to be a useful technique for making crop management decisions. Commercial laboratories using NIR analysis have been tissue-testing crops in the eastern Australian wheat belt since 1993. In healthy, normally developing crops not under stress there is a predictable relationship between nitrogen and fructan. Investigation of the nitrogen and fructan concentrations in commercial crops has confirmed an inverse relationship between these two constituents. The function: Fructan (%)=a+bN%+cN%2 accounted for up to 81% of variation in tissue fructan concentration. In commercial tissue-testing this relationship is used to detect crops under stresses other than nitrogen deficiency. If the fructan concentration deviates by more than 4%, cereal growers are advised that their crop might be subject to other stresses which might reduce its response to applied nitrogen.

Beech nuts (Fagus sylvatica L.) were germinated and grown in soil inoculated with the ectomycorrhizal fungus Laccaria laccata or Paxillus involutus for 18–20 wk. The success of mycorrhizal infection was monitored by measuring the ergosterol contents of the mycorrhizas. Ergosterol levels ranged from 122±23 μg g−1 d. wt (Laccaria mycorrhizas) to 94±36 μg g−1 d. wt (Paxillus mycorrhizas), indicating that ectomycorrhizal symbiosis was established. In root incubation chambers, rates of sulphate uptake and the xylem loading of sulphate of excised mycorrhizas were investigated. Both types of mycorrhizas showed saturation kinetics in external sulphate concentrations from 2·5–1000 μmol l−1. Linearization of these kinetics revealed two phases with low apparent Km (Laccaria mycorrhizas: 15±3 μmol l−1; Paxillus mycorrhizas: 13±3 μmol l−1) and Vmax (Laccaria mycorrhizas: 19±3 nmol h−1 g−1 f. wt; Paxillus mycorrhizas: 25±4 nmol h−1 g−1 f. wt) at low external sulphate concentrations and significantly higher kinetic constants at higher sulphate supplies. Relative xylem loading, i.e. the portion of sulphate loaded into the xylem that was taken up, remained constant over the entire concentration range investigated (c. 4–7% of the sulphate taken up). If trees were supplied for 72 h with different N and sulphur concentrations, both uptake of sulphate and relative xylem loading were unaffected by sulphur availability, but modulated by N supply. Nitrogen depletion diminished the rates of sulphate uptake in Laccaria and Paxillus mycorrhizas. In response to higher N availability combined with sulphur depletion, sulphate uptake of Laccaria mycorrhizas, but not of Paxillus mycorrhizas, increased. Organic compounds considered to be possible signals for the regulation of sulphate uptake were fed to excised mycorrhizas. l-Cysteine but not l-methionine and glutathione (γ-Glu-Cys-Gly) inhibited sulphate uptake of the two mycorrhizas and xylem loading of sulphate was stimulated rather than inhibited by l-Cys in both types. In Paxillus mycorrhizas glutathione had a similar effect. O-Acetyl-l-serine (OAS), a precursor of l-cysteine, stimulated sulphate uptake, but did not affect xylem loading. Apparently, OAS, generated in N metabolism, and l-cysteine, a product of assimilatory sulphate reduction, act as antagonists, together mediating regulation of sulphate uptake.

Immunoelectron microscopy using the anti-pectin monoclonal antibody JIM 7 confirmed earlier observations that pectin degradation is a primary event in the process of host cell wall breakdown during the development of chocolate spot disease (causal agent: Botrytis fabae (Sard.)) of broad bean. Close examination of infected and non-infected Vicia faba L. leaves indicated a loss of JIM 7-labelling, and therefore, methyl-esterified pectin, from swollen walls of infected and contiguous epidermal cells. Modified mesophyll walls also possessed less methyl-esterified pectin than healthy walls. Enzymes which attack methyl-esterified pectin appeared to be most active in regions of host tissue close to sites of fungal infection.

Ultrastructural studies using the enzyme, cellobiohydrolase conjugated to gold (CBH1-Au) revealed that the cellulose microfibrils of outer epidermal walls of non-infected V. faba leaf tissue were heavily masked by other components of the plant cell wall. Such material was most probably pectin because the cellulose microfibrils of swollen epidermal and modified mesophyll walls of infected host tissue were heavily labelled with CBH1-Au. These results were confirmed by double-labelling studies using JIM 7 and CBH1-Au. At early stages of the infection process, limited cellulose degradation was observed in infected leaf tissue.

Double-labelling experiments using the monoclonal antibody BC-KH4 directed against Botrytis matrices and a marker for the plant cell wall (JIM 7 or CBH1-Au) confirmed previous observations that the fungal matrices extended through modified host walls and degenerate cytoplasm. It is suggested that the wall-modifying action of the pectin-degrading enzymes produced during the infection process might facilitate pervasion of matrix material associated with the infection hyphae through host cell walls. Possible role(s) of such matrix material during the post-penetration processes of the V. faba–B. fabae relationship are discussed.