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.

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.

Aspects of the structure and ultrastructure of the fusiform cambial cells of the taproot of Aesculus hippocastanum L. (horse chestnut) are described in relation to the seasonal cycle of cambial activity and dormancy. Particular attention is directed at cell walls and the microtubule and microfilament components of the cytoskeleton, using a range of cytochemical and immunolocalization techniques at the optical and electron-microscopical levels. During the dormant phase, cambial cell walls are thick and multi-layered, the cells possess a helical array of cortical microtubules, and microfilament bundles are oriented axially. In the early stages of reactivation, vesicle-like profiles are associated with the cell walls, whereas arrangement of the cytoskeletal elements remains unchanged. In the succeeding active phase, the cell walls are thin, and cortical microtubules form a random array, although microfilament bundles maintain a near-axial orientation. The observations are discussed in relation to the seasonal cycle of wall structure and cortical microtubule rearrangement within the vascular cambium of hardwood trees. It is suggested that the cell-wall thickening at the onset of cambial dormancy, which is associated with the presence of a helical cortical microtubule array, should be considered to be secondary wall thickening, and that selective lysis of this secondary wall layer during cambial reactivation restores the thinner, primary wall found around active cambial cells.

Isolates of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf et Kernan, and the ectomycorrhizal fungi Suillus variegatus (Swartz ex Fr.) and Pisolithus tinctorius (Pers.) Coker & Couch, along with a Cortinarius sp. and the white rot Phanerochaete chrysosporium Burdsall were examined for the ability to oxidize carbohydrates to their corresponding lactones and to excrete the H2O2 produced thereby. All except Phanerochaete chrysosporium were found to express cellobiose oxidase (cellobiose dehydrogenase, EC 1.1.19.88) and glucose oxidase (β-d-glucose[ratio ]oxygen 1-oxidoreductase, EC 1.1.3.4) when grown on cellobiose and glucose respectively. Production of extracellular H2O2 was visualized during growth on both substrates using ABTS as the chromogen. According to the Fenton reaction, H2O2 will react with hydrated or chelated Fe(II) in the environment to produce hydroxyl (Fenton) radicals, HO·. Mycelial extracts from each of the mycorrhizal fungi produced HO· in the presence of cellobiose and Fe(II), presumably mediated by H2O2 produced by cellobiose oxidase activity in the extracts. Conditions favourable to HO· production were shown to exist in Modified Melin–Norkrans medium, and the data discussed in relation to previously observed lignin degradation by mycorrhizal fungi.

Low phosphorus availability is often a primary constraint to plant productivity in native soils. Here we test the hypothesis that root carbon costs are a primary limitation to plant growth in low P soils by assessing the effect of P availability and mycorrhizal infection on whole plant C budgets in common bean (Phaseolus vulgaris L.). Plants were grown in solid-phase-buffered silica sand providing a constant supply of low (1 μm) or moderate (10 μm) P. Carbon budgets were determined weekly during the vegetative growth phase. Mycorrhizal infection in low-P plants increased the root specific P absorption rate, but a concurrent increase in root respiration consumed the increased net C gain resulting from greater P uptake. The energy content of mycorrhizal and non-mycorrhizal roots was similar. We propose that the increase in root respiration in mycorrhizal roots was mainly due to increased maintenance and growth respiration of the fungal tissue. Plants grown with low P availability expended a significantly larger fraction of their total daily C budget on below-ground respiration at days 21, 28 and 35 after planting (29–40%) compared with plants grown with moderate P supply (18–25%). Relatively greater below-ground respiration in low P plants was mainly a result of their increased root[ratio ]shoot ratio, although specific assimilation rate was reduced significantly at days 21 and 28 after planting. Specific root respiration was reduced over time by low P availability, by up to 40%. This reduction in specific root respiration was due to a reduction in ion uptake respiration and growth respiration, whereas maintenance respiration was increased in low-P plants. Our results support the hypothesis that root C costs are a primary limitation to plant growth in low-P soils.

Brassica rapa L. (rapid-cycling Brassica), was grown in environmentally controlled chambers to determine the interactive effects of ozone (O3) and increased root temperature (RT) on biomass, reproductive output, and photosynthesis. Plants were grown with or without an average treatment of 63 ppb O3. RT treatments were 13°C (LRT) and 18°C (HRT). Air temperatures were 25°C/15°C day/night for all RT treatments.

Ozone affected plant biomass more than did root temperature. Plants in O3 had significantly smaller total plant d. wt, shoot weight, leaf weight, leaf area and leaf number than plants grown without O3. LRT plants tended to have slightly smaller total plant d. wt, shoot weight, root weight, leaf weight, leaf area, and leaf number than HRT plants. For all variables, LRT plants grown in O3 had the smallest biomass, and plants grown in HRT without O3 had the largest biomass.

Ozone reduced both fruit weight and fruit number; LRT also reduced fruit weight but had no effect on fruit number. Ozone reduced photosynthesis but RT had no effect. Conductance and internal CO2 were unaffected by O3 or RT.

These studies indicate that plant growth with LRT might be more reduced in the presence of O3 than growth in plants with HRT, which might be able to compensate for O3-caused reductions in photosynthesis to avoid decreased biomass and reproductive output.

Measurements were made of the rates of nitrogenase activity (acetylene reduction) by cyanobacteria collected from the Baltic Sea at 2-h intervals, over a period of 24 h, and incubated under natural light. By relating the chlorophyll-specific rate of N2 fixation (PN) to the mean photon irradiance (I) at different periods, a PN/I curve was constructed. A mathematical description of this relationship was used in the calculation of rates of N2 fixation at different depths and times of day from continuous measurements of surface irradiance and light attenuation. By relating these calculations to the vertical distribution of chlorophyll due to Aphanizomenon flos-aquae, an estimate of the daily integral of N2 fixation by the population of this cyanobacterium was obtained: it varied from 0·39 to 0·71 mmol m−2 with a mean value of 0·53 mmol−2 over 9 d. Comparisons with similar calculations of the daily integral of photosynthesis over the same period indicated the atomic ratio of N/C fixed is about 0·22, not very different from the N/C ratio expected for the elemental composition of these cyanobacteria. It is demonstrated that when buoyant gas-vacuolate colonies float up during calm periods the increased irradiance that they experience supports an increased rate of N2 fixation. This increase is less than that seen for carbon fixation because photosynthesis is negated by respiration at low irradiances and is less inhibited at high irradiances.

The effects of full-spectrum solar radiation and of solar radiation deprived of total u.v. radiation (λ<395 nm) on the endemic Mediterranean red alga Rissoella verruculosa (Bertoloni) J. Agardh were studied in situ in early summer, from sunrise to sunset. Photosynthetic performance, pigment content and the activities of enzymes related to nutrient uptake, were monitored under both radiation conditions throughout a daylight period.

The doses of solar radiation, measured on the day during which the experiments were carried out (24 June 1997), were 9228·25, 1109·70 and 13·03 kJ m−2 for PAR (λ=400–700 nm), u.v.-A (λ=315–400 nm) and u.v.-B (λ=280–315 nm), respectively. Under these conditions, a clear daily variation in photosynthetic performance was found. However, no significant differences were detected between the two radiation conditions. At noon, strong sunlight impaired O2 evolution by 75%, but complete recovery occurred during the afternoon, reaching similar values to those measured in the early morning. By contrast, photoinhibition of optimal quantum yield (Fv/Fm), and the relative electron transport rate was followed by only a slight recovery during the afternoon. The rate of photosynthesis in air (simulating emersion conditions), estimated by CO2 exchange, showed a negative balance at noon, which was accentuated in plants exposed to the full-solar spectrum.

Significant changes in the activities of nitrate reductase and carbonic anhydrase were found throughout the day under both radiation conditions. In thalli receiving solar radiation deprived of u.v., total CA activity decreased throughout the day. However, in thalli exposed to full-spectrum solar radiation, the activity of the enzyme tended to increase in the afternoon, correlating with an increase in NR activity.

Ascospore germination, thallus initiation, and areole and prothallus development in the lichen Rhizocarpon lecanorinum Anders were examined using light, fluorescence and scanning electron microscopy. The ascospore germ hyphae remain very short and do not form a prothallus-like mycelium. Instead, a compact soredium-like granule develops directly from sporeling contact with a compatible species of Trebouxia. Diffuse initial stages involving non-trebouxioid algae are lacking. The onset of thallus differentiation is marked by the deposition of rhizocarpic acid in an incipient cortical layer within the apical part of the granule. As pigmentation and cortex-formation transform this structure into a typical areole, radiating prothallus hyphae are simultaneously initiated from its basal margin. Most areoles formed subsequently in the marginal prothallus lack subtending melanized hyphae and apparently stem from overgrowth by the prothallus of photobiont cells on, or in, the substratum. Apothecia reach maturity in thalli as small as 2 mm in diameter. It is proposed that the lack of diffuse hyphal growth in sporelings and telescoped morphogenesis of R. lecanorinum are part of a life history strategy geared to precocious, heavy investment in ascospore production. The R. lecanorinum–Trebouxia symbiosis has a number of features which make it well-suited for further studies of the life history and development of prothallus-forming crustose lichens with sexually reproducing mycobionts.

Concentrations of NH4+, NO3−, PO43−, K+, Ca2+ and Mg2+ in snow meltwater resulting from summer snow showers were monitored before and after its passage through monospecific stands of the Antarctic macrolichen Usnea sphacelata R. Br. The sampling was conducted under field conditions near Casey Station in East Antarctica between January and March. Total snow deposition during the 61-d period was 44±1 mm (rainfall equivalent depth) delivering 362±10, 87±2 and 9±1 μmol m−2 of NH4+, NO3− and PO43−, respectively. Meltwater that had percolated through U. sphacelata was depleted in NH4+ and NO3− equating with a retention by the lichen of 87 and 92%, respectively, of the total wet deposition of these ions. Lichen-modified meltwater was slightly enriched in PO43−, but because the volume of the lichen percolate was smaller than that of the original snow deposition, the lichen achieved a net gain of 9% of the total P deposited. Lichen percolate was also enriched in metal cations. Potassium loss associated with the melting of the heaviest snowfall (18 mm) was equivalent to only 0·05% of the total K in the lichen suggesting that ion loss did not signal significant cellular damage. There was also a progressive increase in NH4+ concentration in unmodified meltwater from 3 to 21 nmol ml−1 over a 3-d period whereas levels in the lichen-modified meltwater remained unchanged at [les ]4 nmol ml−1. This enrichment in NH4+ might have resulted from dry deposition onto the melting snow pack of NH3 emitted from nearby penguin rookeries. During the study, tagged thalli of U. sphacelata made a 2% loss in dry mass although they appeared healthy and, at the end of the study, showed an effective quantum yield (ΔF/Fm′) comparable with field material. The results are discussed in relation to the time of year that is likely to be most suitable for lichen growth in this continental Antarctic environment and the potential growth-led demand for N in U. sphacelata.

Axenic cultures of lichen photobionts isolated from bark-inhabiting lichen thalli of the Physcietum adscendentis Ochsner were identified by light microscopy and sequence comparisons of internal transcribed spacer rDNAs to investigate principles of lichenization within a defined lichen sociological unit. The photobiont identity of eight lichen species is reported for the first time (photobiont species in square brackets): Lecania cyrtella (Ach.) Th. Fr. [Trebouxia arboricola Puym.], Lecania naegelii (Hepp) Diederich & v. d. Boom [Dictyochloropsis symbiontica Tscherm.-Woess], Candelaria concolor (Dicks) B. Stein [Trebouxia jamesii (Hildreth & Ahmadjian) Gärtner], Candelariella cf. reflexa (Nyl.) Lettau [T. jamesii], Lecanora spec. [T. arboricola], Phaeophyscia orbicularis (Neck.) Moberg [T. impressa Ahmadjian], Physcia adscendens (Fr.) H. Olivier [T. impressa] and Lecidella elaeochroma (Ach.) M. Choisy [T. arboricola] and could be confirmed for another two species, Physcia stellaris (L.) Nyl. [Trebouxia impressa] and Xanthoria parietina (L.) Th. Fr. [Trebouxia arboricola]. The observation that pioneer lichens without vegetative propagules, growing on smooth bark, had Trebouxia arboricola as photobiont can be explained by the assumption of a free-living population of Trebouxia arboricola. Species of photobionts from Xanthoria parietina were morphologically and genetically different from those of Physcia adscendens and Phaeophyscia orbicularis, respectively; a finding that does not support the previous assumption that Xanthoria parietina takes over its algal partner from a Physcia species, at least at the sites investigated.

Wood degradation by the white-rot basidiomycete Meripilus giganteus (Pers.: Pers.) Karst. was studied in naturally infected and artificially inoculated wood of beech (Fagus sylvatica L.) and large-leaved lime (Tilia platyphyllos Scop.). Semi-thin sections revealed that the secondary walls of most fibres contained internal cavities. Three distinct types of cavity formation, which differed not only between hosts, but also between cell type and location in the annual ring, were identified.

Within discoloured wood of naturally infected beech, the structure of the cavities and their formation by the associated hyphae were reminiscent of a soft-rot. By contrast, cavity formation in artificially inoculated beech and large-leaved lime wood differed from a soft-rot mode of attack as extensive delignification always preceded cavity formation, and neither T-branching, L-bending, nor hyphal growth were found within cell walls. The formation of half-moon shaped cavities in beech wood was present only in tension-wood fibres. From large diameter hyphae, growing within the fibre lumen, numerous fine perforation hyphae extended transversely via helical cracks into the cell wall. Subsequent degradation of cellulose within concentric layers of the tension-wood fibres commenced from the apices of perforation hyphae.

Sections stained with ruthenium red and hydroxlamine-ferric chloride, revealed that M. giganteus preferentially degrades pectin-rich regions of the middle lamellae in xylary ray cells. In large-leaved lime, such regions were uniformly located in the middle lamellae of axial and ray parenchyma. In beech wood, degradation of pectin-rich middle lamellae regions commenced after the delignification of secondary walls and resulted in a conspicuous hollowing of multiseriate xylem rays. Plasticity in wood degradation modes by M. giganteus in large-leaved lime and beech wood reflects variations in cell wall structure and/or prevailing wood conditions.

We examined the roles that seasonal shifts in precipitation and temperature played in the ectomycorrhizal (ECM) colonization of pinyon pine (Pinus edulis Engelm.) at two contrasting sites in northern Arizona. Pinyons growing in ash and cinder soils experienced much greater water and nutrient stress than pinyons growing nearby in sandy-loam soils. Over a one year period, we obtained monthly measurements of ECM colonization, root zone soil moisture and temperature, and air temperature and precipitation. Four major patterns emerged. Firstly, although climate as measured by ambient temperature and precipitation did not vary between the two sites, soil temperature was significantly higher and soil moisture significantly lower at the cinder site than at the sandy-loam site. Secondly, ECM colonization was significantly higher at the cinder site for 5 of 12 months. Thirdly, although nearly 70% of the variation in ECM colonization of pinyons growing in cinder soil was predicted by a combination of soil moisture and soil temperature, these same variables had little predictive power for pinyons growing in sandy-loam soils. Air temperature and precipitation were also significantly correlated with ECM colonization at the cinder site but not the sandy-loam site. Fourthly, a watering experiment showed that ECM colonization significantly increased with supplemental water at the cinder site, but not at the sandy-loam site. Thus, in two sites that did not differ in plant community or climate, ectomycorrhizas in cinder soils were far more sensitive to changes in moisture and temperature than ectomycorrhizas in sandy-loam soils.

Two tetraploid subspecies of Dactylis glomerata L., subsp. reichenbachii (Hausm.) Stebbins et Zohary and subsp. glomerata, occur in the French Alps. The former is confined to dolomitic, south-facing, alpine lawns above 2000 m, whereas the latter occurs in non-dolomitic habitats in subalpine meadows mainly below 1900 m. Previous studies of allozyme variation have shown that genetic introgression between the two subspecies occurs over large areas. By contrast, morphologically intermediate individuals only occur in an extremely narrow area, suggesting that the morphological and physiological differences between the two subspecies is of adaptive significance. A reciprocal clone transplant experiment was set up to examine (1) any genetic differences between subspecies indicative of ecotypic differentiation in relation to habitat characteristics and (2) the level of phenotypic plasticity in the two subspecies. Genetic differentiation was confirmed by a statistically significant taxon × site interaction effect in anova for all traits studied. The glomerata populations produced more tillers, longer leaves and higher culms in all sites, especially in their home environment. However, reichenbachii populations produced more seeds than the glomerata populations in the original reichenbachii environment, suggesting ecotypic differentiation between the two subspecies. This result might also explain why the glomerata subspecies is unable to colonize dolomitic habitats occupied by the reichenbachii subspecies. The reichenbachii populations showed less plasticity than the glomerata populations for leaf length and floriferous tiller number, a result which is discussed in the context of the response of plants from productive and non-productive habitats to environmental variation.

Genetic variation within and between vegetative compatibility groups (VCGs) of Amylostereum areolatum (Fr.) Boid. and Amylostereum chailletii (Pers.: Fr.) Boid. isolates was investigated. DNA fingerprints were made using the M13 core sequence as a primer. A total of 53 isolates of A. areolatum and 57 isolates of A. chailletii from Lithuania, Sweden, Denmark and Great Britain were studied. In all cases isolates belonging to the same VCG showed identical DNA banding patterns, suggesting that VCGs correspond to clones. In A. areolatum the vast majority of the isolates (spread by Sirex juvencus L.) were assigned to dispersive clones, that have wide geographical distribution (i.e. the same genotypes were detected in Lithuania, Sweden and Denmark), with low genetic variation between the different clones. By contrast, A. chailletii population structure was consistent with the spread of airborne basidiospores produced by outcrossing. Only a small fraction of A. chailletii isolates studied, could be assigned to dispersal clones with a local distribution, spread by Urocerus gigas L. Overall, M13 fingerprinting detected low genetic differentiation in both species in the samples we studied.