The treatment of wheat roots with 2,4-dichlorophenoxyacetic acid (2,4-D) led to the formation of tumour-like structures, para-nodules, which were readily colonized by cyanobacteria. The amount of cyanobacteria found on 2,4-D treated roots was 3.6 times higher than that found on untreated roots. Cyanobacteria penetrated the para- nodules by migrating in between loosely arranged cells that covered their surfaces or by penetrating the space at the junction of root and para-nodule. In plants treated with 2,4-D and co-cultivated with cyanobacteria in medium without combined nitrogen, the rate of acetylene reduction was three times that seen in untreated but colonized roots. Addition of 2,4-D itself did not change the rate of acetylene reduction in free-living culture of Nostoc sp. strain 2S9B. In plants treated with 2,4-D and co-cultivated with Nostoc sp. strain 2S9B in medium without combined nitrogen the nitrogen content of roots but not shoots was significantly increased.

The presence and variation of activity of the type 1 ribosome-inactivating protein saporin has been evaluated in cultured roots of the soapwort Saponaria officinalis. Results from western analysis and in vitro protein synthesis inhibition indicate that saporin production is increased in senescing cultures, reaching a maximum value during the late stationary phase. Accordingly, cultures treated with the senescence-related hormone abscisic acid show a significant increase in saporin activity, independently from the culture growth phase. Stress conditions, such as the presence of hydrogen peroxide in the culture medium, had no effect on the modulation of enzymatic activity. The putative regulation of saporin production by abscisic acid and its possible role in accomplishing the ageing programme is discussed.

Uptake of the lipophilic cation, tetraphenylphosphonium (TPP+), which is often used as a membrane potential probe, was studied in the cyanobacterium Synechococcus R-2 (PCC 7942) to determine if the technique was valid for these cells. Lipophilic cation probes are notorious for giving inconsistent estimates of membrane potential (ΔΨ) using the Nernst equation (ETPP+=ΔΨ). It is necessary to correct for the large amounts of TPP+ bound to the surface of the cells. The rate of equilibration of intracellular TPP+ is very fast (t½≈1–3 min), hence the permeability of the plasmalemma to TPP+ (PTPP+≈10–50 nm s−1) is very high. There appears to be an active uptake of TPP+ with a pH optimum at about pHo 7.5. TPP+ does not readily uncouple Synechococcus cells. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and dinitrophenol depolarized ETPP+, but monensin made ETPP+ more negative. Accumulation of TPP+ is not affected by [K+] in the medium in a way consistent with it acting as a reporter of the membrane potential. Addition of K+, NH4+ and HCO3− altered the uptake rate of TPP+ in a way consistent with electrogenic uptake of these ions but the effects were smaller than those found using the 86Rb+/valinomycin and 201TI+ probes. A comparison has been made with ΔΨ determinations using other membrane potential probes. ETPP+ was often close to the value of the membrane potential obtained using other methods under control conditions but effects of experimental treatments are sometimes inconsistent with those found using other ΔΨ probes. The TPP+ method is therefore unreliable and could be seriously misleading.

This paper reports general patterns of relative growth rate and related traits in response to elevated atmospheric CO2 in eight woody species ranging widely in life form, leaf habit, taxonomy and ecology. Young plants of these species, all of comparable ontogenetic phases, were grown simultaneously in large containers with favourable nutrient and water availability in transparent outdoor chambers at 350 and 700 μl l−1 CO2 for one growing season. We found the following consistent responses. (1) All species grew faster at elevated CO2, whereas the following leaf and allocation traits were consistently lower in CO2-enriched environments: specific leaf area (quotient of leaf area and leaf weight), leaf area ratio (quotient of total leaf area and plant weight), weight-based foliar N concentration and, to a smaller extent, leaf weight fraction (quotient of leaf weight and plant weight). (2) There was important interspecific variation in the magnitude of the response of relative growth rate to CO2. Specific leaf area at ambient CO2 explained 88% of the variation in relative growth rate response to CO2 among the eight species. At ambient CO2, relative growth rate itself, was significantly correlated with the relative growth rate response to CO2 only if the leafless species Ulex gallii was excluded from analysis. (3) The four deciduous species had a significantly stronger relative growth rate response to CO2 than the four evergreens. This corresponded with their generally higher specific leaf area. (4) Specific leaf area and leaf habit might be useful for scaling up exercises, as easy-to-measure substitutes for growth responses of (woody) vegetation to elevated CO2. However, the usefulness of such traits in this context needs to be tested in realistic, longer-term manipulative experiments in real ecosystems.

Ozone (O3)-sensitive and tolerant clones of aspen (Populus tremuloides) were exposed to O3 at 1.0, 1.7 and 3.0 times the concentration in ambient air at Ithaca, NY, USA (×1.0, ×1.7 and ×3.0) for 3 months. Visible foliar injury occurred within a few days of the initiation of treatments and developed continuously in ×1.7 and ×3.0 treatments. Leaf senescence began earlier in trees exposed to greater-than-ambient treatments. Gas-exchange variables were measured five times over the growing season. Net photosynthesis was progressively decreased and finally reduced by 40 and 70% on tolerant and sensitive clones, respectively, but stomatal conductance was not affected by the O3 treatments. As stomatal conductance (gs) values did not differ in the two types of aspen, we conclude that this is not the defense mechanism to O3 in aspen. At the end of the season, dry weight was reduced by up to 55 and 35% in sensitive and tolerant clones, respectively. Ozone effects were significant and linear on foliar injury, biomass and growth variables such as height, diameter of stem and total leaf area. The levels of two antioxidants, ascorbic acid and glutathione, were investigated to determine if a clonal difference occurred. Concentrations of the free-radical scavengers did not differ in the clones, nor did they seem to be induced by O3. A cumulative O3 index, SUM06 (the sum of the total μl l−1 of exposure when the O3 concentration exceeded 0.06 μl l−1), was highly correlated with most foliar injury variables and gas-exchange variables except gs in higher O3 treatments. The reduction of carbon gain due to O3 is discussed with regard to foliar damage and biomass decrease.

Regular (monthly) additions of NH4NO3 (4–12 g N m−2 yr−1) were made over a period of 8 yr (1989–98) to areas of moorland in North Wales dominated by the ericaceous shrub Calluna vulgaris. Results from the early stages of the experiment (1990–94) have shown marked and dose-related increases in shoot extension and canopy height in response to the nitrogen treatments, with significantly higher shoot nitrogen contents. The nitrogen-related stimulation in the growth of the C. vulgaris canopy over this period has resulted in large accumulations of litter on the high-nitrogen-treated plots (6.6 kg m−2 in plots treated with 12 g N m−2 yr−1, compared with 3.8 kg m−2 for the water controls). Litter nitrogen concentrations were also significantly increased at the higher rates of nitrogen addition, leading to a doubling of the total return of nitrogen to the litter layer over the experimental period. These changes in vegetation structure were associated with large reductions in the abundance of the bryophyte and lichen species normally present under the untreated canopy. Results since 1994, however, show little increase in shoot extension in response to the nitrogen treatments, with no clear dose response to increasing levels of addition. These findings are associated with a dose-related increase in the susceptibility of the nitrogen-treated areas of the C. vulgaris canopy to late winter injury, characterized as browning of the shoot tips in early to late spring. These results indicate that deleterious effects are now accumulating as a result of the long-term addition of nitrogen to these moorland plots.

A field and laboratory based bioassay has been developed to investigate the effects of the quantity and duration of simulated pollutant nitrogen (N) deposition on root-surface phosphomonoesterase (PME) activities in calcareous and acid grasslands. Seedlings of Plantago lanceolata were transplanted to a calcareous grassland and Agrostis capillaris seedlings were grown in microcosms containing soil from an acid grassland that had received either 7 yr (long-term) N additions or 18 months (short-term) N and phosphorus (P) additions. The bioassay revealed that short-term N treatments had little effect on the enzyme activity, whereas long-term N additions significantly increased PME activity within 7 d of transplanting into the field plots. Root-surface PME activity of A. capillaris was significantly reduced in soil that received additions of P. In the plots receiving long-term additions of N, a strong relationship was observed between extractable soil ammonium and root-surface PME activity. Soil ammonium concentrations accounted for 67% of the variation in PME activity of P. lanceolata in the calcareous grassland, and 86% of the variation in PME activity of A. capillaris in the acid grassland. These results provide evidence that N deposition may have considerable effects on the demand and turnover of P in ecosystems that are approaching or have reached N saturation.

The host range of the soilborne obligate biotroph, Plasmodiophora brassicae was investigated. Evidence is presented that infection by P. brassicae might occur in non-Brassica species, leading to the potential formation of resting spores. Structures resembling P. brassicae were found in the root cortex of Tropaeolum majus, Carica papaya, Reseda alba and Beta vulgaris as demonstrated by scanning electron microscopy. Inoculation of Brassica rapa roots with spores extracted from either T. majus or B. vulgaris roots which had been previously inoculated with P. brassicae led to development of clubroot in the roots of B. rapa. It was also shown that the development of the symptom might be correlated with glucosinolate content, although other host factors are implicated in the B. vulgaris interaction with P. brassicae. In the glucosinolate-containing non-Brassicas, T. majus and C. papaya, the concentrations of benzylglucosinolate increased markedly in roots inoculated with P. brassicae, compared with the controls. There were also increases in concentrations of benzylglucosinolate in leaves of T. majus after P. brassicae infection. However, in R. alba roots, the total glucosinolate content decreased after inoculation with P. brassicae compared with the controls. High root concentrations of 2-OH-2-phenylethylglucosinolate (glucobarbarin) compared with low root indole glucosinolates in this species might limit P. brassicae infection and development. The importance of our investigations in relation to cultivation of non-Brassica species on fields infested with P. brassicae is discussed.

Foliage construction cost (glucose requirement for formation of a unit foliar biomass, G, kg glu kg−1), chemical composition and morphology were examined along a light gradient across the canopies in five deciduous species, which ranked according to increasing shade-tolerance as Populus tremula <Fraxinus excelsior <Tilia cordata = Corylus avellana <Fagus sylvatica. Light conditions in the canopy were estimated by a hemispheric photographic technique, allowing ranking of sample locations according to long-term light input incident to the sampled leaves (relative irradiance). G and foliage carbon concentration increased with increasing relative irradiance in F. excelsior, T. cordata and C. avellana, but wereindependent of irradiance in F. sylvatica and P. tremula. However, if G of non-structural-carbohydrate-free dry mass was considered, it also increased with increasing relative irradiance in P. tremula. A positive correlation between the concentration of carbon-rich lignin and irradiance, probably a result of the acclimation to greater water stress at higher light, was the major reason for the light-dependence of G. Lignin concentrations were highest in more shade-tolerant species, resulting in greatest carbon concentrations in these species. Since carbon concentration and G are directly linked, the leaves of shade-tolerant species were also more expensive to construct. As the result of these effects, G increased faster with increasing leaf dry mass per area which was mainly determined by relative irradiance, in shade-tolerators. Given that shade-tolerant species had lower leaf dry mass per area at common irradiance and that this saturated at lower relative irradiance than leaf dry mass per area in the intolerant species, it was concluded that enhanced energy requirements for foliage construction might constrain species morphological plasticity and the upper limit of leaf dry mass per area attainable at high light.

For a number of generations, we followed the frequency, performance and fecundity of an initial mixture (50[ratio ]50) of triazine resistant (R) and susceptible (S) but otherwise near-isogenic lines of Brassica rapa (for 3 yr) and of Chenopodium album (for 2 yr) in neighbouring experimental garden plots, which differed in aspect (north-facing versus south-facing), shading, and transparent cover from precipitation. Each of eight treatments was replicated four times per species. Seed from each plot was kept separate and sown back in its plot of origin to provide the next generation. For both Brassica and Chenopodium, R frequency changed between generations. In Brassica R frequency declined consistently from an initial 0.5 and, after 2 yr, R plants had disappeared in all replicates in two treatments and averaged <0.126 in the remaining treatments. However, R frequencies in the smaller second generation samples in each year were less consistent. In Chenopodium, R frequency increased consistently from an initial 0.5 in all treatments and replicates during 1995 to average 0.772 on south-facing sites and 0.675 on north-facing sites. However, in 1996 Chenopodium R frequency did not change significantly from that in 1995 for any treatment. Brassica S plants were more fecund that R plants, except in shaded conditions, and south-facing plots produced significantly more S seeds than north-facing plots. Both Brassica plants and plots were more productive in 1996 than in 1995. By contrast, Chenopodium was much less productive in 1996, a season with more climatic extremes between plots than 1995. In 1995, R plants were more fecund than S plants in south-facing plots and in covered conditions, but this difference was not detected in 1996. South-facing Chenopodium plots were more productive than north-facing plots in 1995, but the reverse was the case in 1996. Cover resulted in an increase in seed production in Chenopodium on south-facing plots in 1995, but a decline in 1996. We conclude that selection might act against triazine-resistant plants in B. rapa under the climatic conditions in the UK, but in C. album, which is more susceptible to extreme conditions of light and water stress, triazine-resistant phenotypes might predominate in low stress cool moist conditions. We suggest that a relationship might exist between the overall climatic tolerance of a species, and climate thresholds beyond which herbicide-resistant phenotypes might spread. Thus, in the absence of herbicide, it is not inevitable that the frequency of R phenotypes will decline in field conditions.

The ability of 16 calcareous grassland species to establish in competition with adult neighbours of two different growth forms was assessed in a glasshouse experiment. The questions asked were: (1) how well is establishment ability related to plant traits such as seed mass, DNA content, relative growth rate and adult height; and (2) are the results consistent across different neighbours? Two measures of establishment ability were calculated, survival relative yield and biomass relative yield, for seedlings grown with a dicot. (Leontodon hispidus) and with a graminoid (Festuca ovina). The results depended on which measure of establishment ability was used. When establishment ability was measured in terms of biomass relative yield, seed mass was a good predictive trait, accounting for 38–55% of variation. However, when establishment ability was measured as survival relative yield, adult height was found to be the best predictor, capable of accounting for 20% of variation, although this was not consistent across neighbours. Thus, no clear relationship between plant traits and establishment ability was found: results depended on the measure of establishment ability used, and in the case of adult height, on the identity of the neighbour species. Survival relative yield was strongly associated with target and neighbour species growth form type, with stronger suppression of like than of non-like growth forms. The lack of strong relationships between plant traits and establishment ability, and the dependence on neighbour identity, suggests that the links between plant traits and establishment might be context-dependent; thus an understanding of the mechanisms underlying any such links is essential.

A pathogenic strain of Fusarium oxysporum f. sp. lycopersici transformed with the glucuronidase (GUS) reporter gene was used to study the colonization process of tomato (Lycopersicon esculentum) roots in hydroponic culture. The plants were treated exactly as those used in a different study with a non-pathogenic strain of F. oxysporum in order to compare the two types of colonization process. The pathogenic strain rapidly colonized the root surface, forming a dense network of hyphae – shown by the GUS staining assay – as early as 48 h after inoculation. The first images of the pathogen penetrating into the epidermis of the root were observed 24 h after inoculation. The plant showed defence reactions mainly in the hypodermis, but also in the cortex. Generally these barriers failed to prevent the centripetal growth of the pathogen towards the stele. The GUS activity moved with the actively growing hyphae into the tissues; the stele appeared intensely stained 7 d after inoculation, whereas by this time the hyphae at the root surface did not show any more staining. The hyphae formed a dense network at the apex of the root, but direct contact between hyphae and living cells was prevented by several layers of sloughed cap cells. Although rarely observed, penetration of the pathogen into the apex was possible and led to a rapid destruction of apical cells. The only differences between this pattern of root colonization and that observed for a non-pathogenic strain of F. oxysporum appear to concern the frequency of dead apices and the intensity of fungal colonization in the cortex. When the pathogen passed around the barrier formed in the hypodermis it always reached the xylem, although this barrier and other defence reactions induced at different levels in the cortex always prevented the non-pathogenic strain from reaching the stele. These observations suggest that the main differences between the two types of interaction – between the plant and either the pathogenic or the non-pathogenic strain – are quantitative rather than qualitative.

During their symbiotic phase, white truffles are barely distinguishable morphologically, and molecular probes are needed for their identification. Here we report the design of species-specific primers for two white truffles (Tuber magnatum and T. borchii) on the basis of their ITS sequence. Their efficiency has been successfully tested on fruit bodies of the same species, many related and unrelated fungal species, as well as mycorrhizal roots in direct, nested and multiplex PCR experiments. They have allowed us to identify T. magnatum mycorrhizas for the first time.

The influence of three organic compounds and bakers' dry yeast on growth of external mycelium and phosphorus uptake of the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith (BEG 87) was examined. Two experiments were carried out in compartmentalized growth systems with root-free sand or soil compartments. The sand and soil in the root-free compartments were left untreated or uniformly mixed with one of the following substrates (0.5 mg g−1 soil): bakers' dry yeast, bovine serum albumin, starch or cellulose. Effects of the organic substrates on biomass and hyphal length density of the arbuscular mycorrhizal fungus were examined by using specific fatty acid signatures in combination with direct microscopy. Micro-organisms other than the arbuscular mycorrhizal fungus were measured by fatty acid signatures, and radioactive 33P labelling of the root-free soil was used to determine arbuscular mycorrhizal hyphal phosphorus uptake. In general, hyphal growth of G. intraradices was enhanced by yeast and bovine serum albumin, whereas the carbon sources, starch and cellulose, depressed fungal growth. By analysing the fatty acid 16[ratio ]1ω5 from phospholipids (indicating mycelium) and neutral lipids (indicating storage structures) it was shown that increased fungal growth due to yeast was mainly in vegetative hyphae and less in storage structures. Arbuscular mycorrhizal hyphal phosphorus uptake was decreased by cellulose, but unaffected by the other substrates compared with the control. This means that both growth and phosphorus transport by the arbuscular mycorrhizal fungus were decreased under cellulose treatment. However, the composition of the microbial community varied under different substrate conditions indicating a possible interactive component with arbuscular mycorrhizal hyphal growth and phosphorus uptake.

Arbuscular mycorrhizal (AM) fungi can reduce the incidence and importance of plant root diseases caused by pathogens. The mechanisms involved are not well characterized. We used an in vitro experimental system to test the hypothesis that the extraradical mycelium of AM fungi can interfere directly with microorganisms in the mycosphere and directly or indirectly reduce the population of plant pathogens. This system permitted the isolation of soluble substances released by the extraradical mycelium of Glomus intraradices. The AM fungus was grown on Daucus carota transformed roots in one compartment, while only the extraradical mycelium was allowed to grow in a second compartment. A freezing and centrifugation technique was developed for the extraction and concentration of substances present in the compartment containing only the AM fungal mycelium. Four soil-inhabiting microorganisms were selected, and conidial germination (fungi) or growth (bacteria) of these was studied in the presence and absence (control) of the extract. In comparison with the control, the results indicated that both the growth of Pseudomonas chlororaphis and the conidial germination of Trichoderma harzianum were stimulated in the presence of the AM fungal extract. In contrast, conidial germination of Fusarium oxysporum f. sp. chrysanthemi was reduced while the growth of Clavibacter michiganensis subsp. michiganensis was not affected. The measured effects in general were directly correlated with extract concentration. Differences in pH were noted between the extract containing substances released by the AM fungus and the non-AM control, but no significant influence of the pH on growth or conidial germination was noted, confirming that substances released by the AM fungus in the growth medium is the main factor explaining differential growth of the microorganisms tested. The results suggest that direct interactions exist between AM fungi and soil microorganisms, which might lead to changes in microbial equilibrium detrimental to pathogens.

We performed two glasshouse experiments to determine whether the presence of arbuscular mycorrhizal symbiosis reduces the intensity of intraspecific competition at low concentrations of available phosphorus (P), and whether this effect is modified by a reduction in light intensity. In the first experiment, Trifolium subterraneum cv. Mt. Barker was grown at different densities in controlled conditions of light and temperature, and half of the pots were inoculated with spores of the arbuscular mycorrhizal fungus, Gigaspora margarita. In the second experiment, the plants were grown in similar controlled conditions but the light intensity received by half of the pots was reduced by >50%. The biomass and P content of individual mycorrhizal plants and the biomass response to mycorrhizal infection were drastically reduced as plant density increased. The effects of density on percentage infection, shoot and root P concentrations, and root: shoot ratios were inconsistent. Generally reduction in light intensity did not alter these effects. Mycorrhizal symbiosis increased intraspecific competition intensity through an increase in the availability of soil P. This increase in competition was reflected in the greater size inequality of low density mycorrhizal treatments. Our results emphasize that the main effects of mycorrhizas at the individual level cannot be expected to be apparent at the population level, because they are overridden by density-dependent processes.