Leaves of a mottled stripe disease-susceptible cultivar (B-4362) and of a mottled stripe disease-resistant cultivar (SP 70–1143) of sugar cane (interspecific hybrids of Saccharum) were inoculated with the diazotrophic endophytes, Herbaspirillum rubrisubalbicans or Herbaspirillum seropedicae, via injection into the apex of the stem. At 7 and 20 d.a.i., H. seropedicae could be isolated only from a small necrotic area around the point of inoculation, where there was considerable degradation of host cells, and was not detected in any other part of the leaves. This suggested a hypersensitive response by the host to this bacterium, and no disease symptoms formed on either cultivar. By contrast, H. rubrisubalbicans could be re-isolated from throughout the infected leaves of both cultivars at both harvests and produced widespread disease symptoms on the leaves of cv. B-4362.

Symptoms consisted of necrotic regions near the point of inoculation, and red stripes and red patches along the vertical axis of the leaves, where the bacteria had spread in the primary and secondary veins. The xylem-conducting elements in diseased regions of leaves were filled with bacteria and, at the edges of disease symptoms, the vessels were filled with a gum which stained blue-green with toluidine blue. This material probably contained phenolic compounds, and was produced as a host defence response. Leaves of cv. SP 70–1143 only developed small red stripes near the point of inoculation. These symptoms did not spread along the leaves, and the infected xylem vessels were never seen to be completely full of bacteria. Instead, the vessels contained encapsulated bacterial colonies attached to secondary wall deposition; these colonies were surrounded by blue-green material that might have been host-defence gums. In cv. B-4362, bacteria were abundant in the intercellular spaces of mesophyll adjacent to infected xylem, and also filled sub-stomatal cavities. Immunogold labelling using polyclonal antisera raised against H. rubrisubalbicans gave a weak signal with the bacteria in cv. SP 70–1143, showing that few binding sites were available to the antibodies. By contrast, bacteria in cv. B-4362 reacted strongly with the antibody, suggesting that they had a denser coating of immunoreactive mucus. In the later stages of infection of cv. B-4362, lysed bacteria were seen within degraded plant cells surrounded by a matrix of plant gums and bacterial mucus. This matrix reacted strongly to the H. rubrisubalbicans antibodies. Immunogold labelling using antibodies against nitrogenase component II showed that nitrogenase was expressed by bacteria in the early stages of infection of cv. B-4362, but not in later stages, or by bacteria infecting cv. SP 70–1143.

Among closely related citrus genotypes growing in high phosphorus (P) orchard soils, there is a tendency for less mycorrhizal-dependent (M-dependent) species to have lower rates of root colonization than more M-dependent species. We hypothesized that the less M-dependent the citrus species the more limited is their carbohydrate (CHO) allocation to the M fungus. In a glasshouse study at low and high P supply, lower total incidence of Glomus intraradices FL 208, intensity of vesicles formation, and accumulation of 16[ratio ]1W5cis fungal fatty acid as measures of root colonization were related to lower mycorrhizal dependency (MD; M plant d. wt/non-mycorrhizal (NM) plant d. wt expressed at low P supply) in five citrus genotypes. At high P supply, when host carbon (C) production was not affected by P nutrition, less M-dependent genotypes had consistently lower starch concentrations in their root and shoot tissues than did more M-dependent genotypes, irrespective of M inoculation. At low P supply, M plants were more heavily colonized by G. intraradices and had lower starch levels than the M plants supplied with additional P. At high P, M plants of the more dependent genotypes allocated more C to starch pools relative to the NM plant than the least dependent genotype. Concentration of sucrose in tissues did not vary consistently with the dependency of citrus genotypes or with M inoculation except in high P NM plants, when less M-dependent genotypes had lower levels of sucrose in their roots than the more dependent species. At high P supply, sucrose concentrations were lower in colonized roots than in NM roots. Across citrus genotypes, sucrose in M fibrous roots decreased relative to that in NM roots with increase in root colonization suggesting that more sucrose was allocated for growth and maintenance of G. intraradices in roots of M-dependent species. The concentration of reducing sugars in root tissues varied in relation to MD of citrus genotypes in the same way as that of starch and sucrose, but was less responsive to M colonization. Responses of total non-structural CHO in tissues indicated that more heavily colonized plants at low P expended more C to acquire P than did M plants of similar biomass and P status grown at high P supply. Total CHO pools increased with MD of citrus genotypes, providing evidence than C allocation patterns in the host affect M colonization.

Production of enzymes which degrade plant cell wall macromolecules has been studied in relatively few ericoid fungal isolates, although these polymers are a major component of the organic litter and an important source of nutrients for these fungi. Our aims were to investigate whether the ability to degrade the wall pectic component, only reported for one isolate, is a general feature of ericoid fungi. Of about 35 isolates from different geographic regions, all were capable of growing on pectin as the sole carbon source. Polygalacturonase (PG) activity was detected to a different degree in the culture filtrates and independently of the fungal growth rate. Solid and liquid isoelectric focusing allowed separation and identification of several polygalacturonase isoforms. Among the fungal isolates investigated, those from the northern hemisphere produced mostly acidic isoforms, whereas isolates from South Africa secreted more abundantly basic isoforms. However, purification and biochemical characterization of several PG isoforms from the different isolates revealed an optimal activity in the acidic pH range for all the PG enzymes tested. Polygalacturonase enzymes seem to be an important component of the enzymatic arsenal secreted by ericoid fungi during their saprotrophic life. In addition, they could also play a role during root colonization, since penetration across the plant cell wall is a prerequisite for the establishment of endomycorrhizal symbiosis.

A β-1,4-endoxylanase from the ericoid mycorrhizal fungus H. ericae has been purified to electrophoretic homogeneity using isoelectric focusing, ion exchange and gel permeation chromatography. The enzyme has an isoelectric point of 4·85–5·20 and a molecular weight of 58·4 kDa. The apparent S0·5 of the enzyme for soluble birchwood glucuronoxylan is 3·75 mg ml−1 and the Vmax 468·0 nkatal mg−1 protein. The pH optimum for activity is 4·5 and that for stability is 3·5–4·0; these values are discussed in the context of the pH of the mor humus. The role of wall-degrading activities in the establishment of the ericoid mycorrhizal symbiosis is considered.

The presence of host plants was found to be a key determinant of soil infectivity for endophytes of epacrids growing in south-west Australian jarrah forest carrying a sparse epacrid understorey. A comparable Banksia woodland with higher density of epacrids showed more uniform levels of infective endophytes, presumably because of closer overlap of host-supported patches of endophyte. Disturbed sites in jarrah forest, in which topsoil had been recently returned following bauxite mining, initially showed extremely low endophyte infectivity, probably due to disruption of the hyphal network, absence of host plants and/or increased competition from soil micro-organisms antagonistic to the endophytes. As the system stabilized and epacrids recruited and grew, inoculum potential of the soil re-established and by 12 yr it equalled that of adjacent undisturbed native forest. At disturbed sites with sparsely distributed adult members of the Epacridaceae, endophyte inoculum proved to be adequate only directly adjacent to the epacrids and declined steeply to negligible levels at only 40 cm radially distant from a host plant. The significance of live epacrid root systems on survival of endophyte inoculum in Banksia woodland was examined following removal of shoots of adult plants of the endemic epacrid, Leucopogon conostephioides DC. Coincident with demise of roots of the detopped plants, endophyte inoculum potential of closely adjacent soil declined by 50% during the growing season. However, the effect was short lived and infectivity rose the following year to equal that of undisturbed adjacent woodland as mycelial matrices supported by neighbouring epacrids invaded the depleted study areas.

The presence of extracellular carbonic anhydrase (CA) in relation to medium composition was investigated using cultures of the marine diatom Phaeodactylum tricornutum Bohlin. Large-volume cultures, with low initial cell inocula were grown on ASP-2 (no dissolved inorganic carbon (DIC), 550 μM NO3−), f/2 (2·0 mM DIC, 880 μM NO3−) and modified f/2 (2·0 mM DIC, 20 μM NO3− media. Cells growing on ASP-2 showed extracellular CA in the early stages of growth, whereas extracellular CA was not detected until partial depletion of total DIC in the stationary phase for cultures on f/2 or modified f/2. Both HCO3− and CO2 were important in carbon limitation, extracellular CA being present when the free-CO2 concentration fell below 5 μM, but the HCO3− concentration needed to be below 1 mM. When carbon-replete cells were transferred to carbon-limited conditions, extracellular CA was recorded within minutes, the process being light-dependent and completely inhibited by 3,3,4-dichlorophenyl-1,1-dimethylurea (DCMU). The addition of DIC to carbon-limited cells resulted in a rapid decrease in extracellular CA activity. The membrane-impermeable inhibitor of carbonic anhydrase, dextran-bound sulphonamide (DBS) was used to inhibit extracellular CA activity in relation to photosynthetic rate in carbon-replete and carbon-limited cells. At the lowest DIC concentration (0·10 mM), for cells with maximum external CA activity, DBS gave over 80% inhibition of the photosynthetic rate, demonstrating the key role of external CA in maintaining high photosynthetic rate under conditions of carbon limitation.

It is proposed that the key factor in the regulation of extracellular CA activity is the total flux of inorganic carbon (Ci) into the cell. This determines the Ci flux into the chloroplast and when this is inadequate to support the photosynthetic rate attained by a carbon-replete chloroplast at optimum photon flux density, extracellular CA is activated. This mechanism would explain the observed interaction of CO2 and HCO3− in the regulation of extracellular CA activity.

Abuscular mycorrhizal fungi are thought to survive adverse environmental conditions primarily as spores. Extraradical mycelia of two Glomus species were produced in fine mesh pouches which excluded roots but not hyphae. The mycelia in these pouches were exposed to freezing conditions, either in the field or in a controlled-temperature chamber. Bioassay plants were grown directly in the pouches and mycorrhizal colonization was assessed after 1 month. The mycelia remained infective in frozen soil over winter. This survival was not dependent on either the presence of root pieces or on the connection of mycelia to roots. Spores were not an effective inoculum in these bioassays. Overwinter survival of mycelia would enable plants to become incorporated into functional mycorrhizal associations early in spring.

Re-analysis of data from Salisbury (1974) shows no significant relationship between seed size and habitat conditions, a relationship for which this paper is frequently cited as support. The discrepancy between the results reported here and those found in the original paper is due to Salisbury's ‘evidence’ being a table of congeneric pairs collected specifically because they possessed the target relationship. This paper emphasizes particularly the need for an unbiased sampling of species pairs in comparisons attempting to define ecological relationships. The analysis presented here represents the fourth such instance in which previously assumed robust examples of the relationship between seed size and habitat type are shown not to be so when the appropriate methods are used to account for relatedness among species.

Subterranean clover (Trifolium subterraneum L.) in symbiosis with Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe was grown in compartmented pots. Side compartments were filled with soil where recently added (1 wk) radio-labelled cadmium (Cd) at three levels of non-labelled Cd could be accessed by either roots or arbuscular mycorrhizal hyphae. All treatments were replicated with non-mycorrhizal plants. After a growth period of 52 d roots and shoots were analysed for 109Cd, and lengths of roots and hyphae in respective labelled compartments determined. Cadmium uptake by roots was not significantly influenced by the mycorrhizal status of the plant. Uptake of Cd from hyphal compartments was higher in mycorrhizal than in non-mycorrhizal plants, corresponding to 96, 127 and 131% of that in non-mycorrhizal plants when 1, 10 and 100 mg Cd kg−1 was added, respectively. A large proportion of the increased Cd content of mycorrhizal plants was sequestered in the roots. It is concluded that extraradical hyphae of AM fungi can transport Cd from soil to plants, but that transfer from fungus to plant is restricted due to fungal immobilization. No reduction of hyphal growth into soil with up to 20 mg extractable Cd kg−1 was observed.

Potted specimens of 27 Swedish native herbs and grasses were exposed to three different ozone concentrations, CF (charcoal filtered air), NF (non-filtered air) and NF+ (1·5 × non-filtered air) in open-top chambers from 2 July to 5 August 1994. The species represented a wide range of different plant strategies according to the C-S-R model. The results show that the stress-tolerators, S, had a smaller mean relative growth rate, R¯, during the exposure period and also a smaller accumulation of biomass compared with other strategies. The species with the intermediate strategy, CSR, had a R¯ similar to that of the species with a large component of C, the competitor strategy, and/or R¯ the ruderal strategy, whereas the net accumulation of biomass was smaller in the CSR group. This difference between R¯ and net growth for the CSR species compared to the species of other strategies, can be explained by a slower growth for the CSR group during the establishment phase. In the present investigation the response to ozone was very small regardless of plant strategy, although there was a weak trend towards slower growth with higher ozone concentration, 18 out of 27 species having a greater growth in CF than in NF+. The growth of one typical stress-tolerant species, Festuca ovina L., was stimulated significantly by ozone. Visible injury, most likely caused by ozone, was noticed in three species, Dactylis glomerata L., Dactylis aschersoniana Graebn. and Phleum alpinum L. Although there were no major restrictions to plant growth in terms of water, nutrients or space in the present open-top chamber experiment, the variability in all climatic factors was as large as in the ambient air. This might harden the plants and make them less sensitive to ozone compared to plants grown in the laboratory under controlled and optimal conditions with low variability.

This study was conducted to determine whether xylem abscisic acid (ABA) concentration is altered by mycorrhizal symbiosis of cowpea plants grown in drying soil, and to determine whether stomatal sensitivity to xylem ABA is altered by the symbiosis. We allowed the entire root zone to dry and found that at high soil water contents (θ), mycorrhizal plants had higher stomatal conductance (gs) and lower xylem [ABA] than did non-mycorrhizal plants, but the difference disappeared at low θ, probably because of stomatal closure. The altered gs and xylem [ABA] were apparently not related to plant water status since shoot water potential, xylem sap osmotic potential and shoot water content were similar for mycorrhizal and non-mycorrhizal plants across the range of soil moisture. These differences were also not related to P nutrition or plant size. The relationship of gs to xylem [ABA] was not affected by the symbiosis, indicating that either stomatal sensitivity to xylem ABA was not affected by other xylem constituents, or that more than one xylem constituent was altered by the symbiosis but was offsetting in its effect on gs. We conclude that the symbiosis altered gs non-hydraulically, and that the factor might be xylem ABA.

Seedlings of Pinus sylvestris L. were grown in Plexiglas® observation chambers in limed (CaCO3, pH5·0 and 5·9) and untreated (pH 4·1) peat. The seedlings were either colonized by the mycorrhizal fungus Paxillus involutus (Batsch: Fr.) Fr. Or were non-mycorrhizal. After 18 wk in the observation chambers, 15N-labelled organic nitrogen, as lyophilized and ground mycelium of Suillus variegatus (Swartz: Fr.) O. Kuntze, or ammonium, was added to the peat. The plants were harvested after an uptake period of 14 d.

Irrespective of the nitrogen form added, liming decreased both the content and concentration of 15N in non-mycorrhizal plants, and, to a lesser extent, those in mycorrhizal plants. In mycorrhizal plants the uptake of 15N was not correlated with area colonized by the mycorrhizal mycelium. The amount of KCl-extractable 15N in peat without plants and mycorrhizal fungi decreased with liming. It is proposed that liming induced chemical or microbial immobilization of the added 15N. This is suggested to be the main reason for the decreased uptake of 15N in lime treatments.

This study investigates the phytotoxicity of ozone during periods of natural frosts following long-term fumigation. Heather (Calluna vulgaris (L.) Hull.) plants were fumigated with ozone for 6 months at 70 nl l−1, for 8 h d−1, 5 d a week in open-top chambers. Natural frosting occurred within the chambers towards the end of this period (5–27 December 1995). Plant responses to the ozone/natural frosting episode were recorded in January 1996. We show conclusively that naturally occurring frost exposure results in increased damage and mortality for ozone-treated plants, which could have important implications for the function of heathland ecosystems.

Modern cone scales and seeds of Pinus strobus and Sequoia sempervirens, and their fossil (Upper Miocene, c. 6 Mar) counterparts Pinus leitzii and Sequoia langsdorfii have been studied using pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), electron-microprobe and scanning electron microscopy. Microscopic observations revealed only minor microbial activity and high-quality structural preservation of the fossil material. The pyrolysates of both modern genera showed the presence of ligno-cellulose characteristic of conifers. However, the abundance of (alkylated)phenols and 1,2-benzenediols in modern S. sempervirens suggests the presence of non-hydrolysable tannins or abundant polyphenolic moieties not previously reported in modern conifers. The marked differences between the pyrolysis products of both modern genera are suggested to be of chemosystematic significance. The fossil samples also contained ligno-cellulose which exhibited only partial degradation, primarily of the carbohydrate constituents. Comparison between the fossil cone scale and seed pyrolysates indicated that the ligno-cellulose complex present in the seeds is chemically more resistant than that in the cone scales. Principal component analysis (PCA) of the pyrolysis data allowed for the determination of the discriminant functions used to assess the extent of degradation and the chemosystematic differences between both genera and between cone scales and seeds. Elemental composition (C, O, S), obtained using electron-microprobe, corroborated the pyrolysis results. Overall, the combination of chemical, microscopic and statistical methods allowed for a detailed characterization and chemosystematic interpretations of modern and fossil conifer cone scales and seeds.