The male sterile mutant, ms35, of Arabidopsis thaliana was produced by X-irradiation of seeds. The mutant produces fertile pollen, but is male sterile because the anthers do not dehisce. Anther development in ms35 plants occurs as in wild-type Arabidopsis until shortly after microspores are released from meiotic tetrads. Thereafter, in the wild type, bands of lignified, cellulosic secondary wall thickenings are laid down around the cells of the anther endothecium. In contrast, wall thickenings are not formed in the endothecium of the ms35 mutant. Development of other lignified tissues, for example the vascular tissue of the stamen, occurs normally in ms35 plants. In mutant anthers, as pollen maturation is completed, the stomium is cleaved but the anther wall does not retract to release pollen. The block in anther dehiscence in ms35 plants is specifically correlated with the absence of endothecial wall thickenings. The ms35 mutation represents the first genetic evidence in support of the proposed role of the endothecium in anther dehiscence. The ms35 gene was mapped to the top arm of chromosome 3 (hy2-(4.17±2.31 cM)-ms35-(32.14±5.45 cM)-gl1).

Physiological responses of two provenances of European beech (Fagus sylvatica) were studied in seedlings grown at two [CO2] in combination with four temperature treatments. For the local Danish provenance, the average effect of elevated [CO2] during growth was to increase light-saturated net photosynthesis (An) and instantaneous water-use efficiency or transpiration efficiency (ITE). These increases were strongly related to the temperature treatment. Stomatal conductance (gs) was reduced in seedlings in high [CO2], but there was no statistically significant effect of temperature treatment. Stomatal conductance was 13–26% lower at elevated [CO2] and ITE was 89–156% higher, depending on growth temperature. The effects of [CO2] on An were considerably larger than those shown for many other woody species, but similar to those in other studies on European beech. The absolute value of An for a Romanian provenance of beech was 5–18% lower than in the Danish provenance at low [CO2] and 14–26% lower at high [CO2]. There was no statistically significant interaction between the provenances and [CO2], or between provenance and temperature. A model of the response of An to [CO2] at different temperatures gave predictions close to the measured results, except at the lowest temperature treatment where the model over-predicted the effect of elevated [CO2]. This and measurements of An made at a common, low [CO2] indicated a down-regulation of photosynthesis in the lowest temperature treatment at high [CO2]. Root plus soil respiration on a whole-tree basis (Rtr) was increased by elevated [CO2] at all but the lowest temperature, but no effect was seen of [CO2] on root respiration per unit root d. wt. Mean Rtr on any given date was significantly correlated with An, except at the lowest temperature treatment. It is hypothesized that low temperature limited the ability of the roots to use photosynthates resulting in a feedback inhibition of An when elevated [CO2] was combined with low temperature.

The results presented in this paper describe the short- and long-term toxicity of arsenate in Silene vulgaris. Short-term toxicity, measured as inhibition of root elongation, depended on phosphate nutrition, arsenate being much less toxic at high phosphate supply. At low phosphate levels more arsenic was taken up by the plants. Under chronic exposure, toxicity (measured as inhibition of biomass production) did not increase with time. In addition, the accumulation of phytochelatins (PCs) as a function of toxicity and duration of exposure was studied. Short-term PC accumulation (over a 3 d period) was positively correlated with exposure. Isolation of peptide complexes from prolongedly exposed plants showed that PC2, PC3 and PC4 were present, although the latter not until at least 3 d exposure. Arsenic co-eluted mainly with PC2 and PC3. Fractions containing PC4 were devoid of As, probably due to dissociation of the complexes during extraction or elution. The breakdown of PCs after arresting As exposure was very slow. This could explain the continuous accumulation of PCs throughout longer periods of As exposure.

Clones of ectomycorrhizal fungi can colonize new areas through production of vegetative mycelium or spore dispersal, but the relative importance of these processes in nature is not known. In this study, sporocarps of an ectomycorrhizal fungus, Suillus grevillei, were mapped and sampled from a Larix kaempferi stand at the foot of Mt Fuji. DNA was extracted directly from each sporocarp, and DNA polymorphism was analysed by polymerase chain reaction (PCR) amplification of inter-simple sequence repeat (ISSR) regions primed by (GTG)5, (GCC)5 and (GACA)4. Different sensitivities to detect polymorphism were found among the three primers, with (GACA)4 showing the highest sensitivity. Forty seven sporocarps were analysed by the three ISSR primers and divided into 34 genets based on combination of PCR fingerprints. In the population 28 genets were represented by individual sporocarps. In most cases, sporocarps grown in aggregation (within a circle of 50 cm diameter) showed some different ISSR band patterns. These results suggest that genets of S. grevillei at the test site are relatively small. The genetic similarities between the 34 genets were also calculated and similarity groups were determined by the criterion that all similarity F values of genets within a group were not <80%. In general, the genets within a similarity group located close to each other. The results of multiple different but highly related genets in a small area suggest that the population of S. grevillei in this stand is not spread and maintained by clonal mycelium extension but is reproduced by spore dispersal.

To predict changes in the range and density of plant species as a consequence of elevated atmospheric CO2, it is essential to characterize the effect of elevated CO2 on key components of plant life history stages such as rate of establishment and maturation of individuals, reproductive output and offspring fitness. We measured vegetative response, phenology, reproductive output and seed quality of four annual C3 plant species, grown from seed to senescence under ambient and elevated (ambient+200 ppm) CO2 and ambient and reduced (33% shade) light. Few previous studies have included all stages of a plant's life as well as characteristics of the next generation. Elevated CO2 had no effect on the vegetative attributes of Cardamine hirsuta, Spergula arvensis or Poa annua whereas Senecio vulgaris produced longer leaves and greater biomass. Both Senecio and Poa had faster maturation times. The vegetative response of Senecio was not translated into increased seed output, although seed mass and carbon[ratio ]nitrogen ratios were significantly increased. By contrast, Poa showed no vegetative response to elevated CO2, but had significantly increased seed production. Thus we found no evidence for a simple translation from vegetative response to elevated CO2 into reproductive response. There was also no consistent light-mediated response to elevated CO2 among the four species. However, the effect of reduced light (33% shade) on vegetative and reproductive output was consistent across the four species and significantly stronger than the effect of elevated CO2. On the basis of this glasshouse study, we predict that Poa would be most likely of the four species to show significant increases in population size and migration potential, as a result of increased reproductive output, under elevated atmospheric CO2. However, this response may be relatively small compared with variation in growth and reproduction as a result of environmental heterogeneity in resources such as light.

Branch bags were used to expose branches on mature Sitka spruce trees to either ambient [CO2] (A) or elevated [CO2] (E) for 4 yr. This paper reports the effects of this treatment on the growth, development and phenology of the branches, including shoot expansion, shoot numbers, needle dimensions, needle numbers and stomatal density. The effect of elevated [CO2] on the relationship between leaf area and sapwood area was investigated. Exposure to elevated [CO2] doubled photosynthetic rates in current-year shoots and, despite some down-regulation, 1-yr-old E shoots also had higher rates of photosynthesis than their A counterparts. Thus, the amount of assimilate fixed by E branches was substantially more than that fixed by A branches; however, this increase in the local production of assimilate did not lead to an increase in non-structural carbohydrate or stimulate growth or meristematic activity within the E branches. There was a very consistent relationship between leaf area and stem cross-sectional area that was not influenced by [CO2]. However, unbagged branches had thicker stems than bagged branches, resulting in a slightly lower ratio of leaf area to cross-sectional area. The implications of the results for the modelling of growth and allocation and the potential utility of the branch bag technique are discussed.

The growth of the marine flagellate Isochrysis galbana was followed in batch cultures at four concentrations of dissolved inorganic carbon (DIC), from C- and N-replete lag phase into C- and/or N-deplete stationary phase. Organic buffers were omitted from the growth medium, and culture pH was maintained at 8.30±0.05 by the addition of acid or alkali. The responses of the flagellate to N stress included an increase in the C[ratio ]N ratio, and decreases in the ratios of glutamine (Gln)[ratio ]glutamate (Glu) and Chl a[ratio ]C, and the cell Chl a quota. Conversely, the responses to C stress included a decrease in the C[ratio ]N ratio, and increases in the ratios of Gln[ratio ]Glu and Chl a[ratio ]C, and the cell Chl a quota. The relationship between carbon-specific growth rate (C-μ), and the concentration of extracellular DIC, [DIC]ext, exhibited Michaelis–Menten type kinetics with a half saturation constant, KG(DIC), of 81 μM. Comparative studies of the diatom Phaeodactylum tricornutum showed similar results, although the value of KG(DIC) was lower at 30 μM.

The study reported was conducted to establish the impacts of photochemical oxidants (primarily ambient ozone, O3) on the yield of watermelon (Citrullus lanatus) at a site on the east coast of Spain. Fruit yield and quality were monitored in plots established in a commercial watermelon field exposed, in open-top chambers (OTCs), to non-filtered air (NFA; near-ambient levels of ozone) or charcoal-filtered air (decreased levels of photochemical oxidants including O3; CFA), or to ambient air (AA), during the 1988 and 1989 growing seasons. Ambient levels of O3 were found to exceed present UN-ECE (United Nations Economic Commission for Europe; Convention on Long-Range Transboundary Air Pollution) critical level guidelines for the protection of crop yield by approx. twofold in 1988 and by approx. fivefold in 1989. Plants exposed to NFA and AA developed visible O3 injury on the upper surface of sun-exposed older leaves, and fruit yield (annual marketable fruit weight and number) was found to be depressed in OTCs ventilated with NFA in comparison with those receiving CFA. Consistent with inter-annual variations in O3 exposure, greater yield losses were experienced in 1989 (39%) than in 1988 (19%), an effect mediated predominantly by a decline in fruit number rather than average fruit weight. Exposure to ambient levels of ozone also slightly decreased fruit quality (4–8% decline in soluble solids content). Leaf gas exchange measurements made in the field in 1988 revealed effects of O3 on fruit yield and quality to be associated with a decline in the net CO2 assimilation rate per unit leaf area under light saturation (Asat) and stomatal conductance to water vapour (gs), and enhanced rates of dark respiration. A/ci curves (where A is the net CO2 assimilation rate per unit leaf area and ci is the mole fraction of CO2 in the leaf intercellular air space) constructed for plants grown in laboratory-based closed chambers, and exposed to an accumulated O3 exposure similar to that experienced by plants in the field, suggested that the likely cause of the decline in photosynthetic capacity was (1) a decrease in the amount and/or activity of Rubisco and (2) an impaired capacity for regeneration of ribulose 1,5-bisphosphate, which was not mediated through changes in the photochemical efficiency of photosystem II (Fv/Fm, where Fv is variable chlorophyll a fluorescence and Fm is maximum chlorophyll a fluorescence). No shift in the relative stomatal limitation to photosynthesis was observed under the influence of O3, suggesting that the decline in gs induced by the pollutant in both field and laboratory was the result, and not the cause, of the decrease in Asat. Ozone exposure also caused a decrease in C isotope discrimination (approx. 0.5‰), a shift that revealed a departure from predicted theory based on supporting leaf gas exchange measurements. The study demonstrates that ambient levels of photochemical oxidants on the Spanish Mediterranean coast are high enough to adversely influence the yield and physiology of an economically important crop grown in the region, and the magnitude of the effects was greater than would be predicted from exposure–yield-loss relationships for grain and fodder crops in central and northern Europe. Implications for the derivation of UN-ECE ozone standards are discussed, along with the mechanistic basis of the observed yield decreases.

Sunflower seedlings (Helianthus annuus hybrid Select) were grown in a complete nutrient solution in the absence or presence of Cd2+ (10 and 20 μM). Analyses were performed to establish whether there was a differential effect of Cd2+ on mature and young leaves. After 7 d the growth parameters as well as the leaf area had decreased in both mature and young leaves. Accumulation of Cd2+ in the roots exceeded that in the shoots. Seedlings treated with Cd2+ exhibited reduced contents of chlorophyll and CO2 assimilation rate, with a greater decrease in young leaves. The photochemical efficiency of photosystem II (PSII) was not altered by Cd2+ treatment in either mature or young leaves, although during steady-state photosynthesis in young leaves there was a significant alteration in the following parameters: quantum yield of electron transport by PSII (ΦPSII), photochemical quenching (qP), non-photochemical quenching (qNP), and excitation capture efficiency of PSII (Φexc).

This paper considers the extent to which differences in pollen tube growth rates can provide prezygotic reproductive isolation between Mimulus nasutus and its presumed progenitor, Mimulus guttatus. Mimulus nasutus is partially cleistogamous, but its larger chasmogamous flowers offer appreciable opportunity for outcrossing. Mimulus nasutus was found to have smaller pollen grains and shorter styles than M. guttatus. No differences were observed in pollen grain germination on conspecific and heterospecific stigmas. However, pollen tube growth rates of M. nasutus were found to be much slower than those of M. guttatus in the styles of that species. Consequently, any M. nasutus pollen transferred to an M. guttatus stigma was found to be competitively disadvantaged in an M. gutattus style. By contrast, no difference in pollen tube growth rate was detected between the species when growing in M. nasutus styles, possibly because M. nasutus styles are unable to support fast pollen tube growth. We tested the prediction from the pollen tube studies that a 50[ratio ]50 mix of M. guttatus and M. nasutus pollen would produce 50% hybrid seeds when M. nasutus was the maternal parent, and near to 0% hybrid seed when M. guttatus was the maternal parent. The results were found to support this prediction. We conclude that pollen–pistil interactions can effect strong reproductive isolation between these species, as M. guttatus pollen tubes have a competitive advantage over those of M. nasutus in an M. guttatus style, but not in an M. nasutus style.

The effects of excess nitrogen (N) on the frost hardiness and freezing injury of bark and buds were tested in 2-yr-old sessile oaks (Quercus petraea) and pedunculate oaks (Q. robur) that had been grown outdoors in sand culture with normal or luxurious N supply during the growing season. Some trees from both N treatments were subjected to drought stress in summer, whereas others were adequately watered. Between January and March, whole plants were exposed to artificial freezing treatments at −25 °C of different durations (10 d, 21 d, or two periods of 4 d interrupted by an 8 d frost-free period, referred to as a freeze–thaw cycle). The frost hardiness and freezing injury of the bark were determined by two different versions of the electrolyte leakage method (calculation of an index of injury, I−25, from the relative conductivity after a fixed time; and measurement of the electrolyte leakage rate). In addition, the vitality of bark and buds was assessed visually with the 2,3,5-triphenyltetrazolium chloride (TTC) method. In comparison with the control, the oaks that had been luxuriously supplied with N exhibited distinctly higher N concentrations and lower C:N ratios in their leaves and bark, higher relative shoot increments, delayed leaf discoloration in autumn, and earlier budbreak in spring. Oaks kept outdoors during the winter were frost-hardy from December until the end of February without showing differences between species or N treatments. In comparison with the pedunculate oak, the sessile oak was more susceptible to freezing injury induced by prolonged artificial freezing or by the freeze–thaw cycle. Increased freezing injury in trees with high N supply was detected only by the TTC test of the bark of sessile oaks subjected to the freeze–thaw cycle. In all oaks, except for the pedunculate oaks grown with normal N, the freeze–thaw cycle resulted in significantly lower frost hardiness, as indicated by increased I−25 values. This treatment also led to increased freezing damage of bark and buds, as revealed by the TTC test. In drought-stressed oaks, the electrolyte leakage rate was significantly elevated after the freeze–thaw cycle. It is concluded that, in the sessile and pedunculate oaks, the water supply before frost stress and the course of the temperature in winter have a greater effect on frost hardiness and freezing damage than excess N.

Seedlings of 12 provenances of European beech (Fagus sylvatica) were exposed to ambient, non-filtered air (NF) or NF+50 nl l−1 ozone (NF50) for 8 h d−1 in open-top chambers (OTCs), from 1 June to 4 October 1995. In 1996 exposure was continued from 31 May to 1 October at four levels: charcoal-filtered air (CF), NF, NF50 and NF+100 nl l−1 ozone (NF100). Provenances were grown for both seasons in outside reference plots. All treatments were replicated. Ozone did not affect gas exchange in the provenances until late in the second season. NF100 reduced photosynthesis by 18% in August 1996 compared to CF. In September, photosynthesis was reduced by 22% in NF50 and by 29% in NF100. After two seasons, ozone reduced the root:shoot ratio by 24% when comparing CF and NF100; this was caused by reductions in the root biomass. Ozone did not affect height growth or stem diameter, and there were no ozone×provenance interactions for any growth parameter. There was, however, a significant ozone×provenance interaction for photosynthesis, showing northwest European provenances to be more sensitive to ozone than southeast European provenances when comparing dose–response estimates. This is interpreted in terms of genetic adaptation of the photosynthetic apparatus to regional growing conditions. Seedlings in the chambers grew 45% taller, and had 28% more shoot biomass and 29% smaller root biomass, resulting in a 44% reduction of root:shoot ratios compared to seedlings outside. Increased temperature and decreased PAR inside the chambers relative to the outside were probably the main causes for the differences. The magnitude of the chamber effects in OTCs raises doubts about conclusions drawn from ozone exposures in such chambers. This and previous ozone experiments with OTCs may have reached inaccurate conclusions concerning the size of ozone responses due to chamber effects.

Most recent reviews of plant salinity response have included the concept of a nutritional disturbance as one likely mechanism by which shoot growth might be inhibited. None the less, few studies of dicotyledonous plants have presented data on nutrient transport into the most intensively growing shoot tissues. In this paper net nutrient deposition was followed for 3 d in 8 sequential, growing leaves of Lactuca sativa, which were grown either in conditions of moderate salinization, or in a growth-stimulating concentration of NaCl. The nutrient deposition was studied from 0.7 to 3.7 d following completion of stepwise salinization. This deposition was followed in immature leaves, which had attained only 1–2% of ultimate leaf mass by the completion of the study. In such young leaves development is still dominated by cell division. The transport of Ca2+ specifically to the youngest leaves was reduced by more than twice as much as was K+ transport. Transport of the other major divalent cationic nutrient, Mg2+, was not decreased for these leaves. The factors of increase for Na+ and Cl− after 3.7 d after completion of salinization averaged 152 and 62% over control levels for the three youngest leaves (for Na+ and Cl−, respectively). Though significant, these increases were only 27 and 14% as great as increases in three leaf sets of more developed growing leaves. Decreases in net K+ deposition and leaf K+ concentration were not greater for the youngest than they were for the oldest leaves. Net S deposition was reduced 44% more in younger than older growing leaves, but for most leaves not beyond the level expected due to reduced sink strength. The reduction in net P deposition also seemed more related to reduced sink strength, but was reduced to approx. 50% in both younger and more developed growing leaves. While Fe concentration was not reduced by salinization at any developmental stage, Zn2+ net transport and Zn concentration were both reduced in the two youngest leaves (57 and 70%, respectively). Given the moderate treatment imposed (Na:Ca ratio of 22) the results suggest that Ca2+ transport to the youngest leaves is probably highly sensitive to salinization of the root medium and is perhaps a key physiological response in the inhibition of leaf growth.

The effects of enhanced UV-B radiation were investigated in the carnivorous plant Pinguicula vulgaris in a field experiment performed in Abisko, North Sweden (68° 21′ N, 18° 49′ E, 380 m above sea level). Potted plants were exposed to either ambient or ambient plus supplemental UV-B radiation, simulating a 15% ozone depletion. No effect was observed on either the epicuticular (external) or cellular (internal) UV absorbing capacity of the leaves. However, the anthocyanin content was more than doubled by supplemental UV-B radiation. In laboratory experiments, the anthocyanin rich, UV-B treated leaves were less susceptible to a low temperature/high light photoinhibitory treatment, as judged by in vivo chlorophyll fluorescence measurements. Yet, this potential benefit did not considerably affect the growth of the plant in the field (leaf area and dry mass, reproductive dry mass, flowering frequency, senescence rates, dry mass of winter buds). However, there was a marginally significant increase in root dry mass and in the root to shoot ratio, which may underlie the significant increase in the nitrogen content of the leaves. We suggest that P. vulgaris is resistant against UV-B radiation damage and that the possible negative effects of additional UV-B radiation on the growth of these plants may have been effectively counterbalanced by the lower risk of photoinhibition, due to the concomitant increase in anthocyanins.

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.

The nodulation status and nodule morphology of 62 taxa of Leguminosae in a rain forest in French Guiana are reported according to the taxonomy of the family. The N2-fixing species are then fitted into ‘functional groups’ according to their behaviour towards illumination, in order to evaluate their importance in the global dynamics of the stand. The results showed that 67% of the observed species were nodulated (50, 71 and 77% of the Caesalpiniaceae, Mimosaceae and Papilionaceae, respectively). In the Caesalpiniaceae, nodule-like structures were reported in the genus Crudia and in the species Senna quinquangulata, although this needs to be confirmed. All the nodules studied in this subfamily were astragaloid and mucunoid. In the Mimosaceae, the ability of a new genus (Balizia) to form nodules was reported, as well as nodulation on aerial roots in Inga stipularis. The nodules studied were mainly mucunoid. In the Papilionaceae, nodulation on aerial roots in Poecilanthe hostmannii and on conventional roots of the genus Paramachaerium were reported for the first time. All types of nodular structures were found in this subfamily but the structures were quite uniform at the tribal level. These are consistent with suggestions that nodule morphology has a taxonomic value. Eight functional groups of N2-fixing species are proposed, ranging from light dependance to shade tolerance. These results indicate the important role played by N2-fixing species in the global dynamics of the stand and that N inputs by N2 fixation were continuous along the gradient of energetic resources that characterizes the silvigenetic process. The interactions between the photosynthetic capacities of the species and the ability to fix N2 in low light conditions are discussed.

In studies with a laboratory isolate of the fungal pathogen Stagonospora (Septoria) nodorum three different isolates of bacteria were closely associated with the fungus. Bacteria were also closely associated with fresh isolates of S. nodorum obtained from artificially and naturally infected field material. Although a range of bacteria was isolated, only one type of bacterium was found to be associated with each isolate of S. nodorum. In co-inoculation studies with pycnidiospores of the fungus on detached leaves, some of the bacterial isolates significantly increased the pathogenicity of the fungus, particularly Xanthomonas maltophilia, Sphingobacterium multivorum, Enterobacter agglomerans and Erwinia amylovora. Evidence is presented indicating that one of the ways that the ‘helper bacteria’ may assist in the establishment of infections is by the production of lipases that were not detected in germinating fungal spores.

Young trees of European beech (Fagus sylvatica) acclimated for one growing season to ambient (c. 367 μl l−1) or elevated CO2 levels (c. 660 μl l−1) were exposed during the subsequent year to combinations of the same CO2 regimes and ambient or twice-ambient ozone (O3) levels (generated from the database of a rural site). By the end of June, before the development of macroscopic leaf injury, the raised O3 levels had not affected the light and dark reactions of photosynthesis. However, acclimation to elevated CO2 had resulted in lowered chlorophyll and nitrogen concentrations, whereas photosynthetic performance, examined over a wide range of parameters from light and dark reactions, remained unchanged or showed only slight reductions (e.g. apparent electron transport rate, ETR; apparent quantum yield of CO2 gas exchange, ΦCO2; apparent carboxylation efficiency, CE; and photosynthetic capacity at light and CO2 saturation, PC). In August, after the appearance of leaf necroses, plants grown under ambient CO2 and twice-ambient O3 conditions declined in both the photosynthetic light reactions (optimum electron quantum yield, Fv/Fm, non-photochemical energy quenching, NPQ, reduction state of QA, apparent electron quantum yield, ΦPSII, maximum electron transport rates) and the dark reactions as reflected by CE, ΦCO2, as well as the maximum CO2 uptake rate (i.e. PC). CE, ΦCO2 and PC were reduced by c. 75, 40 and 75%, respectively, relative to plants exposed to ambient CO2 and O3 levels. By contrast, plants exposed to twice-ambient O3 and elevated CO2 levels maintained a photosynthetic performance similar to individuals grown either under ambient CO2 and ambient O3, or elevated CO2 and ambient O3 conditions. The long-term exposure to elevated CO2 therefore tended to counteract adverse chronic effects of enhanced O3 levels on photosynthesis. Possible reasons for this compensatory effect in F. sylvatica are discussed.

The role of cytosolic pH (pHc) in growing germ tubes of the filamentous fungus Magnaporthe grisea was analysed by confocal ratio imaging of the pH-sensitive fluorescent dye 5(6)-carboxyseminaphthorhodafluor-1 (SNARF-1). The cytosol of these cells was successfully loaded with the acetoxymethyl ester of the dye and the pHc was visualized and quantified during conidium germination, germ tube growth and appressorium induction by simultaneous dual-emission confocal ratio imaging. Calibrations of the free acid in vitro and calibrations in vivo produced results indicating a similar dynamic response in the pH range 6.0–8.0 for both methods. The pHc in growing germ tubes was consistently pH 7.2±0.1 during all developmental stages analysed. Only slight changes in pHc (<0.1 pH unit) were found in response to alkaline external pH (pH 8.0). No changes in pHc occurred in response to an acidic extracellular pH (pH 6.0) or to the presence of nutrients. There was no observation of either pronounced gradients or changes in pHc in growing germ tubes accompanying conidium germination, germ tube growth or early appressorium formation.