Effects of aluminium in acid streams on growth and sporulation of aquatic hyphomycetes

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S0269-749

I (97)00054-7

Environmental Pollution, Vol. 96, No. 3, pp. 289-298, 1997 ~) 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0269-7491/97 $17.00 + 0.00

ELSEVIER

EFFECTS OF ALUMINIUM IN ACID STREAMS ON GROWTH A N D SPORULATION OF AQUATIC HYPHOMYCETES Anne-Carole Chamier* and Edward Tipping Institute of Freshwater Ecology, The Ferry House, Ambleside, Cumbria LA22 OLP, UK (Received 10 December 1996; accepted 24 March 1997)

Abstract

The buffering capacity of stream water is related to its alkalinity. Around pH 5.7 alkalinity is zero and below this value in Northern Hemisphere streams, changes in the structure of freshwater communities have been observed. There is a general impoverishment of the biota (Sutcliffe and Carrick, 1973; Groom and Hildrew, 1989). In the case of salmonid fish, a combination of acidity and high levels of inorganic aluminium have been found to be lethal; though these effects can be ameliorated by high calcium-ion concentrations (Mason, 1990). Some aquatic invertebrates suffer osmoregulatory problems in acid waters owing to the combination of low pH and elevated aluminium levels (Sutcliffe and Hildrew, 1989; Twitchen, 1990). However, the deleterious effects of anthropogenic acid waters on freshwater communities may also be due to indirect, food-web interactions (Groom and Hildrew, 1989). Allochthonous, organic litter is a major energy source for heterotrophic streams (Kaushik and Hynes, 1971). Decomposition of litter by aquatic fungi (particularly hyphomycetes) and bacteria enhances its nutritional value to stream detritivores (Sinsabaugh et al, 1985; Chamier and Willoughby, 1986; Findlay et al., 1986; Chamier, 1991). Rates of microbial degradation of leaf litter are significantly reduced in streams of pH _<5.7 compared with circumneutral streams (Mackay and Kersey, 1985; Ailard and Moreau, 1986; Chamier, 1987: Mulholland et al., 1987), associated with significantly lower levels of microbial colonisation (Chamier, 1987: Groom and Hildrew, 1989). Accumulation of aluminium on leaves in acidic streams increases with submersion time, and on some leaf species is three to four times higher after 8 weeks than on leaves in circumneutral streams (Palumbo et al., 1987; Chamier et al., 1989; Groom and Hildrew, 1989). These studies suggested a direct effect of acid-water chemistry on microbial metabolism. In this study, we set out to investigate levels of monomeric aluminium on alder leaves (Alnus glutinosa (L.) Gaertn), which accumulate aluminium when submerged in acid streams (Chamier et al., 1989), and to test the effects of this species of aluminium at pH 5.0 on the growth, sporulation and pectinase production of four species of aquatic hyphomycete fungi.

We investigated, by field and laboratory experiments, the effects of aluminium in an acid stream (pH 5.0) on the growth and sporulation of aquatic hyphomycete fungi which degrade organic litter. The stream water had monomeric aluminium (Aim) concentrations of 9.113.4 ~tM - - f i f t y times higher than a nearby circumneutral stream. Alder leaves submersed in the stream accumulated AI, most of which was tightly bound. Growth rates of four species of aquatic hyphomycetes were altered by inclusion of Alto in the culture medium. On a polypectate substrate, and on low-phosphate medium with glucose, growth rates increased significantly. On a low-nutrient substrate of homogenized alder leaves, growth rates were inhibited by aluminium. The pattern of mycelial growth was found to be different on a polypectate medium including Aim, compared with a control without aluminium. There was a significant increase in hyphai radial growth and a decrease in the hyphal growth unit. The effect resembled the growth of a starved fungal colony. Treatment with Aim decreased pectinase production by the four fungal species tested. The capacity of these species to sporulate was reduced by flooding culture plates with Aim solution. These deleterious metabolic effects were most severe in isolates taken from circumneutral streams and less marked, though significant, in species originating from acid streams. © 1997 Elsevier Science Ltd Keywords." Aquatic hyphomycetes, aluminium, acid streams, fungal metabolism, hyphal growth patterns.

INTRODUCTION Some upland streams in the River Duddon catchment (English Lake District) resemble acid streams in North America and Europe, whose acidity has been exacerbated in the past century by the atmospheric deposition of strong acids arising from anthropogenic emissions. Characteristically, the stream catchment area has acidic, base-poor soils and bedrock. The stream water has very low buffering capacity, but has high levels of labile, monomeric aluminium (Sutcliffe, 1983; Tipping, 1990). *Address for correspondence: Achandunie House, Ardross, by Alness, Ross-shire IV17 0YB, UK. 289

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A.-C. Chamier, E. Tipping

MATERIALS AND METHODS

The stream The study site was Mosedale Beck, (Grid Ref. NY246018,) a tributary of the River Duddon, Cumbria with mean pH 5.0. Details of the site together with major ion concentrations over the autumn/winter period are given in Carrick and Sutcliffe (1983) and Chamier (1987, 1989). Leaf packs Freshly fallen alder leaves were collected in nets placed under a stand of alder trees. The leaves were air-dried at room temperature and 1.5 g were placed in a monolayer in separate litter bags (15× 10cm) with l-mm mesh to exclude grazing invertebrates. Three bags of leaves were tied into plastic gutter tubing (50 cm long; 11 cm diam.) so that the bags did not overlap. Fifteen tubes in five sets of three were lashed to stones in the stream on 23 November 1990. Heavy stones were placed on top of the tubes, which lay in the line of the water current. Samples were taken at 2 or 3-weekly intervals when one tube was removed and placed in a sterile polythene bag. At the same date, two water samples of 250 ml were taken from the stream in acid-washed, stoppered glass bottles to be analysed for pH and monomeric aluminium (Alm). On each sampling date, the stream water temperature was measured with a glass-mercury thermometer. Aiuminium measurements (1) The concentration of monomeric aluminium in 2ml of stream-water sample was measured spectrophotometrically by a 4 rain reaction with pyrocatecol violet (Seip et al., 1984). Measurements were read against a standard curve for Aim (0, 0.01 (×2), 0.02, 0.05, 0.1(×2)/ZM) made for each set of samples. (2) Dilute acid-labile aluminium taken up by sample leaves was estimated by placing a leaf-disc (10mm diam.) in 2ml 10-3M HCI for 24h (DAA124). The leafdisc was removed and the supernatant reacted for 4 min with pyrocatecol violet, as above. Six discs were used which had been cut randomly from leaves taken from the three sample bags. Each disc was treated individually. (3) Total aluminium (Altot) on leaf discs, cut from samples as above, specifically for this purpose, was determined by flame atomic absorption spectroscopy, following acid digestion of the samples (details in Chamier et al., 1989).

area (Chamier, 1987). This isolate came from Upper Crosby Gill. • Alatospora constricta Dyko - - an isolate from the study stream, Mosedale Beck, mean pH 5.0. • Articulospora angulataf, tetracladia Petersen - - an isolate taken from Gaitscale Gill, a tributary of the River Duddon, mean pH 4.9 (Chamier, 1987).

Media Minimal mineral salts solution (MMS): in I litre distilled water: 1 g(NH4)SO4; 0.2 g Na2HPO4; 1.0 g KH2PO4; 0.5g MgSO4; 0.2g FeSOa.7H20; 1.0ml trace elements solution. Low phosphate MMS - - as above with PO4 reduced 1/I00. A g a r - 15g litre -I. Carbon sources: 10 g litre -1 glucose added through a sterilizing millipore filter into autoclaved medium; or 5g litre -~ sodium polypectate (Napp). Aluminium: appropriate dilutions were made into all media using an 0.01 M--stock solution of Al(NO3)3. The range of concentrations of Aim was based on calculations derived from measurements of A1 on alder leaves given in Chamier et al. (1989). The pH of all media was adjusted to 5.0 with 1 M HCI or 1 ra NaOH. Leaf/water agar: 10 g litre-~ wet weight of alder leaves that had been leached in running water for 4 days and blotted dry were homogenised for 1 min at high speed in a blender in Mosedale Beck water to which was added 20 g agar - - all adjusted to pH 5.0 as above. Agar media were dispensed into standard, sterile Petri dishes. Replication There were three replicate plates for each species in every treatment. The culture inoculum was cut with a 7 or 5-mm cork borer from the growing edge of the colony. Three types of experiment were conducted.

Fungi The following species, maintained on 1.5% (w/v) malt extract agar, were used for growth and sporulation experiments:

Experiment 1 Unpolluted streams usually have very low levels of phosphate. Most synthetic media have unnaturally high levels of phosphate which, at pH 5~6, complexes with aluminium to give aluminium phosphate, making the phosphate inaccessible, so that observed effects could be 'phosphate' effects rather than those due to aluminium. High phosphate concentrations have been found to suppress the effects of inhibitors of degradative fungi (Da Costa, 1972). Experiment l, therefore, was designed to compare aluminium effects on the fungi at very low and high phosphate concentrations. They were grown either on a complex polysaccharide substrate, requiring enzymatic degradation (Napp), or a simple sugar (glucose). The pectic polymers in leaf cell walls are degraded by all aquatic hyphomycete species tested (Chamier, 1985).

Tricladium splendens Ingold - - a species found in the circumneutral streams of the Duddon catchment. This isolate was taken from Upper Crosby Gill, mean pH 6.8. Varicosporium elodeae Kegel - - a species widespread in circumneutral and acid streams in the

Experiment 2 In Experiment 1, enhancement of growth occurred in the presence of Alm. This could have been the result of complexation with the medium, or of stimulation of growth from the extra nitrate in AI(NO3)3. Experiment 2 was designed to explore these possibilities.

•

•

Aluminium and aquatic hyphomycetes Ten mm cores were taken from the four species of fungi and placed in 5 ml sterile solution of 8, 40 or 80/zm NaNO3 or 8, 40 or 80/ZM Al(NO3)3 with sterile distilled water as controls. The bottles containing these inocula were shaken for 42 h. Each core was then plated onto half-strength M M S + l g (NH4)SO 4 agar with Napp as a carbon source (5g litre-I), pH 5.0. The colonies were grown for 13 days at 15°C, measured (diameter), then flooded with sterile Al(NO3)3 solution - - 0.5, 1 or 2 mM, for 2 days. The aluminium solution was then discarded and the colonies left to grow for a further 12 days, then remeasured. Controls were flooded with sterile distilled water. The purpose of this methodology was to exclude AI from the medium, thus avoiding complexation during manufacture. Unlike many aquatic fungi, hyphomycetes can take up nitrate from solution (Thornton, 1963), a feature exploited in the first half of the experiment, where the influence of A1 was compared with Na. The second treatment with A1 solution was designed to test the effect of additional AI to growing colonies, as would occur on a leaf in an acid stream. Experiment 3 To test the effect of increasing aluminium concentration on fungi growing on a natural substrate, A1 was added to leaf-water agar so that the final concentrations of A1 were: 0, 0.5, 1.0, 1.5, 2.0 and 2.5mM. To exclude the effects of the nutrients in the inoculum medium, measurements of growth were made from days 13 to 27. Sporulation Aquatic hyphomycetes can be induced to sporulate if submerged in water. At the end of Experiment 2, cores (10-mm diam.) were cut from the experimental cultures just behind the growing edge of the colony. The cores were submersed for 6 days at 15°C in 5 or 7ml sterile water. The bottles containing the samples were shaken gently on a shaker for 10 s. One millilitre of spore suspension was placed in a Sedgewick-Rafter counter and the number of conidia in 60 × 1 litre squares was counted. This was repeated three times. Temperature All growth and sporulation experiments were conducted at 15°C. This is about the optimum temperature for growth and sporulation of aquatic hyphomycetes (Thornton, 1963; Koske and Duncan, 1973). Although measured stream temperatures were much lower than the experimental temperature, growth of colonies at stream temperatures is so slow as to be impractical. We would not expect the effects we observed to be affected by temperature, but cannot prove it here. Pectinase assay Colonies growing on Napp-medium plates were flooded with a 1% (w/v) cetrimide solution and left for 24h. Cleared zones in the medium around the colonies were measured (radius) (Chamier and Dixon, 1982). There were three replicate plates per treatment.

291

Hyphal growth unit (HGU) These were measured for A. angulata using the method of Trinci (1974). Conidia in sterile water were spread on plates of 10ml Napp medium (MMS with 1/10 PO4) overlaid with sterile, uncoated cellophane (300P, kindly donated by BCL Cellophane Ltd) and incubated for 15 h at 15°C. The control plate had no AI in the medium; the test plate had 0.5 mM AI; pH 5.0 for both. A single germling with about four tips was chosen and photographed every half-hour for 8 h. Ambient temperature was 20°C. The total length of hyphae on each photograph was measured by an image analyser, Quantimet 970, and the total number of hyphal tips counted.

RESULTS Table 1 presents routine measurements made on the water of Mosedale Beck over the winter of experimentation. The pH was consistently around 5.0 and monomeric aluminium concentrations were 9-13.4 #M compared with 0.19tzM in a nearby circumneutral stream, Hardknott Gill. Measurements of aluminium on alder leaf discs (10mm diam.) taken from leaves that had been submersed in Mosedale Beck for given periods are presented in Fig. 1. Four treatments were made on these leaf discs to test the leachability of aluminium: (a) pyrocatecol-violet reagent for4 min (removes Aim); (b) the same for 1 h; (c) 10-3M HCI for 1 h; (d) the same for 24h. Neither (a) nor (b) produced positive results. Treatment (c) removed some AI. Treatment (d) provided the results given in Fig. 1 as DAA124 which, after 12 weeks in the stream, accounts for about 30% of the total aluminium on alder leaves. The results of Experiment 1 are presented in Table 2. A 3-way ANOVA was used to test the three main factors involved: aluminium, carbon source and species. The rate of radial increase (colony growth), Kr, was calculated from the mean colony measurements on the three replicate plates. The standard errors of the means were 0 - + 0.94 mm. There was a significant difference (p < 0.001) between Al-present and Al-absent (controls); but the magnitude and direction of the difference depended on the carbon Table I. Water temperature, pH and monomeric aluminium concentration of water in Mosedale Beck over the experimental period which began on 23 November 1990. During that period, pH and Aim were also measured in water from Hardknott Gill, a contrasting, nearby tributary of the River Duddon

Sample date Start 3 weeks 6 weeks 8 weeks 10 weeks 12 weeks Hardknott Gill

Temp.°C

pH

#M Aim

4.5 2.0 4.0 3.0 3.0 3.0

5.05 5.01 4.95 4.98 5.02 4.9 6.69

9.13 8.96 13.37 12.59 10.05 10.26 0.19

A.-C. Chamier, E. Tipping

292 50 _--.= < 40

31)

20

10

2

4

6

8

10

12 WEEKS

Fig. 1. Aluminium in alder leaves submersed in an acid stream (pH 5.0) for 12 weeks. ( 3 - - 0 Total aluminium (Altot),/zg per leaf disc. 0 - - 0 Labile aluminium from a leaf disc submersed for 24 h in 10-3M HCI (DAAI24), #g per leaf disc. Area of leaf disc: 79.3 sq mm. Air-dried weight of leaf disc: 3.5 mg + 0.35.

source (p<0.001). For Napp with standard PO4, the Al-present plates had higher growth by 0.34 mm day-l, on average, than the Al-absent plates. For Napp with low PO4, the Al-present plates again had higher growth, but by 0.26 mm day-l on average. For glucose with standard PO4, the Al-present plates had slower growth than the Al-absent plates - - an average of 0.2 mm day -l lower. However, for glucose with low PO4, the Al-present plates again had higher growth, by 0.51 mm day -1, than the controls with A1 absent. To test whether the Aim content of the low-phosphate Napp medium in Experiment 1 had an effect on the capacity of the fungi to sporulate, discs of mycelium were submerged in stream water of contrasting pH

(Table 3). Analysis of the data was made by a 3-way ANOVA of the three factors involved: stream water, aluminium and species. Because the observations were counts, these were square-rooted before analysis to homogenise the variance. There was no significant difference between aluminium absent or present; nor any significant interactions between aluminium and the two other factors. However, there was stream-species interaction (p<0.001). The table of species' means in each stream and the standard errors of differences between these means, indicate that counts of A. constricta are not significantly different between the two streams; but that those of V. elodeae, T. splendens and A. angulata are different; the first two having higher counts in Hardknott Gill, but the last having higher counts in Mosedale Beck. Table 4(a) presents the results of part 1 of Experiment 2 - - where the four fungal species were plated onto Napp medium after 2 days in a Na or A1 bathing solution. Two 2-way ANOVAs were carried out with two factors: solution and species. In this analysis the different amounts of Na and AI were ignored and assumed to be replicates, in order to obtain some between-replicate variability. Kr measurements were calculated as above, with the same standard error range. There was a significant difference (p<0.001) in Kr between the Na-grown and the Al-grown cultures - the Al-grown being smaller than the Na-grown for all species. However, the magnitude of the difference was species-dependent. For the three species, T. splendens, A. constricta and A. angulata, the difference was 0.06, 0.08 and 0.04 mm day-l, respectively; but for V. elodeae the difference was 0.12 mm day -~. For the three species, V. elodeae, T. splendens and A. constricta, the growth in the Na inoculum was identical to that in the control inoculum; but for A. angulata, the growth in A1 inoculum was identical to the control. The concentration of AI made no significant difference to results in this part of Experiment 2. In part 2 of Experiment 2, the plates from part 1 were flooded with AI solutions for 2 days and after a further 12 days growth, Kr was measured again. (Varicosporium elodeae sporulated during flooding and was subject to a spore count rather than a Kr measurement, Table 6). A paired t-test was carried out to compare the beforeflooding with the after-flooding rates of growth. Two 2-way ANOVAs were carried out on the change in growth rate with two factors: solution and species. The

Table 2. Radial growth rate, Kr, (mm week -I over 5 weeks) of four species of aquatic hyphomycetes grown on agar plates made up with standard phosphate or low phosphate (1]100) minimal mineral salts solution and either Napp (5 g litre-I) or glucose (! 0 g litre-1) as the carbon source; pH 5.0. Control plates contained no aluminium; test plates contained 500/~M Aim. There were three replicate plates per treatment. Kr was calculated on the mean measurement of colony radii. Standard errors of means: 0 - ± 0.94 mm

Kr (mm week -1) Carbon source Napp Napp low PO4 Glucose Glueoselow PO4

V. elodeae Control AI 6.8 3.9 3.0 2.5

7.21+6% 4.21+8% 2.75-8% 3.15+26%

T. splendens Control AI 5.0 4.1 1.5 1.5

5.35+7% 4.35+6% 1.38-8% 1.9 +27%

A. constricta Control AI 4.0 3.0 2.6 3.2

4.3 +8% 3.24+8% 2.37-9% 3.84+20%

A. angulata Control AI 6.1 3.5 2.2 2.8

6.47+6% 3.75+7% 2.0 - 10% 3.6 + 13%

Aluminium and aquatic hyphomycetes Table 3. Spore counts from cultures grown on low PO4 medium and Napp substrate with or without 500 pM AI= in Experiment I. Ten mm discs of mycelium were submersed in 7 ml sterile water from Hardknott Gill (pH 6.7) and Mnsedale Beck (pH 5.0). Counts are of the number of spores in 60 x llz litre chambers of a Sedgewick-Rafter counter

Species V. elodeae V. elodeae + AI T. splendens T. splendens + A1 A. constricta A. constricta + A1 A. angulata A. angulata + AI

Hardknott Gill

Mosedale Beck

186 180 52 48 96 106 110 114

100 80 26 24 98 116 130 134

different amounts of N a and A1 were ignored in these analyses and assumed to be replicates in order to obtain some between-replicate variability. The results showed that flooding significantly increased (p <0.001) the rate of growth. For the species tested, the increase was greater in the AI inoculum, 0.15mm day -t on average, than in the N a inoculum, 0.06mm day -1. The increase in growth differed significantly ( p < 0 . 0 0 1 ) between the three species: an increase of < 0.09 m m d a y - t for T. splendens and A. angulata; and an increase of 0.17mm day -1 for A. constricta. The effects of AI concentration were inconsistent. Where effects were significant, AI concentration was above 8/LM (i.e. 40 and 80/LM). When pectinase activity was tested on the cultures in Experiment 2 (Table 5), T. splendens plates, other than controls, showed negligible activity. By contrast, A. constricta and A. angulata showed some enzyme inhibition after treatment with AI compared with controls, but severely deleterious effects appeared only in cultures treated with the highest concentrations of A1 overall.

293

The final capacity of cultures in Experiment 2 to sporulate was tested (Table 6). As V. elodeae sporulated during flooding, counts were made of germinated spores on the plates. To analyse the results, a 2-way A N O V A was carried out with two factors: inoculum and species. The results showed that there was a significant interaction of inocula with species (p < 0.001). For V. elodeae and T. splendens, the Na inoculum had lower counts than the control inoculum; and the AI inoculum had lower counts than the N a inoculum. For A. constricta and A. angulata, there was no significant difference between the N a inoculum and the control; but the AI inoculum gave lower counts than the other two. Since inhibition of growth rate is a more usual response of fungi to toxins than stimulation, it was decided to compare the hyphal growth unit ( H G U ) of A. angulata grown on Napp, p H 5.0, with and without 0.5mM Aim. Figures 2(a) and (b) illustrates the differences in growth between mycelial colonies of germlings subjected to the two treatments. Figure 3 quantifies these differences. On the medium with Aim, there is accelerated mycelial extension and, after 6h, a significant increase in the production of new hyphal tips. The H G U of this colony decreases after 6 h, compared with that of the control. After 22 h, differences between the colonies were very marked. That treated with A1 was twice as extensive radially as the untreated control. The latter had evenly distributed branches and hyphae, whereas in the former, intensive branching was concentrated centrally around the conidium with more sparsely distributed, very extended leading hyphae. Finally, in Experiment 3, the effects of increasing Aim concentrations on growth were tested on a very low nutrient medium - - leaf and stream-water agar. The Kr was measured between 13 and 27 days of growth to exclude the effects of nutrient supply from the inoculum core (Fig. 4). The results were analysed by 2-way

Table 4. (a) Initial radial growth rate, K,, (mm day i) for 13 days of cultures in Experiment 2, where the inoculum discs were submersd either in NaNO3 or AI(NO3)3 solutions for 42 h then plated onto an Napp substrate. (b) Subsequent K, of cultures in (a) after being flooded for 2 days with AIm solutions and grown for a further 12 days, with percentage change from the initial Kr. There were three replicate plates per treatment. Kr was calculated on the mean of colony radii measurements. Standard errors of means: 0- + 0.94 mm

(a) lnoculum

Initial K~ V. elodeae

T. splendens

A. constricta

A. angulata

Water (control) 8/zM Na 40 tZM Na 80/zu Na

0.85 0.85 0.85 0.9

0.58 0.58 0.58 0.58

0.46 0.46 0.46 0.46

0.73 0.77 0.77 0.77

8 #M A1 40 #M AI 80/zM AI

0.77 0.73 0.73

0.54 0.54 0.5

0.38 0.38 0.38

0.73 0.73 0.73

(b) lnoculum

T. splendens

Kr after 2 days flooded with AI solution + 12 days % change A. constricta % change A. angulata

% change

Water (control) 8/zM Na 0.5mM AI 40/ZMNa 1 mM AI 80/*M Na 2mM A1

0.79 0.64 0.64 0.64

+ 35 + 10 + 10 + 10

0.61 0.61 0.54 0.54

+ 32 + 32 + 17 + 17

0.73 0.77 0.77 0.82

0 0 0 +6

8/LMAI 0.5mM AI 40/zM AI I mM AI

0.64 0.64 0.64

+ 18 + 18 +28

0.61 0.61 0.61

+60 +60 +60

0.79 0.86 0.89

+8 + 18 +22

80/zi Al 2 m i Al

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A.-C. Chamier, E. Tipping

Table 5. After the treatments described in Table 4(a) and (b), the culture plates were tested for pectinase activity. They were flooded with a 1% (w/v) solution of Cetrimide and the radius of the cleared zone of substrate around the fungal colony is given

lnoculum

AI solution

T. splendens

Water (control) 8/xM Na 40 #M Na 80 ~tM Na

0.5 mM 1 mu 2 mM

3 mm (strong) V. weak V. weak V. weak

8 #M AI 40 #M A! 80/ZM A!

0.5 mM 1 mM 2 mM

V. weak V. weak V. weak

A. constricta 9 mm 7 mm 7 mm 7 mm

,4. angulata

(strong) (strong) (strong) (strong)

5 mm 3 mm 3 mm 3 mm

5 mm (strong) 5 mm (strong) Diffuse, weak

(strong) (strong) (strong) (strong)

3 mm (strong) 3 mm (strong) Diffuse, weak

Table 6. Sporulation of fungal cultures after 2 days flooding with Aim solution (V. elodeae), followed by 12 days growth in Experiment 2. A 10-mm diam. core was cut from the edge of the colony and incubated in 5 ml sterile distilled water for 6 days to induce sporulation. Counts of the number of spores in 60x 1 /~1 chamber of a Sedgewick-Rafter counter

lnoculum

AI solution

V. elodeae spores/plate

T. splendens

A. constricta

A. angulata

Water (control) 8 #M Na 40#M Na 80 #u Na

0.5 mM 1 mu 2 mM

379 ± 15 183 -4-12 101 ±8 79 ± 6

80 82 4 2

242 224 232 268

274 263 266 270

8 #M AI 40 #M AI 80 #M AI

0.5 mM I mM 2 mM

65 ± 5 69 + 5 68 + 4

0 6 0

42 51 60

200 168 186

A N O V A using two factors: aluminium concentration and species. The two-way interaction between concentration and species was used as the residual term. There was a significant difference in growth rate between the four levels of aluminium concentration (p<0.001). Between 0.0 and 0.5, the rate fell by an average of about 30/ZM d a y - l ; between 0.5 and 1.5 by about 150#M d a y - I ; and between 1.5 and 2.5 by about 130/ZM day -t. There was also a significant difference between species (p < 0.01). The highest growth was for A. constricta, followed by A. angulata, F. elodeae and T. splendens.

DISCUSSION Monomeric aluminium levels in Mosedale Beck were consistently high over the winter of experimentation; about fifty times higher than those in a nearby circumneutral stream. However, methods which specifically measure A1m were ineffective in removing this form from alder leaves which had been submersed in Mosedale Beck for periods up to 12 weeks. It required fairly drastic treatment (24h in 10 -3 M HCI) to remove measurable quantities of inorganic AI which, initially, represented about 17% of the total AI on experimental leaves; and about 30% after 12 weeks. Total AI values roughly doubled every 2 weeks - - confirming results from a previous study (Chamier et al., 1989). There, it was indicated that whilst some of the total Al may be due to adhering aluminium/silicate particles, most is monomeric aluminium taken up by the leaf and degradative micro-organisms. Sites for adsorption or complexation would increase as degradation of cell-wall

polymers in the leaf progressed and microbial biomass increased. Salim and Robinson (1985a) found that undecayed leaves placed in a solution of aluminium adsorbed the element at high levels for 1 or 2 days; after which little more was taken up. Therefore, the increase in aluminium levels on alder leaves with time detected in our studies, together with the evidence that it is tightly bound, indicates a progressive increase in sites for uptake. Pectic polymers, for example, are readily available in primary plant cell walls and are a major substrate for aquatic hyphomycetes (Chamier and Dixon, 1982). Endopolygalacturonases and pectin esterases secreted around p H 5.0 by these organisms would depolymerise the galacturonic acid chains and offer increasing numbers of carboxyl groups for cationic binding. Aluminium complexes with organic acids (Cronan et al. 1986; Tipping et al., 1988a,b; Furrer et al. 1990) have suggested that organic acids complexed with A1 (III) are protected from decomposition. In a previous study, aluminium levels on alder leaves increased as calcium levels declined (Chamier et al., 1989). Leaves with less labile calcium (e.g. oak) attract much lower levels of aluminium. Salim and Robinson (1985b) found that whereas most divalent cations competed with A1 for uptake by leaves and reduced the levels of A1 removed from solution, calcium did not compete. In alder leaves, leaching of calcium from cell walls, where it exists as calcium pectate, may involve exchange with aluminium and/or protons. The effects of the aluminium in stream water and leaves on aquatic hyphomycetes would depend on the extent to which it is directly adsorbed onto hyphal surfaces, or accumulated in the cytoplasm by uptake from the water;

Aluminium and aquatic hyphomycetes

295

(a)

I

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Fig. 2. Hyphae of germlings of A. angulata at To (above) and after 8-h growth (below) grown on Napp medium (a) and Napp with 0.5 mM AI (b). Details in Materials and Methods. or the extent to which A1 complexation with substrate molecules either inhibits uptake or interferes stereochemically with degradative mechanisms, effectively starving the organism. Experiment 1 contrasts the effects of AI complexation with a polymeric substrate, with AI effects on a noncomplexing substrate, glucose. There is, on either substrate, an interaction between AI, the carbon source and the level of phosphate; though the presence of A1 always brings about a difference significant from that of the control. On the polymeric substrate, higher levels of phosphate stimulate growth compared with glucose; but the presence of AI 'stimulates' growth still further on the former medium. At low phosphate levels, lower growth rate is associated with a lower AI 'stimulation'. As natural streams have very low phosphate levels, the latter situation would be more relevant to this study. The 'stimulation' of growth by A1 appears to be a starvation response. The radial growth rate of fungi can be increased when a colony is starved (Trinci, personal communication); but our results cannot distinguish between increase in colony diameter and increase in biomass. On the simple-sugar substrate, glucose, higher PO4 had an inhibitory effect on growth rate compared with the polymeric carbon-source. There are two possible explanations for this: (1) unnaturally high levels of phosphate depress growth of aquatic hyphomycetes, a phenomenon encountered by Iqbal (1972) and Chamier (1980). On the glucose substrate, aluminium has an inhibitory, toxic effect which reinforces the high PO4 effect, but on low PO4, AI, appears to produce a starvation

effect. (2) On the polymeric substrate without A1, enzymic depolymerisation works through a respiratory mechanism which utilises phosphate, thereby lowering its concentration and its inhibitory effect. Haug (1984), however, found that AI binds to the plasma membrane and affects the action of ATPases and ion uptake (Haug and Caldwell, 1985) which would interfere with enzymatic activity, resulting in starvation. Similar effects have been found to affect P metabolism in higher plants (Wright and Donahue, 1953; Jens~n et al., 1989). These results are similar to those obtained by Greger et al. (1992a,b) when investigating A1 effects on the unicellular, green alga Scenedesmus obtusiusculus. High PO4 precipitated with AI to form AIPO4 at cell surfaces and interfered with ion transport across membranes. This caused depression of growth. The intercellular level of AI was higher in low PO4 and precipitated with PO4 within cells, interfering with their division processes. The interpretation of the A1 effect on fungal growth on Napp as being due to reduced availability of substrate and slow starvation, and to effects brought about by hyphal adsorption or accumulation, is supported by the spore counts given in Table 3 and by the pectinase assays reported in Table 5. Growth rates measured after bathing mycelial inoculure with sodium or aluminium nitrate solutions (Experiment 2), indicate that fungi can adsorb and absorb aluminium from stream water and that it causes inhibition of growth. Subsequent growth, after flooding with AI solutions of increasing concentration, resulted in severe inhibition of T. splendens, with cessation of pectinase production. The capacity of this species to sporulate was almost wholly curtailed by aluminium.

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Fig. 4. Kr (ttm day -l) of fungal species grown on leaf/water agar at increasing Aim concentrations, with percentage overall decline. Measurements were made from 13 to 27 days' growth. 0 - - 0 A. constricta (-35%) 0 - - 0 A. angulata (-22%) O - - - 0 V. elodeae (-54%) O - - - O T. splendens (-66%).

Deleterious effects appear to arise from adsorption and absorption from solution rather than via the substrate. Tricladium splendens is a species common in circumneutral streams and is not found in the acid streams of the Duddon catchment. Initial inhibition of growth of V. elodeae by AI led to a reduction by one-sixth of its capacity to sporulate where there had been double AI treatment, and increasing sensitivity where initial treatment had been with sodium nitrate. These effects could only have been due to adsorption and absorption. This species occurs in circumneutral streams of the Duddon catchment, and at low levels in acid streams (Chamier, 1987). O f the two species taken from acid streams, A. constricta was more sensitive to AI in growth, sporulation and pectinase production than A. Angulata. However, unlike T. splendens, they showed starvation-type growth after flooding (i.e. higher Kr than controls) suggesting a different metabolic response to AI. There was no inhibition of sporulation where the inoculum had been bathed in Na, and a modest inhibition of enzyme activity by aluminium. Articulospora angulata, in particular, showed only minor metabolic effects of absorption and adsorption of AI from solution.

It has been suggested throughout this discussion that the apparent stimulation of colony growth by AI is a starvation response. The growth pattern of A. angulata was changed by the presence of AI in the growth medium (Figs 2 and 3), with an increased Kr, but a reduced HGU. The appearance of the colony treated with AI after 22-h observation, with intensive branching concentrated around the spore, and very extended leading hyphae with few branches, suggested exploratory hyphae escaping from a depleted nutrient source, as described by Dowson et al. (1989) for mycelium growing on a patchily distributed resource. However, this is a response elicited where nutrient levels are comparatively high and there is some substrate availability. Aluminium produces an inhibitory effect on leaf/ water agar, where nutrient levels and substrate availability are very low (Fig. 4). Again, the effect is most marked on the species common in circumneutral streams. The deleterious effects of AI on the aquatic hyphomycete species tested were all in the order T. splendens > V. elodeae > A. constricta > A. angulata. Similarly, in experiments to test the toxicity of cadmium to aquatic hyphomycetes, Abel and B/irlocher (1984) found species' differences in tolerance to cadmium.

Aluminium and aquatic hyphomycetes Species originating in hard-water streams were more sensitive to cadmium than those originating in softwater streams. A further example of species-specific response is that of algae taken from an acid stream of low alkalinity which were found to be less sensitive to AI than species taken from an alkaline stream of high alkalinity (Lindemann et al., 1990). Similarly, Tipping and H o p w o o d (1988) estimated that the liverwort Nardia compressa (Hook.) Gray, which grows in the acid streams of the Duddon catchment, contains levels of A1 comparable with the highest values found on submersed alder leaves in our study. Some ability to tolerate AI must be a condition of survival for organisms in streams subject to acid deposition. Although aquatic hyphomycete species in anthropogenic acid streams show greater tolerance of A1 than species from circumneutral streams, conditions are nonetheless, sub-optimal. They survive in this habitat because they can sporulate. A species like V. elodeae from a circumneutral stream could tolerate acid conditions less well; but it grows and sporulates very quickly, which may account for its ubiquitous presence at low levels in acid streams. Species common in circumneutral and alkaline streams, like T. splendens, would not survive in a stream like Mosedale Beck. If species from acid streams are less affected by the uptake of A! from stream water than by bonding of A1 with leaf cell-wall molecules, the consequences would be more pronounced on leaf species like alder, with labile calcium and very high levels of A1, than on a species like oak, with little leaching of calcium and less than half the levels of A1 (Chamier et al., 1989). A well-replicated field study (Chamier, 1987) showed that this is indeed so. Levels of colonization by aquatic hyphomycetes were consistently higher on oak than on alder; and whereas weight-loss (degradation) of alder in acid streams was four times slower than in circumneutral streams, degradation of oak was only twice as slow. The effects of aluminium on aquatic hyphomycetes appear to act through two pathways. Species common in circumneutral/alkaline streams appear to be more affected by uptake of AI by hyphal adsorption and absorption; resulting in deleterious metabolic effects on sporulation and on enzyme production, which induces starvation. Species originating in acid streams appear less affected in sporulation and enzyme production by hyphal uptake of AI; but more affected by stereochemical blocking of polymeric substrates by ion-exchange with AI, which interferes with degradative mechanisms and results in starvation. However, the responses are complex and depend, as well, on the availability of other elements, including calcium and phosphate.

ACKNOWLEDGEMENTS For help and/or advice with this study, we thank: Jean Lishman, Colin Woof, Bernard Simon, Benjamin James, Dr John Hilton, Dr Grahame Hall and particularly Margaret Hurley of the I.F.E.; Peter Dixon and Stephen

297

Janes of Royal Holloway College, University of London; Professor A. P. J. Trinci and Dr M. Wiebe of the University of Manchester; Dr Gillian Butler of the University of Birmingham and Dr Simon Archer of Imperial College, London for a critical reading of the manuscript.

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