C. T. INGOLD President 1953 and 1971
Vol. 85, Part 2
September 1985
[ 193 ]
Trans. Br. mycol. Soc. 85, (:Z), 193-199 (1985)
Printed in Great Britain
EFFECTS OF SULPHIDE ON SURVIVAL OF AERO-AQUATIC AND AQUATIC HYPHOMYCETES By J. I. FIELD AND J. WEBSTER Department of Biological Sciences, University of Exeter Sulphide concentrations have been measured at the mud surface in two ponds and an adjacent river within which aero-aquatic fungi grow. Studies on the survival of aero-aquatic and aquatic Hyphomycetes on beech leaf disks under anaerobic conditions and subjected to sulphide concentrations ranging from 5-20 mg 1-1 at pH 4 and pH 7 for 24 h indicated that the latter were more sensitive to sulphide concentration. Although not confined to such habitats, aeroaquatic fungi are frequent and abundant on leaves, twigs and other liner dredged from the mud surface of stagnant ponds and ditches (Fisher & Webster, 1981; Abdullah & Fisher, 1984). Many ofthem are capable of growth at low levels of dissolved oxygen (Fisher & Webster, 1979) and some can survive prolonged periods under strictly anaerobic conditions (Field & Webster, 1983). In contrast many Ingoldian aquatic Hyphomycetes frequent wellaerated habitats such as rapidly-flowing streams (Webster & Descals, 1981), and appear unable to survive for long periods in the absence of oxygen. Many of the habitats of aero-aquatic Hyphomycetes smell of hydrogen sulphide. This gas is a strong reducing agent and its presence is indicative of oxygen deficiency within the sediment and in the water immediately above. It is therefore of interest to determine whether aero-aquatic Hyphomycetes are more tolerant of high concentrations of hydrogen sulphide and of the bisulphide ion to which they may be rather frequently exposed in their natural habitats than are the Ingoldian aquatic Hyphomycetes. There is considerable evidence for the toxicity of soluble sulphides within the range 0'1-1 '0 mg 1-1 to the roots of rice (Hollis et al., 1975) and also to Citrus roots (FDrd, 1973). Animal mortality has been reported at: sulphide concentrations around 1 mg 1-1 orIess (Oseid & Smith, 1974; Smith, Oseid, Kimball & EI-Kandelgy, 1976). There have been very few studies of the effects of H 2S on fungi at ecologically probable concentrations. In this paper we report on the concentrations of sulphide measured in two small ponds and an adjacent estuary, and on the effects of sulphide concentration in the range 5-20 mg I-Ion the survival of mycelium of six aero-aquatic and three Ingoldian Hyph.omycetes in beech leaf disks. The study Df sulphide equilibria in aquatic
systems is complex. Hydrogen sulphide is moderately soluble in water (4'132 g 1-1 at 20°C) and behaves as a weak electrolyte; in solution, it undergoes a primary and a secondary dissociation according to the equations: H2S~H++HS-, HS-~H++S-.
The proportion of H 2S and HS- is dependent on H+ concentration. If the pH is lowered more H 2S molecules are present while a higher pH increases the proportion of HS- ions. At pH 7 the proportions of H 2S and HS- ions are about equal. Estimates in the literature of the amount of hydrogen sulphide occurring in the water phase of various aquatic habitats vary enormously; from < 0'001 up to 0·64 p.p.m. in rice paddy fields (Allam, 1971; Allam, Pitts & Hollis, 1972), from < 1 up to 30 p.p.m. in submerged soils (Ayotade, 1977), and up to 700 p.p.m. in the interstitial water of some sandy saline sediments (Fenchel & Riedl, 1970). The total sulphide content of submerged soils, mainly in the form of ferrous sulphide, may reach concentrations in the order of 150 p.p.m. (Allam et al., 1972), while concentrations in excess of 2000 p.p.m. have been reported (Harter & McLean, 1965). Some of these p.p.m. figures represent mg 1-1 of soil solution, but in others, it is not clear how they relate to a weight of soil or sediment. MATERIALS AND METHODS
Measurement of sulphides in solution Two Orion electrodes were used, model 94-16, a silver/sulphide electrode and model 90-02, a double junction reference electrode. Both were linked to a Pye digital pH/mV meter, set to read millivolts. For the analysis ofsulphide, anti-oxidant buffer (SAOB) (Orion Research, 1969) was used to
Vol. 85, Part 1, was issued 16 August 1985 7
MYC
85
194
Sulphides and aquatic fungi
adjust the ionic strength of the standard and sample solutions. Calibration was performed using A.R. sodium sulphide (Na 2 S . 9H20) dissolved in SAOB titrated against a standard solution of lead nitrate.
Sites investigated Efford lake is a small brackish pond, of variable depth to about 3 m, surface area ca 1 ha, near a salt marsh by the R. Erme, Devon at SX 619491. A drain to the river permits estuarine water to flood the pond at high tides. The pond is surrounded by trees including Quercus petraea (Matt.) Liebl, Fagus sylvatica L., Rhododendron ponticum L., and Betula pendula Roth., and the bank vegetation inclUdes Scirpus maritimus L., Lythrum salicaria L., and Iris pseudacorus L. The organic detritus was clearly anaerobic and smelt strongly of H 2 S. The only aero-aquatic fungus isolated from the submerged litter was Pseudaegerita foliicola ined. Sequer's Bog is set in an irregular marshy depression amid trees near to Sequer's Bridge, at SX 632517. It has an area of about 900 m 2, and any water it contains is usually less than 0'5 m deep. It contains willow (Salix cinerea L.) scrub, and generally rather few other marsh plants but is entirely surrounded by trees which include Alnus glutinosa (L.) Gaerm., Acer pseudoplatanus L., Quercus petraea, Fagus sylvatica, Ulmus procera Salisb. and several Salix spp. which provide an abundant litter layer. Many species of aero-aquatic Hyphomycetes were isolated from this site, including BeverwykeUa pulmonaria (Beverwijk) Tubaki, Helicodendron conglomeratum Glen-Bott, H. luteoalbum Glen-Bott, H. triglitziense (Jaap) Linder, Helicoon fuscosporum Linder, H. richonis Boudier and Pseudaegerita foliicola. The site obtains water by drainage from the surrounding fields and woodland, and usually dries out in the summer, though tends to remain quite marshy; at other times, it is variably flooded. River Erme Site is at the edge of the river, about 30 m from Sequer's Bog. Sulphide levels in the bottom water were measured here to provide comparative data for those taken at the other two sites. The site lies in a sheltered niche at the edge of the river, and is protected from some of the water flow by fallen alder logs. In dry spells, the water level is generally quite low, though it is greater when the river is in flood, and is usually between 0'2 and 0'5 m. The water is fresh at all states of the tide. No leaves were collected from this site, but fungi isolated from wood included Aegerita candida Pers., Aegeritospira tortuosa (Bourd. & Galzin) ined., Candelabrum brocchiatum Tubaki, Clathrosphaerina
zalewskii van Beverwijk, Peyronelina glomerulata Fisher, Webster & Kane, Pseudaegerita cf. corticalis and Spirosphaera flori/ormis van Beverwijk. The flora of aero-aquatic fungi thus differs completely from that found in Sequer's Bog.
Collection of sulphide samples in the field Water samples were collected at or just above the litter-water interface at approximately monthly intervals between Nov. 1979 and Sept. 1980. In deeper water, a brass water-sampler was used, and the sample was collected in a 100 cm 3 bottle located inside the sampler. Where the water was shallow, samples were taken using a large syringe. At each site, three samples were taken from the edge ofthe water, and a further three samples about 3 m from the shore; three samples only were taken from the edge of the river. Care was taken in sampling that the bottles were absolutely full before the cap was replaced. Sulphide, especially hydrogen sulphide, in water samples collected in this way is stable for only a very short period, even if considerable precautions are taken to prevent oxidation, as a consequence of the inevitable disturbance and exposure to air and aerated water. To minimize this effect, allowing only time for most of the sediment to settle, 50 cm3 of the water sample was pipetted into 50 cm3 of 50 % SAOB in a 100 cm3 bottle, ensuring that the tip of the pipette was kept below the fluid level. This stabilized the sulphide for a reasonable period. Care was taken to avoid the inclusion of too much sediment in the sub-sample as this could have an unpredictable effect on the results. The sulphide level was determined as soon as possible thereafter (within 2-3 h).
Fungi used Aero-aquatics isolated from Sequer's Bog included B. pulmonaria and H. conglomeratum from oak (Q. petraea) leaves, H. luteo-album and Helicoon fuscosporum from beech (Fagus sylvatica) leaves and H. triglitziense from an oak (Quercus sp.) leaf. Pseudaegeritafoliicola II was isolated from a beech leaf at Sequer's Bog, and isolate I from an oak (Quercus cf. cerris) leaf in Efford Lake. The sulphide tolerances of these species were compared with those of three species of Ingoldian aquatic Hyphomycetes. Freshly-isolated Anguillospora rosea Descals (ined.) isolated from foam and two isolates of Tricladium splendens Ingold and a laboratory culture of A rticulospora tetracladia were used.
J. I. Field and J. Webster Treatment of leaf disks Beech leaf disk inocula for each species were prepared as described by Field & Webster (1983). Tolerance offungi to H 2 S and HS- was tested in buffer solutions at pH 4 and 7. The buffer used must be capable of operating at both pH levels and be non-toxic. A suitable growth rate of mycelium from leaf disks could be obtained using a buffer containing 3,3-dimethylglutaric acid and sodium hydroxide (McKenzie, 1969). The buffers were sterilized by autoclaving at 121° for 15 min. This treatment was found not to affect the pH. To avoid problems associated with the oxidation of hydrogen sulphide in the buffer solutions, these were deoxygenated before use as described by Field & Webster (1983). Medical fiat bottles were autoclaved and allowed to cool and fifty inoculated leaf disks placed in each. Air was then displaced from the bottles by passing nitrogen through for a few minutes before they were filled with deoxygenated buffer. Each bottle was filled to the brim with buffer and the cap screwed on tightly with great care to ensure that no air was trapped inside. During the dispensation of the buffer, deoxygenated white spot nitrogen was continually bubbled into the fiask from which it was being withdrawn. Adjustment of sulphide concentration Medical fiat bottles had previously been fitted with metal screw-caps through which a tiny hole had been drilled to permit a hypodermic needle to pass through. Each cap contained a rubber liner which was sealed to the inside of the cap using aquarium sealant (silicone rubber) which was able to withstand autoclaving. Hydrogen sulphide from a gas cylinder was fed through a tube into a football bladder which acted as a reservoir and also provided a small positive pressure to prevent the ingress of air into the system. A connecting tube was fitted with a T -piece and a short length of tubing closed with a self-sealing cap. Hydrogen sulphide in the required quantities was withdrawn by a gas syringe and introduced into the medical fiat bottle by a hypodermic needle inserted through the hole in the cap which was then sealed with aquarium sealant. The medical fiats were laid on their sides and left for 24 h in the light at 20° ± 2°. At the end of this period, the sulphide concentration in each fiat was determined and the leaf disks harvested and thoroughly washed in sterile distilled water. The 50 disks from each bottle were then plated out onto agar, the aero-aquatic Hyphomycetes onto
195
0'1 % MEA and the Ingoldian aquatic Hyphomycetes onto 2 % MEA. The numbers of disks from which mycelium grew were recorded. The results were expressed as the mean of 4 replicates (see Table 2). Disks from which mycelium failed to grow out were replated, to prevent colonization from adjacent disks. All the plates were kept in plastic bags to avoid desiccation and were kept in the light at 20° ±2° for up to 2 months. Preliminary experiments suggested that most species were able to withstand 2 mg 1- 1 sulphide, and that at levels of 5 mg 1- 1 of sulphide and above, death followed after 24 h exposure. This was therefore selected as an appropriate experimental time. It seemed likely that some sulphide must penetrate the leaf disks within this period and possibly even the fungal mycelium itself. An upper limit of 20 mg 1- 1 was used in these experiments and although appreciably higher than anything detected in the water of our sites it is within the sulphide level which has been reported from similar habitats elsewhere (Ayotade, 1977). Most sulphide production takes place within the sediment in shallow waters (Hallberg, 1973), so that leaves buried in the sediment are exposed to higher sulphide levels than those actually measured in the water immediately above.
RESULTS
Field concentrations of sulphide As expected, the sulphide levels at the river site were lower than those obtained in either Efford Lake or Sequer's Bog (Table 1). Only in May was there an appreciable rise in the sulphide level in the river, when it reached a peak of 1·6 mg 1-1 • There had been little rainfall and the water level and river fiow rate were low. Rise in temperature could have also contributed to the rise in sulphide level. In Efford Lake, the highest sulphide levels were again found about this time, reaching their maximum of 7'5 mg 1-1 , and relatively high levels persisted throughout the summer months. It was not possible to relate these figures to comparable ones from Sequer's Bog, as by this time the bog had dried up. The highest levels of sulphide measured in the bog (2-8-3'2 mg 1-1) were found in the Sept. and Nov. samples. These corresponded to a secondary maximum in Efford Lake (6-5 mg 1- 1 ), and may be due to an inftux of leaf litter. The lowest sulphide levels generally corresponded to periods of high rainfall, especially between Dec. 1979 and Mar. 1980; this effect was less noticeable in Efford Lake. 7-2
Sulphides and aquatic fungi
196
Table 1. Soluble sulphide concentrations from natural habitats (mg 1-1)
Date
18.11.79 30 .11.79 16.12·79 lS·01.80 03.02.80 18.02.80 18.03·80 17.04.80
Posn. Edge
3m Edge 3m Edge 3m Edge 3m Edge
3m
Edge 3m Edge
3m
Edge
3m
14.0 5.80
18.oS·80 27·0S.80 17·06.80 18,07·80 22.08.80 29.09·80
Edge 3m Edge
3m Edge
3m Edge
3m Edge
3m Edge
Temp.
10 10 11 11 9 9 2 2 8'5 8'5 7'5 7'5
3 3 13 13
S-
6,6
1'1 3'2 1,8 2·8 < 0'1 0'2 < 0'1 0'2 < 0'1 0'1 0'3 0'7 0'1 O'S 0'7 1'4
6'4 6'S
6'4 6'7 6'7 7'0 7'0 6·8 6'4 7'0 6'8 6'S 6'S
14'5 14'5
3m
1S'S 15'5
s-
7'4
< 0'1
11
7'2
0'1
11'S
7'1
<0'1
7
6,8
< 0'1
8'S
7'S
<0'1
9
6'9
<0'1
Temp.
pH
S-
9'S
7'4 7'6 7'1 7'3 7'3 7'4 7'4 7'6 7'4 7'6 7'6 7'7 7'2
0'4 0,6 0'7 0'9 0,8 1'0 0'4 0·8 0,6 3'8 1'4 S'O 2'9
7'7
7'S
1'7 4'0 0'9 3'0 0'4 2'0 1'7 3'0 2'0
10 9 9'5
8 8'S
7
7'3
< 0'1
9 9'5
7'1
11
<0'1
12 12'S
15"5
6'9
o'S
19 19'5
6'3 6'S
0'3 0,6
Dry Dry
3m
pH
11
Temp.
Dry Dry Dry Dry Dry Dry
lS 1S
Edge
pH 6'S 6'S
Etrord Lake
R. Enne
Sequer's Bog
6'3 6'4 6'9 6'9
0,6 1'4 1·6 2,8
Survival of mycelia in leaf disks Mycelium grew out from every disk after the experimental period at both pH levels in control solutions containing no sulphide. There was a reduction in the survival of all species of aero-aquatic and Ingoldian aquatic Hyphomycetes tested (except for H. triglitziense) with increasing concentrations of sulphide (Table 2). The results have been analysed using the analysis of variance after an arcsine transformation, to provide some evaluation of the effects of pH and the sulphide treatments, together with any interaction between these. In practically every case the interaction effect was significant, but small. In most cases the pH effect was not significant, the only exception being A. tetrac/adia. The sulphide effect was highly significant in all cases except for H. trig/itziense. Helicodendron luteo-album and Pseudaegerita foliicola proved to be the most susceptible of the aero-aquatic fungi to high sulphide concentrations.
13
6'7
1·6
14
6'9
0'7
13
7'0
0'1
15
16
7'3 7'9 7'4 8'0
14
6'9
0'1
16'5
7'5
17
8'2 7'4 8'3 8'1 8'3
14 18 16 18
13
7'1
0'1
1S'S
14
7'0
0'2
17 17 17
6'S
It is apparent from Table 2 that the Ingoldian Hyphomycetes tested are more sensitive than the aero-aquatics. Within the 24 h period of exposure a sulphide concentration of 5 mg 1-1 had no effect on survival of the aero-aquatics, and some slight, though variable effects on the Ingoldian Hyphomycetes. At higher concentrations the differences were more noticeable. Amongst the Ingoldian aquatic Hyphomycetes tested, the least tolerant of the higher sulphide concentrations was A. tetrac/adia. The two isolates of T. splendens differed in their response, especially at the higher sulphide concentrations, isolate I being the more tolerant. It is interesting that this strain was isolated from a stagnant drainage ditch, the other having been isolated from foam in the rapidly-flowing R. Teign. In our experience T. splendens fruits readily in moist chambers on twigs and leaves removed from stagnant waters. The relation between the interaction effects between sulphide concentration and pH needs
J. I. Field and J. Webster
197
Table 2o Survival ofaero-aquatic and Ingoldian aquatic Hyphomycetes in SO leafdisks after sulphide treatment (mean of 4 replicates, ±SoEoMo) Sulphide concentration (mg 1-1) to
S
20
pH 4
pH 7
pH 4
pH7
pH 4
pH7
so"o±OOo
SOoo±o·o so·o±o·o So"o±OOo so"o±o"o So"o±OOo so·o±o"o so"o±OOo
40"O±t·t 3S"3±t09 30 'S±t"7
4S"4±O"4 4S"S±o'6 42"S±t"7
3t'S±2"3
49°S±o04
So"o±o"o
27"S±t"O 27"S±t"3 33'0 ±t"3
4t"S±toS 4 t ·S±O"S 4 t "S±t'4
2t"S±O'S 26·S±toS 3°0 ±ooS 47"S±O"S tS03±o"6 S'S±o09 SoS±t"t
t6'S±t"2 U'O±2"S
39'3±2"O
2"S±0·6
32°O±3'S
27'S±2'9 4S'O±o09
t3"3±t·t
3003± t'7
ooS±O'4 3'3±o04 t·S±o"S
6'S±t"2 2°S±0"S t3°S±t'4 6'3±0"4
Aero-aquatic Hyphomycetes Beverwykella pulmonaria Helicodendron conglomeratum Ho luteo-album Ho triglitziense Helicoon fuscosporum Pseudaegerita foliicola I Po foliicola 11
sooo±o·o SOOo±o"o So"o±o"o so·o±ooo so·o±o"o Sooo±o·o
Ingoldian Hyphomycetes Anguillospora rosea Ao tetracladia Tricladium splendens I To splendens 11
49°3±o"3 44'3±o'4 so·o±ooo 49'O±o'S
49'S±o"3 49'S±o03 so"o±o·o so·o±o·o
careful interpretationo If the interaction is ignored, then the pH effect becomes highly significanto Reference to Table 2 suggests that responses at pH 4 are substantially lower in every case than those at pH 7°Further studies covering a more detailed and perhaps wider range ofpH values are needed to gain insight into the nature of this effect.
DISCUSSION
The assessment of sulphide levels both in the field and in the laboratory is beset with difficulties. Measurements can be affected by pH, temperature and .ionic strength of the solutions to be tested in relation to those of the calibration standards, so that it is not always clear how field measurements for sulphide concentration can be related to the sulphide content of a soil or soil solution. The variations reported in the literature for the dissociation constants also a:dd to the difficulties of extrapolation from the laboratory to the field. All the measurements reported here were carried out under laboratory conditions, in solutions where temperature and pH were controlled as far as was practicable and the background ionic strength of the solutions was maintained. These were resolved by the use of antioxidant buffer, which had the further advantage that it prevented the alteration of sulphide to more oxidized or electropositive forms. The use of hydrogen sulphide in place of the more generally adopted sodium sulphide was advantageous. Although it is an unpleasant chemical to use, it allowed the injection of small and easily
36 0S±o·6
6"S±t"6 So"o±o"o 24"S±toS
t003±t"t t6'3±2'2
controlled amounts of sulphide under anaerobic conditions, without the complications involved in the preparation of sulphide solutions which are easily oxidized. Only one-third to one-half of the injected sulphide was subsequently recorded as providing sulphide ions in solution. The reaction was consistent, and it was quite possible to predict the volume of hydrogen sulphide to be injected in order to achieve a particular sulphide level in the reaction vessels° Two suggestions which could account for the discrepancy are that some had reacted with the buffer, and/or that there was some oxidizing reaction with the leaf disks. The former seems more probable, and there is some evidence that hydrogen sulphide does react with 3,3dimethylglutaric acid. The buffering ofthe solution does not appear to be affected, at least over the period of the experimento From the results obtained, it would appear that water immediately above the liner layer in Sequer's Bog, where aero-aquatic Hyphomycetes are common, has a variable sulphide level, but that this rarely exceeds 3 mg 1-1. Because the pH varies between 6°3 and 7°0, the proportion of sulphide present as hydrogen sulphide will vary from about 60 % to about So %. In Efford Lake, with a pH variation of 7°1-8°3, appreciably more bisulphide ion will be present. Also, at this site, the total sulphide concentration is higher, up to 7 S mg 1-1. This is of some interest, in that isolate II of P. foliirola would have experienced rather more HIS and quite a lot more HS- than isolate I from Sequer's Bog. However, the results indicate that the tolerances of these two isolates are rather 0
Sulphides and aquatic fungi similar, except at pH 7 and the highest sulphide level, where isolate II appears to survive better. This investigation suggests that several species of aero-aquatic Hyphomycetes are quite tolerant, and Helicodendron triglitziense very tolerant, of up to 20 mg 1-1 of soluble sulphide, both at pH 4 and 7. Such concentrations are higher than the maximum level of 3'2 mg 1-1 of soluble sulphide found in the water of Sequer's Bog, and the 7'5 mg 1-1 found in Efford Lake, though not as high as the sulphide levels which have been reported from mud layers and from some other natural habitats (Haner & McLean, 1965; Fenchel & Riedl, 1970; Allamet al., 1972; Ayotade, 1977)· The mycelia of the Ingoldian aquatic Hyphomycetes were shown to be less able to tolerate hydrogen sulphide and the bisulphide ion than the aero-aquatic Hyphomycetes. In general, all species of both aero-aquatic and Ingoldian aquatic Hyphomycetes were more tolerant of sulphide at pH 7 than at 4. This suppons the view that undissociated hydrogen sulphide is more toxic than the bisulphide ion, although the latter clearly has some toxicity, otherwise the results for pH 7 and 20 mg 1-1 soluble sulphide should be comparable to those for the same species at pH 4 and 10 mg 1-1 soluble sulphide, when the levels ofundissociated hydrogen sulphide would be approximately equal. Previous research on animals has suggested that the primary biochemical' lesion' of sulphide is the reversible inhibition of cytochrome c oxidase (cytochrome aaa), which is the terminal enzyme of the electron transport chain and directly responsible for the utilization of oxygen by cells during aerobic respiration (Smith, Kruszyna & Smith, 1977 ; WHO, 1981; Torrans & Clemens, 1982). A respiratory transpon chain containing a range of cytochromes has been found in at least a few species of all groups offungi (Boulter & Derbyshire, 1957) and these are fundamentally similar to those of both higher plants and animals (Lindenmayer, 1965; Unestam & Gleason, 1968). Undissociated hydrogen sulphide is more inhibitory to cytochrome c oxidase than is the bisulphide ion (Smith et al., 1977), although the bisulphide ion itself appears to have some toxicity (Broderius, Smith & Lind, 1977). This could be a panial explanation of why, at a panicular value for dissolved sulphide, more of the species tested were able to survive at pH 7 than at pH 4. There is some evidence that undissociated hydrogen sulphide passes across membranes more easily than does the bisulphide ion (Pop, 1936; Ford, 1973; Broderius et al., 1977). Clearly, in at least some aero-aquatic Hyphomycetes, and especially in H. triglitziense, there must be some mechanism which is able to protect
the fungus from the effects of sulphide toxicity. There appear to be three possible ways in which this could operate: they may be able to avoid sulphide uptake, or only take it up very slowly: they may be able to detoxify the sulphides in some way; they may be able to tolerate the consequences of cytochrome c oxidase inhibition. The last possibility requires that either they must be able to reduce their energy requirements so that any unaffected cytochrome c oxidase is sufficient for respiration, or that there is an alternative electron transport pathway which is either not sensitive, or not so sensitive, to sulphide. Nothing appears to be known about these different possibilities in fungi. It is interesting to note that those species of aquatic Hyphomycetes which best survived exposure to sulphide also survived well under conditions of anaerobiosis (Field & Webster, 1983). In the susceptible species, and especially ArticuIospora tetracladia, new mycelial growth took longer to detect after exposure to the higher concentrations of dissolved sulphide. The reason for the delay of new mycelial growth after sulphide treatment is not clear. It seems likely that some cells could recover from the effects of sulphide more rapidly than others. In these species, a functional mitochondrial electron transport pathway, including a noninhibited cytochrome c oxidase, is presumably essential to provide the energy needed for active growth. Thus, after the removal of sulphide, the time needed by the species to re-establish a functional mitochondrial electron transport chain must delay energy production and thus the synthesis of components necessary for growth. Alternatively, the high levels of sulphide could result in the death of some mycelial cells, with the conse~uence that the number of viable cells remaining in each leaf disk could be quite small, and the time taken for new mycelium to become visible could be considerable. The operation of both these mechanisms could result in some very variable times before mycelium was seen to re-emerge. The greater tolerance of the aero-aquatic fungi to anaerobic conditions and high sulphide concentrations make it probable that, as available oxygen becomes depleted in stagnant ponds, and sulphide levels in the litter tend to rise, the aero-aquatic Hyphomycetes will survive in preference to the other aquatic fungi, such as the Ingoldian aquatic Hyphomycetes. They probably persist as thickwalled mycelium in leaves and twigs, and will only begin to grow actively when the oxygen level stans rising. It is worth noting that the combination of a relatively high sulphide level,anaerobic conditions and a variable salinity appears to be detrimental even to the aero-aquatic Hyphomycetes, as under
J. I. Field and J. Webster these conditions, which were found in the bottom of Efford Lake, only Pseudaegerita foliicola was recorded. We are grateful to S.E.R.C. for a research studentship. We also acknowledge the competent technical assistance of Mr R. A. Davey, Miss Denise Howe, Mr G. Wakley and Mr D. Askew. We thank Dr R. B. Ivimey-Cook for help with statistical analysis, and Dr S. K. Abdullah and Dr P. J. Fisher for helpful discussion. We are indebted to A. J. B. Mildmay-White, Esq., for his kindness in permitting us to work on the Flete Estate at Holbeton, and to the South West Water Authority for the provision of weather and water analysis data for the field studies, and for their advice on recording sulphide levels in the field. REFERENCES
ABDULLAH, S. K. & FISHER, P. J. (1984). Aero-aquatic fungal flora of two static water habitats in Devon.
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(Received for publication
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