- Email: [email protected]
Anaerobic survival of aquatic fungi
Trans. Br. mycol. Soc. 81 (2) 365-369 (1983) Printed in Great Britain
ANAEROBIC SURVIVAL OF AQUATIC FUNGI By J. 1. FIELD AND J. WEBSTER Department of Biological Sciences, University of Exeter, U.K. Aero-aquatic Hyphomycetes were tested for their ability to survive on beech leaf disks under anaerobic conditions for periods of up to 12 months. Helicodendron triglitziense, H. conglomeratum and H. giganteum showed 100 % survival, while H. luteo-album and H. hyalinum showed reduced survival. Survival of aero-aquatic Hyphomycetes, Ingoldian aquatic Hyphomycetes and members of the Saprolegniales was also compared on hemp seeds under anaerobic conditions. Aero-aquatic Hyphomycetes showed the greatest percentage survival. Articulospora tetracladia, Achlya colorata (isolate lacking oospores) and Dictyuchus sterilis failed to survive even 3 months. No significant difference was found in the survival of Tricladium splendens from a stagnant drainage ditch and from a fast-flowing river. Only species of Saprolegniales with oospores survived anaerobic conditions. Aero-aquatic Hyphomycetes are conidial fungi commonly found in freshwater habitats, particularly in stagnant ponds, ditches or slow-running streams, and are capable of vegetative growth on submerged plant material (Webster & Descals, 1981). Experiments by Fisher (1977) indicate that many aero-aquatic fungi are adapted to exist in anaerobic or micro-aerobic situations, although colonization of leaf detritus is slow under microaerobic conditions. It is of interest to compare the survival under anaerobic conditions of some aero-aquatic Hyphomycetes with several species of Ingoldian aquatic Hyphomycetes and with members ofthe Saprolegniales, which in general are found in more aerated waters. Ingoldian aquatic Hyphomycetes occur abundantly in non-polluted fast-flowing streams and rivers and in well-aerated lakes (Webster & Descals, 1981). Dick (1971), working on mud samples from Lake Marion in Canada, concluded that 'most Saprolegniaceae are primarily fungi of the emergent littoral and lentic/littoral interface and very few are clearly lentic'; that is, most are to be found in the lake margin muds, rather than the deeper, less aerobic, lentic muds. Collins & Willoughby (1962) and Willoughby & Collins (1966) have drawn similar conclusions from a study of water and mud samples from Blelham Tarn, in the English Lake District. Counts of Saprolegniales from mud samples near the centre of the lake taken from the hypolimnion where the dissolved oxygen content may be zero, were lower than those for the water above, or in the lake margin. MATERIALS AND METHODS
Species of aero-aquatic Hyphomycetes were isolated from three sites close to Exeter, Devon;
Helicodendron triglitziense (jaap) Linder and H. giganteum Glen-Bott were obtained from a drainage
ditch in a wood on Woodbury Common (SY 027840), and H. hyalinum Linder from the adjacent Bystock Reservoir (SY 034843). H. luteo-album Glen-Bon and H. conglomeratum Glenn-Bott were found on plant material taken from a stagnant ditch in Stoke Woods (SX 915958). All these species colonize a variety of decaying leaves, especially of oak (Quercus spp.) and beech (Fagus sylvatica), although H. hyalinum is usually found on pine (Pinus spp.), Of the Ingoldian aquatic Hyphomycetes, Anguillospora rosea Descals ined. and Articulospora tetracladia Ingold were isolated from foam collected at the outfall of Bystock Reservoir. Tricladium splendens Ingold (isolate I) was isolated from Quercus leaves collected from the bottom of a stagnant drainage ditch on Woodbury Common (SY027840) and T. splendens (isolate II) from foam collected at Fingle Bridge, Drewsteignton (SX 743899). Of the Saprolegniales, Saprolegnia sp. (affinity S.ferax) and Dictyuchus sterilis Coker were produced on baited hemp seeds, from twigs collected from the surface waters of a drainage ditch on Woodbury Common; Achlya colorata Pringsheim (isolate I) was a laboratory stock culture, while A colorata (isolate II) was collected from Bystock Reservoir. Saprolegnia sp. and A. colorata I readily produced oogonia in distilled water in 5-10 days at 15°±2°. A. colorata II often failed to produce oogonia, or took several weeks for a small number to be formed. No oogonia were ever observed on D. sterilis. Aero-aquatic Hyphomycetes were cultured on 0"1 % Oxoid malt extract agar (MEA), and an inoculum prepared by homogenizing a piece of the culture in sterile distilled water. Each inoculum was added to a sterilized flask containing a large number
Anaerobic survival of aquatic fungi of j mm diam beech leaf disks and 150 em" of beech leaf decoction (Fisher & Webster, 1979). Flasks were placed on a rotary shaker at 15° ±2°, in the dark and were shaken daily for 10-15 min. Colonisation of the disks took place over a period of 6-8 weeks. One week prior to use, a check was made by plating out a sample of 100 disks onto 0'1 % MEA to confirm that all were colonized. Colonized disks were then collected under aseptic conditions and washed thoroughly with sterile distilled water to remove loose surface mycelium before use. The experiment was set up in 5 em Petri dishes contained in four GasPak (BBL, Becton Dickinson, Wembley, Middlesex) anaerobic jars. For each species, four Petri dishes were used, each containing 50 disks. To each dish 15 em" of deoxygenated beech leaf decoction was added, prepared by autoclaving it and allowing it to cool with oxygen-free nitrogen bubbling through it. This was obtained by passing white-spot nitrogen (BOC, London) over BASF R3-11 catalyst heated to 140-150° to remove residual oxygen. As a further precaution against the inclusion of oxygen-sterile filtered sodium formaldehyde sulphoxylate (1'0 g 1-1) was used as a reductant (Tabak & Cooke, 1968) at the rate of l ' 5 ern" added to half of the dishes. No change in the pH of the leaf decoction was detected on addition of the reductant. Each sealed anaerobic jar was set up to include a methylene blue indicator strip and the GasPak envelope containing water and the chemical required to generate the anaerobic atmosphere. The GasPak gradually produces carbon dioxide and hydrogen, the latter combining with the oxygen in the jar catalysed by paUadiumised alumina in the safety screen under the lid of the jar. After 12-24 h at room temperature, the jar became anaerobic (methylene blue indicator went colourless). Jars were kept in the dark at 15° ±2°. One jar was opened every 13 weeks and the disks removed and plated out onto 0'1 % MEA and the presence or absence of outgrowing mycelium was recorded for each disk. Disks from which no growth was observed were removed and replated, to prevent their colonisation by spores or mycelium from adjacent disks. Comparative experiments were conducted with species of aero-aquatic Hyphomycetes, Ingoldian aquatic Hyphomycetes and Saprolegniales. Experimental procedure was similar to that described above, but differed in two ways: sterile distilled water was used instead of beech leaf decoction in the Petri dishes; and 25 hemp seeds were used in place of leaf disks for all fungi because Saprolegniales do not grow well on leaf disks.
Preparation of hemp seed inocula
Hemp seeds were sterilized by autoclaving in distilled water, washed and re-sterilized in a flask. Colonisation by aero-aquatic and aquatic Hyphomycetes was by mycelial inocula as described above. For the Saprolegniales, sterilized hemp seeds were placed on cultures grown on Corn Meal Agar (CMA) and left for 3 days. Seeds were then transferred to sterile distilled water and grown for a further period of 11 days, during which time oospores had developed in A. colorata I and Saprolegnia sp. On removal from the anaerobic jar at the end of the experiment, the hemp seeds inoculated with aero-aquatic Hyphomycetes were plated onto 0'1 % MEA, those with Ingoldian aquatic Hyphomycetes onto 2 % MEA and the Saprolegniales onto CMA. All media were made up with 0·6 % agar to make them slightly sloppy; this was found to enhance re-growth, especially of the Saprolegniales. RESUL TS
The results are summarised in Tables 1-3 . Results for Petri dishes containing beech leaf disks with and without reductant were so nearly identical that the four sets are treated as replicates. Three species, Helicodendron triglitziense, H. conglomeratum and H. giganteum, produced mycelium from all disks, even after 12 months in an anaerobic jar (Table 1). Results were identical for H. triglitziense on hemp seeds (Table 2), showing that for this species, the substratum makes no difference to its ability to survive under the conditions of the experiment. H. luteo-album on disks showed 100% survival after 3 and 6 months in an anaerobic jar but reduced to 94 % after 9 months and to 76 % after 12 months. H. hyalinum was less able to survive anaerobic conditions; all disks produced mycelium after 3 months, and 99 % after 6 months, but this declined to 77 % after 9 months and to 18 % after 12 months. This species survived much better on hemp seeds and even after 12 months, 91 % of seeds produced mycelium. This suggests that the richer food store of the hemp seed allows more mycelium to be produced, and therefore improves the chances of survival and regrowth from a segment of mycelium. Ingoldian aquatic Hyphomycetes and Saprolegniales were less tolerant of the anaerobic conditions in the jars (Table 2). Some showed no re-growth at all, even after only 3 months anaerobiosis. These were Articulospora tetracladia, Achlya colorata II and Dictyuchus sterilis. Of the remaining species, only Anguillospora rosea showed more than 5 % growth from the hemp seeds on CMA after 12
J. I. Field and J. Webster Table
1.
Percentage survival of aero-aquatic Hyphomycetes on beech leaf disks
(Mean of four replicates ± S.E.M. calculated using the binomial distribution.) Months in anaerobic jar
Helicodendron triglitziense H. co~101neratUtn H. giganteum H. luteo-album
H. hyalinum
3 l00±O 100±0 100±0 100±O 100±O
6
9 l00±O 100±O 100±O 94±2 77±3
100±O 100±O 100±O 100±O 99±1
12 l00±O 100±O l00±O 76±3 18±3
Table 2. Survival (%) of Saprolegniales, aero-aquatic and aquatic Hyphomycetes on hemp seeds (Mean of four replicates ± S.E.M. calculated using the binomial distribution.) Months in anaerobic jar
Helicodendron triglitziense H. hyalinum Anguillospora rosea
Tricladium splendens T. splendens II
~
Articulospora tetracladia
Saprolegnia sp.
}
Achlya colorata I A. colorata II } Dictyuchus sterilis
with oospores no oospores
3 100±O 100±O 100±0 84±4 52±5 O±O 84±4 2±1 o±o o±o
6 100±O 99±1 95±2 52±5 45±5 o±o 68±5 9±3 o±o o±o
9 100±O 99±1 36±5 5±2 7±3 o±o 26±4 1±1 o±o o±o
12 100±O 9 1±3 15±4 2±1 5±2 O±O 4±2 4±2 o±o o±o
months in the jar. The survival of this species was appearing vacuolate or devoid of contents, after consistently higher throughout, showing 100 % anaerobic incubation. However, some oospores on survival after 3 months, dropping to 15 % after 12 each hemp seed appeared intact, which suggests months. After 3 months anaerobiosis, Tricladium that it is the ability of these to germinate which splendens I, taken from the stagnant drainage ditch, detertnines whether regrowth occurs or not. showed a greater percentage survival than T. Several oogonia, within which an oospore appeared splendens II, taken from flowing aerated water. to have germinated, were teased out from the Although in the former case 84 % of the hemp seeds surrounding mycelium, but oospore germination produced mycelium, as compared to 52 % in the was difficult to detertnine with certainty, as the latter, the difference was not significant at the 5 % growing mycelium invariably obscured its origin level. The percentage survival of both isolates from the oospore. Time take for mycelium to develop from the leaf declined further after longer incubation under anaerobic conditions until, after 12 months, T. disks or hemp seeds may depend on the duration splendens I showed only 2 % survival and T. of the anaerobic incubation period. However, this effect may also vary with the species and the splendens II 5 % survival. Only two members of the Saprolegniales survived substrate (leaf disks or hemp seeds) (Table 3). even 3 months anaerobiosis. In Saprolegnia sp., Regrowth of mycelium of H elicodendron triglitziense 84 % of the hemp seeds produced mycelium after seems little affected by the length of time of 3 months, falling to 4 % after 12 months. The incubation or the substratum, growing out within percentage survival of Achlya colorata I was erratic, 1-3 days when removed from anaerobic conditions. but never exceeded 9 %. No evidence of the growth For H. hyalinum the period of anaerobic incubation of new mycelium from the old was found, and much affects the time taken for mycelial re-growth. The of the mycelium was nearly devoid of contents, or longer the period of anaerobiosis, the longer is the was empty, after anaerobic incubation. It is of time taken for re-growth to occur. This effect is interest that the only two species which survived shown whether the substratum is leaf disk or hemp were those producing oospores (Table 2). Many seed, even though the time taken for re-growth after oospores of both species had broken down, ,prolonged incubation may differ on the two
368
Anaerobic survival of aquatic fungi
Table 3. Time for mycelium to develop from leaf disks or hemp seeds incubated anaerobically for 3 and months
12
Time (days) 3
months
12
months
Earliest
Latest
Earliest
Latest
1 1 1
1 2 2
1 1 2
1 2
1
2
1
2
4 14
1 1 2
1 2 6
2
10
2
10
2
10
9
9
Leaf disks Helicodendron triglitziense H. conglomeratum H . giganteum Hi luteo-album H. hyalinum Hemp seeds H. triglitziense H . hyalinum Anguillospora rosea Tricladium splendens I T. splendens II Saprolegnia sp. Achlya colorata I
7 7 8 8 6 12
3 25 30
3 21 21 10 15 26 23
proportion of the cells survive anaerobic conditions, and the longer period before mycelium becomes visible reflects the time needed for the few surviving cells to develop fresh hyphae. Alternatively prolonged anaerobiosis may induce a proDISCUSSION found change in the physiology of the cells, such as In discussing these results, it is important to a state of dormancy, from which they take several distinguish between the type of cell which is days to recover. It is, of course, possible that both capable of survival. In the Helicodendron spp. used, these phenomena may be involved. As discussed earlier, there is evidence that only no chlamydospores have been observed; survival on and within the leaf is apparently exclusively as those members of the Saprolegniales which possess mycelium. This is for the most part thick-walled, oospores are capable of prolonged anaerobic and becomes brown on ageing in beech leaf survival and, since these fungi usually make.rapid decoction in air. After a period inside the anaerobic growth in air, we believe that oospores which have jar, the mycelium appears vacuolate and is often ' been kept under anaerobic conditions for 12 empty. It is from this vacuolate mycelium that new months may take longer to germinate; also, that the mycelium is produced on exposure to air, either if proportion of oospores which survive prolonged left in the beech leaf decoction or if plated onto agar. anaerobiosis is low. Preliminary tests, not performed on all the fungi The advancing mycelium is hyaline and often granular, with small vacuoles. There is no evidence used, have shown that when inoculated disks or to suggest that the mycelium actually grows during hemp seeds which had been kept for 12 months its time under anaerobic conditions. Experiments under anaerobic conditions were placed in sterile in which spores were immersed in 0'4 % liquid malt distilled water through which air was being extract showed that germ-tube lengths after 3 bubbled, more rapid growth of mycelium took months anaerobiosis were comparable to those place than when the material was incubated on agar. produced during the time it takes for a jar to become Possibly this is due to the more rapid diffusion of anaerobic (12-24 h). Similarly, the survival of the oxygen to the surviving fungal cells. These Ingoldian Hyphomycetes appears to be in the form observations parallel those of Fisher (1977) who of thick-walled vacuolate mycelium in the hemp found that the number of species of aero-aquatic seeds, as we have no evidence of chlamydospore fungi which could be detected on beech leaf disks which had been immersed in static water habitats formation in the species used . The reasons for the delay in mycelial emergence in the field, was increased if the disks were in certain fungi after prolonged anaerobiosis are not incubated in aerated distilled water. One of the most interesting facts to arise from this known. One possibility is that only a small substrata. An extension of the time taken for re-growth was also found in H. luteo-album, A. rosea and T. splendens.
J. I. Field and J. Webster and related work is the overall resilience of the aero-aquatic Hyphomycetes and their adaptation to their usual freshwater habitat, often on plant material in micro-aerobic or anaerobic situations in mud. Although they show no marked ability to grow, they can certainly survive for long periods under anaerobic conditions in the laboratory. Thus they can probably remain viable in anaerobic situations in the field until oxygen again becomes available and they can resume growth. High levels of hydrogen sulphide can also exist in anaerobic muds, and preliminary experiments suggest that the aero-aquatic Hyphomycetes can survive relatively high concentrations of this also, at least for short periods. The mycelium of some aero-aquatic Hyphomycetes also appears to be very tolerant to prolonged drying, both in the laboratory and in the field (Fisher, 1978), and thus they are also able to survive periods of drought during which ponds and ditches may dry up. Some species have even been isolated from moist leaf litter on land, although their preferred habitat is in static water (Fisher & Webster, 1981). We are grateful to Mr R. A. Davey for excellent technical assistance and to Dr M. W. Dick for helpful advice on the identification of the Saprolegnia.
REFERENCES
COLLINS, V. G. & WILLOUGHBY, L. G . (1962). The distribution of bacteria and fungal spores in Blelham Tam, with particular reference to an experimental overturn. Archiv fUr Mikrobiologie 43, 294-307. DICK, M. W. (1971). The ecology of Saprolegniaceae in lentic and littoral muds with a general theory of fungi in the lake ecosystem. Journal of General Microbiology 65, 325-337. FISHER, P. J. (1977). New methods of detecting and studying saprophytic behaviour of aero-aquatic Hyphornycetes from stagnant water. Transa ctions of the British Mycological Society 68, 407-411 . FISHER, P. J. (1978). Survival of aero-aquatic Hyphomycetes on land. Transactions of the British Mycological Society 71, 419-423 . FISHER, P. J. & WEBSTER, J. (1979). Effect of oxygen and carbon dioxide on growth of four aero-aquatic Hyphomycetes. Transactions of the British Mycological Society 72, 57-61. FISHER, P. J. & WEBSTER, J. (1981). Ecological studies on aero-aquatic Hyphomycetes. In The Fungal Community : Its Organisation and Role in the Ecosystem (Ed . D. T. Wicklow & G . C. Carroll), pp . 709-730. New York and Basel: Marcel Dekker, Inc. TABAK, H. H. & CooKE, W. B. (1968). Growth and metabolism of fungi in an atmosphere of nitrogen. Mycologia 60, 115-140. WEBSTER, J. & DESCALS, E. (1981) . Morphology, distribution and ecology of conidial fungi in freshwater habitats. In Biology of Conidial Fungi, vol. 1 (ed. G . T . Cole & B. Kendrick), pp. 295-355. New York and London: Academic Press. WILLOUGHBY, L. G. & COLLINS, V. G. (1966). A study of the distribution of fungal spores and bacteria in Blelham Tam and its associated streams. Nova Hedwigia 12,149-171.
(Received for publication 18 November 1982)
ANAEROBIC SURVIVAL OF AQUATIC FUNGI By J. 1. FIELD AND J. WEBSTER Department of Biological Sciences, University of Exeter, U.K. Aero-aquatic Hyphomycetes were tested for their ability to survive on beech leaf disks under anaerobic conditions for periods of up to 12 months. Helicodendron triglitziense, H. conglomeratum and H. giganteum showed 100 % survival, while H. luteo-album and H. hyalinum showed reduced survival. Survival of aero-aquatic Hyphomycetes, Ingoldian aquatic Hyphomycetes and members of the Saprolegniales was also compared on hemp seeds under anaerobic conditions. Aero-aquatic Hyphomycetes showed the greatest percentage survival. Articulospora tetracladia, Achlya colorata (isolate lacking oospores) and Dictyuchus sterilis failed to survive even 3 months. No significant difference was found in the survival of Tricladium splendens from a stagnant drainage ditch and from a fast-flowing river. Only species of Saprolegniales with oospores survived anaerobic conditions. Aero-aquatic Hyphomycetes are conidial fungi commonly found in freshwater habitats, particularly in stagnant ponds, ditches or slow-running streams, and are capable of vegetative growth on submerged plant material (Webster & Descals, 1981). Experiments by Fisher (1977) indicate that many aero-aquatic fungi are adapted to exist in anaerobic or micro-aerobic situations, although colonization of leaf detritus is slow under microaerobic conditions. It is of interest to compare the survival under anaerobic conditions of some aero-aquatic Hyphomycetes with several species of Ingoldian aquatic Hyphomycetes and with members ofthe Saprolegniales, which in general are found in more aerated waters. Ingoldian aquatic Hyphomycetes occur abundantly in non-polluted fast-flowing streams and rivers and in well-aerated lakes (Webster & Descals, 1981). Dick (1971), working on mud samples from Lake Marion in Canada, concluded that 'most Saprolegniaceae are primarily fungi of the emergent littoral and lentic/littoral interface and very few are clearly lentic'; that is, most are to be found in the lake margin muds, rather than the deeper, less aerobic, lentic muds. Collins & Willoughby (1962) and Willoughby & Collins (1966) have drawn similar conclusions from a study of water and mud samples from Blelham Tarn, in the English Lake District. Counts of Saprolegniales from mud samples near the centre of the lake taken from the hypolimnion where the dissolved oxygen content may be zero, were lower than those for the water above, or in the lake margin. MATERIALS AND METHODS
Species of aero-aquatic Hyphomycetes were isolated from three sites close to Exeter, Devon;
Helicodendron triglitziense (jaap) Linder and H. giganteum Glen-Bott were obtained from a drainage
ditch in a wood on Woodbury Common (SY 027840), and H. hyalinum Linder from the adjacent Bystock Reservoir (SY 034843). H. luteo-album Glen-Bon and H. conglomeratum Glenn-Bott were found on plant material taken from a stagnant ditch in Stoke Woods (SX 915958). All these species colonize a variety of decaying leaves, especially of oak (Quercus spp.) and beech (Fagus sylvatica), although H. hyalinum is usually found on pine (Pinus spp.), Of the Ingoldian aquatic Hyphomycetes, Anguillospora rosea Descals ined. and Articulospora tetracladia Ingold were isolated from foam collected at the outfall of Bystock Reservoir. Tricladium splendens Ingold (isolate I) was isolated from Quercus leaves collected from the bottom of a stagnant drainage ditch on Woodbury Common (SY027840) and T. splendens (isolate II) from foam collected at Fingle Bridge, Drewsteignton (SX 743899). Of the Saprolegniales, Saprolegnia sp. (affinity S.ferax) and Dictyuchus sterilis Coker were produced on baited hemp seeds, from twigs collected from the surface waters of a drainage ditch on Woodbury Common; Achlya colorata Pringsheim (isolate I) was a laboratory stock culture, while A colorata (isolate II) was collected from Bystock Reservoir. Saprolegnia sp. and A. colorata I readily produced oogonia in distilled water in 5-10 days at 15°±2°. A. colorata II often failed to produce oogonia, or took several weeks for a small number to be formed. No oogonia were ever observed on D. sterilis. Aero-aquatic Hyphomycetes were cultured on 0"1 % Oxoid malt extract agar (MEA), and an inoculum prepared by homogenizing a piece of the culture in sterile distilled water. Each inoculum was added to a sterilized flask containing a large number
Anaerobic survival of aquatic fungi of j mm diam beech leaf disks and 150 em" of beech leaf decoction (Fisher & Webster, 1979). Flasks were placed on a rotary shaker at 15° ±2°, in the dark and were shaken daily for 10-15 min. Colonisation of the disks took place over a period of 6-8 weeks. One week prior to use, a check was made by plating out a sample of 100 disks onto 0'1 % MEA to confirm that all were colonized. Colonized disks were then collected under aseptic conditions and washed thoroughly with sterile distilled water to remove loose surface mycelium before use. The experiment was set up in 5 em Petri dishes contained in four GasPak (BBL, Becton Dickinson, Wembley, Middlesex) anaerobic jars. For each species, four Petri dishes were used, each containing 50 disks. To each dish 15 em" of deoxygenated beech leaf decoction was added, prepared by autoclaving it and allowing it to cool with oxygen-free nitrogen bubbling through it. This was obtained by passing white-spot nitrogen (BOC, London) over BASF R3-11 catalyst heated to 140-150° to remove residual oxygen. As a further precaution against the inclusion of oxygen-sterile filtered sodium formaldehyde sulphoxylate (1'0 g 1-1) was used as a reductant (Tabak & Cooke, 1968) at the rate of l ' 5 ern" added to half of the dishes. No change in the pH of the leaf decoction was detected on addition of the reductant. Each sealed anaerobic jar was set up to include a methylene blue indicator strip and the GasPak envelope containing water and the chemical required to generate the anaerobic atmosphere. The GasPak gradually produces carbon dioxide and hydrogen, the latter combining with the oxygen in the jar catalysed by paUadiumised alumina in the safety screen under the lid of the jar. After 12-24 h at room temperature, the jar became anaerobic (methylene blue indicator went colourless). Jars were kept in the dark at 15° ±2°. One jar was opened every 13 weeks and the disks removed and plated out onto 0'1 % MEA and the presence or absence of outgrowing mycelium was recorded for each disk. Disks from which no growth was observed were removed and replated, to prevent their colonisation by spores or mycelium from adjacent disks. Comparative experiments were conducted with species of aero-aquatic Hyphomycetes, Ingoldian aquatic Hyphomycetes and Saprolegniales. Experimental procedure was similar to that described above, but differed in two ways: sterile distilled water was used instead of beech leaf decoction in the Petri dishes; and 25 hemp seeds were used in place of leaf disks for all fungi because Saprolegniales do not grow well on leaf disks.
Preparation of hemp seed inocula
Hemp seeds were sterilized by autoclaving in distilled water, washed and re-sterilized in a flask. Colonisation by aero-aquatic and aquatic Hyphomycetes was by mycelial inocula as described above. For the Saprolegniales, sterilized hemp seeds were placed on cultures grown on Corn Meal Agar (CMA) and left for 3 days. Seeds were then transferred to sterile distilled water and grown for a further period of 11 days, during which time oospores had developed in A. colorata I and Saprolegnia sp. On removal from the anaerobic jar at the end of the experiment, the hemp seeds inoculated with aero-aquatic Hyphomycetes were plated onto 0'1 % MEA, those with Ingoldian aquatic Hyphomycetes onto 2 % MEA and the Saprolegniales onto CMA. All media were made up with 0·6 % agar to make them slightly sloppy; this was found to enhance re-growth, especially of the Saprolegniales. RESUL TS
The results are summarised in Tables 1-3 . Results for Petri dishes containing beech leaf disks with and without reductant were so nearly identical that the four sets are treated as replicates. Three species, Helicodendron triglitziense, H. conglomeratum and H. giganteum, produced mycelium from all disks, even after 12 months in an anaerobic jar (Table 1). Results were identical for H. triglitziense on hemp seeds (Table 2), showing that for this species, the substratum makes no difference to its ability to survive under the conditions of the experiment. H. luteo-album on disks showed 100% survival after 3 and 6 months in an anaerobic jar but reduced to 94 % after 9 months and to 76 % after 12 months. H. hyalinum was less able to survive anaerobic conditions; all disks produced mycelium after 3 months, and 99 % after 6 months, but this declined to 77 % after 9 months and to 18 % after 12 months. This species survived much better on hemp seeds and even after 12 months, 91 % of seeds produced mycelium. This suggests that the richer food store of the hemp seed allows more mycelium to be produced, and therefore improves the chances of survival and regrowth from a segment of mycelium. Ingoldian aquatic Hyphomycetes and Saprolegniales were less tolerant of the anaerobic conditions in the jars (Table 2). Some showed no re-growth at all, even after only 3 months anaerobiosis. These were Articulospora tetracladia, Achlya colorata II and Dictyuchus sterilis. Of the remaining species, only Anguillospora rosea showed more than 5 % growth from the hemp seeds on CMA after 12
J. I. Field and J. Webster Table
1.
Percentage survival of aero-aquatic Hyphomycetes on beech leaf disks
(Mean of four replicates ± S.E.M. calculated using the binomial distribution.) Months in anaerobic jar
Helicodendron triglitziense H. co~101neratUtn H. giganteum H. luteo-album
H. hyalinum
3 l00±O 100±0 100±0 100±O 100±O
6
9 l00±O 100±O 100±O 94±2 77±3
100±O 100±O 100±O 100±O 99±1
12 l00±O 100±O l00±O 76±3 18±3
Table 2. Survival (%) of Saprolegniales, aero-aquatic and aquatic Hyphomycetes on hemp seeds (Mean of four replicates ± S.E.M. calculated using the binomial distribution.) Months in anaerobic jar
Helicodendron triglitziense H. hyalinum Anguillospora rosea
Tricladium splendens T. splendens II
~
Articulospora tetracladia
Saprolegnia sp.
}
Achlya colorata I A. colorata II } Dictyuchus sterilis
with oospores no oospores
3 100±O 100±O 100±0 84±4 52±5 O±O 84±4 2±1 o±o o±o
6 100±O 99±1 95±2 52±5 45±5 o±o 68±5 9±3 o±o o±o
9 100±O 99±1 36±5 5±2 7±3 o±o 26±4 1±1 o±o o±o
12 100±O 9 1±3 15±4 2±1 5±2 O±O 4±2 4±2 o±o o±o
months in the jar. The survival of this species was appearing vacuolate or devoid of contents, after consistently higher throughout, showing 100 % anaerobic incubation. However, some oospores on survival after 3 months, dropping to 15 % after 12 each hemp seed appeared intact, which suggests months. After 3 months anaerobiosis, Tricladium that it is the ability of these to germinate which splendens I, taken from the stagnant drainage ditch, detertnines whether regrowth occurs or not. showed a greater percentage survival than T. Several oogonia, within which an oospore appeared splendens II, taken from flowing aerated water. to have germinated, were teased out from the Although in the former case 84 % of the hemp seeds surrounding mycelium, but oospore germination produced mycelium, as compared to 52 % in the was difficult to detertnine with certainty, as the latter, the difference was not significant at the 5 % growing mycelium invariably obscured its origin level. The percentage survival of both isolates from the oospore. Time take for mycelium to develop from the leaf declined further after longer incubation under anaerobic conditions until, after 12 months, T. disks or hemp seeds may depend on the duration splendens I showed only 2 % survival and T. of the anaerobic incubation period. However, this effect may also vary with the species and the splendens II 5 % survival. Only two members of the Saprolegniales survived substrate (leaf disks or hemp seeds) (Table 3). even 3 months anaerobiosis. In Saprolegnia sp., Regrowth of mycelium of H elicodendron triglitziense 84 % of the hemp seeds produced mycelium after seems little affected by the length of time of 3 months, falling to 4 % after 12 months. The incubation or the substratum, growing out within percentage survival of Achlya colorata I was erratic, 1-3 days when removed from anaerobic conditions. but never exceeded 9 %. No evidence of the growth For H. hyalinum the period of anaerobic incubation of new mycelium from the old was found, and much affects the time taken for mycelial re-growth. The of the mycelium was nearly devoid of contents, or longer the period of anaerobiosis, the longer is the was empty, after anaerobic incubation. It is of time taken for re-growth to occur. This effect is interest that the only two species which survived shown whether the substratum is leaf disk or hemp were those producing oospores (Table 2). Many seed, even though the time taken for re-growth after oospores of both species had broken down, ,prolonged incubation may differ on the two
368
Anaerobic survival of aquatic fungi
Table 3. Time for mycelium to develop from leaf disks or hemp seeds incubated anaerobically for 3 and months
12
Time (days) 3
months
12
months
Earliest
Latest
Earliest
Latest
1 1 1
1 2 2
1 1 2
1 2
1
2
1
2
4 14
1 1 2
1 2 6
2
10
2
10
2
10
9
9
Leaf disks Helicodendron triglitziense H. conglomeratum H . giganteum Hi luteo-album H. hyalinum Hemp seeds H. triglitziense H . hyalinum Anguillospora rosea Tricladium splendens I T. splendens II Saprolegnia sp. Achlya colorata I
7 7 8 8 6 12
3 25 30
3 21 21 10 15 26 23
proportion of the cells survive anaerobic conditions, and the longer period before mycelium becomes visible reflects the time needed for the few surviving cells to develop fresh hyphae. Alternatively prolonged anaerobiosis may induce a proDISCUSSION found change in the physiology of the cells, such as In discussing these results, it is important to a state of dormancy, from which they take several distinguish between the type of cell which is days to recover. It is, of course, possible that both capable of survival. In the Helicodendron spp. used, these phenomena may be involved. As discussed earlier, there is evidence that only no chlamydospores have been observed; survival on and within the leaf is apparently exclusively as those members of the Saprolegniales which possess mycelium. This is for the most part thick-walled, oospores are capable of prolonged anaerobic and becomes brown on ageing in beech leaf survival and, since these fungi usually make.rapid decoction in air. After a period inside the anaerobic growth in air, we believe that oospores which have jar, the mycelium appears vacuolate and is often ' been kept under anaerobic conditions for 12 empty. It is from this vacuolate mycelium that new months may take longer to germinate; also, that the mycelium is produced on exposure to air, either if proportion of oospores which survive prolonged left in the beech leaf decoction or if plated onto agar. anaerobiosis is low. Preliminary tests, not performed on all the fungi The advancing mycelium is hyaline and often granular, with small vacuoles. There is no evidence used, have shown that when inoculated disks or to suggest that the mycelium actually grows during hemp seeds which had been kept for 12 months its time under anaerobic conditions. Experiments under anaerobic conditions were placed in sterile in which spores were immersed in 0'4 % liquid malt distilled water through which air was being extract showed that germ-tube lengths after 3 bubbled, more rapid growth of mycelium took months anaerobiosis were comparable to those place than when the material was incubated on agar. produced during the time it takes for a jar to become Possibly this is due to the more rapid diffusion of anaerobic (12-24 h). Similarly, the survival of the oxygen to the surviving fungal cells. These Ingoldian Hyphomycetes appears to be in the form observations parallel those of Fisher (1977) who of thick-walled vacuolate mycelium in the hemp found that the number of species of aero-aquatic seeds, as we have no evidence of chlamydospore fungi which could be detected on beech leaf disks which had been immersed in static water habitats formation in the species used . The reasons for the delay in mycelial emergence in the field, was increased if the disks were in certain fungi after prolonged anaerobiosis are not incubated in aerated distilled water. One of the most interesting facts to arise from this known. One possibility is that only a small substrata. An extension of the time taken for re-growth was also found in H. luteo-album, A. rosea and T. splendens.
J. I. Field and J. Webster and related work is the overall resilience of the aero-aquatic Hyphomycetes and their adaptation to their usual freshwater habitat, often on plant material in micro-aerobic or anaerobic situations in mud. Although they show no marked ability to grow, they can certainly survive for long periods under anaerobic conditions in the laboratory. Thus they can probably remain viable in anaerobic situations in the field until oxygen again becomes available and they can resume growth. High levels of hydrogen sulphide can also exist in anaerobic muds, and preliminary experiments suggest that the aero-aquatic Hyphomycetes can survive relatively high concentrations of this also, at least for short periods. The mycelium of some aero-aquatic Hyphomycetes also appears to be very tolerant to prolonged drying, both in the laboratory and in the field (Fisher, 1978), and thus they are also able to survive periods of drought during which ponds and ditches may dry up. Some species have even been isolated from moist leaf litter on land, although their preferred habitat is in static water (Fisher & Webster, 1981). We are grateful to Mr R. A. Davey for excellent technical assistance and to Dr M. W. Dick for helpful advice on the identification of the Saprolegnia.
REFERENCES
COLLINS, V. G. & WILLOUGHBY, L. G . (1962). The distribution of bacteria and fungal spores in Blelham Tam, with particular reference to an experimental overturn. Archiv fUr Mikrobiologie 43, 294-307. DICK, M. W. (1971). The ecology of Saprolegniaceae in lentic and littoral muds with a general theory of fungi in the lake ecosystem. Journal of General Microbiology 65, 325-337. FISHER, P. J. (1977). New methods of detecting and studying saprophytic behaviour of aero-aquatic Hyphornycetes from stagnant water. Transa ctions of the British Mycological Society 68, 407-411 . FISHER, P. J. (1978). Survival of aero-aquatic Hyphomycetes on land. Transactions of the British Mycological Society 71, 419-423 . FISHER, P. J. & WEBSTER, J. (1979). Effect of oxygen and carbon dioxide on growth of four aero-aquatic Hyphomycetes. Transactions of the British Mycological Society 72, 57-61. FISHER, P. J. & WEBSTER, J. (1981). Ecological studies on aero-aquatic Hyphomycetes. In The Fungal Community : Its Organisation and Role in the Ecosystem (Ed . D. T. Wicklow & G . C. Carroll), pp . 709-730. New York and Basel: Marcel Dekker, Inc. TABAK, H. H. & CooKE, W. B. (1968). Growth and metabolism of fungi in an atmosphere of nitrogen. Mycologia 60, 115-140. WEBSTER, J. & DESCALS, E. (1981) . Morphology, distribution and ecology of conidial fungi in freshwater habitats. In Biology of Conidial Fungi, vol. 1 (ed. G . T . Cole & B. Kendrick), pp. 295-355. New York and London: Academic Press. WILLOUGHBY, L. G. & COLLINS, V. G. (1966). A study of the distribution of fungal spores and bacteria in Blelham Tam and its associated streams. Nova Hedwigia 12,149-171.
(Received for publication 18 November 1982)