Sporulation responses of some ‘aquatic hyphomycetes’ in flowing water

[ 601 ]

Trans. Br, mycol. Soc. 74 (3) 601-605 (1980)

Printed in Great Britain

SPORULATION RESPONSES OF SOME 'AQUATIC HYPHOMYCETES' IN FLOWING WATER By P. F. SANDERS*

AND

J.

WEBSTER

Department of Biological Sciences, University of Exeter

Seventeen species of 'aquatic hyphomycetes ' were tested for their ability to sporulate at different rates of water flow in small chambers. Eight species were able to sporulate almost as prolifically at low flow rates as at more rapid rates; nine species showed a lower flow rate limit below which sporulation was significantly reduced. The members within each group generally show a similarity in habitat range, the second group of species being found more commonly in aquatic environments whilst those of the first group are more commonly recorded from non-aquatic habitats. , Aquatic hyphomycetes' are found abundantly in clean, fast flowing streams and rivers in most parts of the world, although they have been occasionally reported from other habitats (Bandoni, 1972; Park, 1974). They are important members of the community, breaking down plant material, especially deciduous tree leaves, that are shed into water (Kaushik & Hynes, 1971). The fungi are not a natural taxonomic group but show many similar adaptations which make them successful in this environment. They generally produce complex conidia which are more efficiently trapped - at least on smooth surfaces - than are more regularly shaped spores (Webster, 1959). Water is usually required for normal sporulation to occur and sporulation can be enhanced in some species by agitation of submerged culture disks, or shake culture pellets, with compressed air or by mechanical stirring (Younis, 1966; Webster & Towfik, 1972). This effect was not due to an increase in oxygen level, because a similar effect was shown when nitrogen was used in place of compressed air. Webster (1975) further showed that Varicosporium elodeae produced three times as many conidiophores in highly turbulent conditions as in less turbulent ones and suggested that an increase in hyphal branching, caused by agitation, could be responsible for this . Earlier initiation of conidiophores and more rapid conidium development are other possible contributing factors which might be involved in this response. This response has been investigated further by subjecting pure cultures of some ' aquatic hyphomycetes' to a range of rates of flowing water to determine the effect of water flow on sporulation. * Present address: North East River Purification Board, WoodsideHouse, Persley, Aberdeen, Scotland.

MATERIALS AND METHODS

The fungi used in the study were isolated from local streams and maintained in culture on 1 % malt extract agar. They were sub-cultured every 2 months using a suspension of conidia so that capacity to sporulate was maintained. The same technique was used to inoculate Petri dish cultures required for experiments. Of the 20 species initially tested, 17 gave consistent results and were used in further experiments. Petri dishes bearing an even layer of mycelium were used and after allowing 1 to 2 months of growth at 15 °C strips of culture were cut out, using two razor blades, mounted 1'5 mm apart, and placed inside the flow chambers. Flow chambers were constructed from Perspex to allow observation of the fungi inside them and to allow water to flow across the face of the culture strip at a steady rate . These chambers were based on the design by Descals, Nawawi & Webster (1976), with modifications to ensure that water passed across the face of the whole culture strip at an even velocity (Fig. 1). Two sides of the chambers were formed from coverslips, sealed to the Perspex using molten paraffin wax to give an air and water-tight joint. The cavity of the chamber was accurately constructed using a metal jig, the final size of the face of the culture exposed to the flow being 1'5 x 10 mm. Constant rates of flow were maintained using a 101 reservoir, feeding a 2 1 constant head flask. Four flow chambers were fed from this constant head by siphon, the height of the flow chamber determining the rate of water flowing across the fungus culture strip. Glass distilled water was used in all experiments, and the apparatus was cleaned and lightly autoclaved after each experi-

0007-1536/80/2828-6280 $01.00 © 1980The British Mycological Society

602

Sporulation of aquatic hyphomycetes

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Fig. 1. Plan (above) and elevation (below) of the Perspexflow chamber.The inflow and outflow are 1 mm internal diam capillary glasstubing. (a) Inflow; (b) fungus culture strip; (c) chamber; (d) coverslip sealed with molten wax; (e) outflow. ment. All experiments were carried out at 15°, and four replicates of each treatment were made . Flow rates of 0, 5, 10, 50, 100 and 200 em" h- 1 were normally used with each species; occasionally 400 and 800 em" h- 1 flows were also used. Volume flows were measured by collecting the outflow over a 30 min period, and these values were converted to linear flow rates using a calibration curve obtained by introducing conidia or small particles into the constant head flask and timing their passage across the face of the culture strip. All the flow values will be given as volume flow rates, in preference to linear rates. The peak of sporulation generally occurred within 60 to 72 h, after which the spore output decreased. Conidia were collected by filtration of the outflow for a period of 84 h after the start of the flow treatment, using sintered glass crucibles and glass fibre filters, similar to the method of Webster, Moran & Davey (1976). Detached conidia within the chamber were removed every 1Z h by tapping the chamber gently whilst under microscopic observation. Filters were cut into quarters and stained in cotton blue in lactic acid and the conidia counted by microscopic examination of a regular grid pattern of fields at x 150

representing 5 % of the filter area. From this count the total number of conidia was found by calculation. RESUL TS AND DISCUSSION

The results of the experiments are shown in Table 1 , and Figs 2 and 3 show the course of spore production in two species over the 84 h period under different conditions of water flow. Two groups of species can be distinguished on the basis of sporulation response to water flow: Group I: eight species showed no significant increase in sporulation when water flow rates were increased from 5 ern" h- 1 to 100 or zoo cm3 h- 1 • With the exception of Anguillospora longissima and Lemonniera terrestris these species all sporulated at zero flow rates. The group is exemplified by Heliscuslugdunensis (Fig. z) , In this species the sporulation response was at about the same level at all water flow rates above 5 em" h- 1• Group II : nine species showed significant increases in sporulation when water flow rates were increased from 5 em" h- 1 to 100 or zoo em" h- 1 . This group is exemplified by Lemonniera aquatica (Fig. 3). In all species sporulation is weak at flow

P. F. Sanders andJ. Webster

603

Table 1. Sporulation response of a range of 'aquatic hyphomycetes' to different rates of water flow (Numbers indicate total spore output in 84 h, as a mean of 4 replicates, with standard deviations .) Flow rate (cmt/h) Species 0 5 Group I Anguillospora 0 86 35 longissima (de Wild.) ±2341*** Ingold Heliscus lugdunensis 125°0 54025 Sacco & Therry ± 13262** ±3923 Lemonniera comuta 17 60 ° Ranzoni ±422*** Lemonniera terrestris 155 3425 Tubaki ±728*** ±99 Pyricularia aquatica 8540 15° Ingold ±3 107*** ±13 1 Tetracladium 154 00 74° marchalianum ±3461*** ±333 de Wild . Tricladium splendens 32 17°95 Ingold ±21t ±25 67*** Varicosporium 60 445 72 elodeae Kegel ± 1142*** ±5 6t Group II Alatospora acuminata Ingold Anguillosporacrassa Ingold Articulospora tetracladia Ingold Clauariopsis aquatica de Wild. Lemonniera aquatica de Wild. Lunulosporacurvula Ingold Tetrachaetum elegans Ingold Tetracladium setigerum (Grove) Ingold Tricladium chaetocladium Ingold

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775 ±284*** 115 ±88

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3435 ±2927* 226 50 ±3 823*** 25° 19 ±3 266*** 3°95 ±622*** 3° ±28t 900 ±33 1***

10

50

100

200 1022 5 ±2524

9660 ±2764

11545 ±2933

8765 ±2121

50760 ± 2189O 1890 ±5 17 34 65 ±891 8820 ± 2813 12 5° 0 ±3 243*

54°15 ± 18374 1980 ±598

523°0 ±20113

4100 ±1023 10005 ±3374 14 105 ±3065

324° ± 2 134 10 364 ±3883 168 9° ±3307

19 625 ±3539 54545 ± 1°479

194°5 ±4107 59560 ± 109 25

8 ±10t 480 ±311 2°9° 4 ±6208*** 355°5 ±16469** 45 25° ±5 102** 28 705 ±37 17** 8810 ± 1689*** 4° ±37t 14175 ±2817***

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1780 ±5 82

924° ±4476 16090 ±3033 2°39° ±4 195 53 160 ± 122 37

6465 ±1305

6340 ±1345

4°420 ±48 24 74°45 ±275 80** 6601 5 ±8742** 28 090 ± 4 14° 10 700 ±3903 1065 ±3 15**

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- , Not tested. t Counts made in chamber by visual estimation due to the production of few spores. Using a 2 tailed t test the difference between the preceding value is significant at: *** P < 0'001, ** P < 0'01, * P < 0'°5 ·

Sporulation of aquatic hyphomycetes rates of 5 em" h-l, and very weak or non-existent at zero flow rate. The essential difference in response of the two groups is that the Group I spec ies are generally

capable of sporulation at zero flow rates, but above very low flow rates their sporulation is not enhanced by increasing flow, whilst in Group II species sporulation is poor at zero flow rates, and

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Fig. 2 . Sporulation response of H eliscus lugdunensis in different rates of flowing water. e, 0 em" h- 1; 0 , 5 em" h- 1 ; _ , 10 ern! h- 1 ; 0 , 50 ern " h - 1 ; A, 100 ems h- 1• Points are means of four replicates with standard deviations shown. 21

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Fig. 3. Sporulation response of Lemonn iera aquatica in different rates of flowing water . e, 0 ern " h- 1 ; 0 , 5 em" h- 1 ; _ , 10 ern" h - 1 ; 0 , 50 ern" h- 1 ; A, 100 em" hr ' . Points are means of four replicates with standard deviations shown.

P. F. Sanders and J. Webster continues to improve as water flow rate increases. The higher volume flow rates used in these experiments correspond to linear flow rates likely to be encountered in a stream, whilst the lower flow rates are similar to those found in ditches or drainage channels with slow-moving water. The ability to sporulate in static water shown by most Group I species as compared with most Group II species correlates with their known range of habitats. All the species included in Group I, with one exception (Lemonniera cornuta) have been reported from terrestrial litter (Bandoni, 1972; Scourfield, 1940). Some can sporulate on dry agar surfaces, for example H eliscus lugdunensis, Varicosporium elodeae (Nilsson, 1964; Petersen, 1962). All are active or survive well in terrestrial conditions (Sanders & Webster, 1978). The Group II species,· with the exception of Tetracladium setigerum, seem to have little tolerance to these conditions, although some species survive for long periods in dry leaf litter (for example Articulospora tetracladia and Clavariapsis aquatica). Many of these 'aquatic hyphomycetes' have been shown to have ascomycetous or basidiomycetous perfect stages (Webster & Descals, 1979), and these are found in both groups I and II (for example Anguillospora crassa and H. lugdunensis). Younis (1966) and Webster & Towfik (1972) found that sporulation could be enhanced by mechanical agitation or by forced aeration. The results of this work on cultures in flow chambers are, presumably, a demonstration of the same effect. They used Lemonniera aquatica, Articulospora tetracladia, Tetrachaetum elegans, Heliscus lugdunensis and Varicosporium elodeae in their experiments and found that all these species showed a similar increase in sporulation when an aeration rate of 100 em" min- I was compared with 1000 em" min-I. Sporulation was increased in all these species by a factor of approximately two at the higher aeration rate. H. lugdunensis and V. elodeae are both Group I species and did not show a corresponding sporulation response in the flow chambers. It is not clear why these results should differ, unless the conditions of aeration at 100 em" min- I approximate to a flow rate in the chambers of below 5 ern" h- I. It is possible that these results indicate ecological groups which are adapted to a greater or lesser extent to the aquatic environment, with some species being not exclusively aquatic in nature, but able to sporulate in standing or slowly moving water, or even water films over leaves.

605

We would like to thank Dr E. Descals for his help with the design of the flow chamber. This work was supported by a grant to P. F. Sanders from the Science Research Council. REFERENCES BANDONI, R. J. (1972). Terrestrial occurrence of some aquatic hyphomycetes, Canadian Journal of Botany 50, 2283-2288. DESCALS, E., NAWAWI, A. & WEBSTER, J. (1976). Development studies in Actinospora and three similar aquatic hyphomycetes. Transactions of the British Mycological Society 67, 207-222. KAUSHlK, N. K. & HYNES, H. B. N. (1971). The fate of the dead leaves that fall into streams. Archive fur Hydrobiologie 68, 465-515. NILSSON, A. (1964). Freshwater hyphomycetes, taxonomy, morphology, and ecology. Symbolae Botanicae Upsalienses, Uppsala 18, 1-130. PARK, D. (1974). Aquatic hyphomycetes in nonaquatic habitats. Transactions of the British Mycological Society 63, 183-187. PETERSEN, R. H. (1962). Aquatic hyphomycetes from North America: I. Aleuriosporae (Part I) and Key to the Genera. Mycologia 55, 117-151. SANDERS, P. F. & WEBSTER, J. (1978). Survival of aquatic hyphomycetes in terrestrial situations. Transactions of the British Mycological Society 71, 231-237. SCOURFIELD, D. J. (1940). The microscopic life of the 'leaf carpet' of woods and forests. Essex Naturalist 26, 231-246. WEBSTER, J. (1959). Experiments with spores of aquatic hyphomycetes. I. Sedimentation and impaction on smooth surfaces. Annals of Botany (London) 23, 595-611. WEBSTER, J. (1975). Further studies of sporulation of aquatic hyphomycetes in relation to aeration. Transactions of the British Mycological Society 64, 119- 127. WEBSTER, J. & DESCALS, E. (1979). The teleomorphs of water-borne hyphomycetes from fresh water. In The Whole Fungus, vol. II (ed. W. B. Kendrick), pp. 419-451. Ottawa, Canada: National Museum of Natural Sciences. WEBSTER, J., MORAN, S. T. & DAVEY, R. A. (1976). Growth and sporulation of Tricladium chaetocladium and Lunulospora curvula in relation to temperature. Transactions of the British Mycological Society 67, 491-549. WEBSTER, J. & TOWFIK, F. H. (1972). Sporulation of aquatic hyphomycetes in relation to aeration. Transactions of the British Mycological Society 59, 353-364. YOUNIS, F. M. (1966). Sporulation in aquatic hyphomycetes. M.Sc. Thesis, University of Sheffield.

(Received for publication 25 June 1979)