Effect of inoculum size on conidial germination in Aspergillus flavus

Notes and brief articles sporulation of both fungi are shifted to lower values, around S° (or possibly lower) for T. chaetocladium and around 10° for L. curvula. The interpretation of these experiments is not straightforward. Although the optima for growth and sporulation for L. curvula are some S° higher than for Tricladium, stream temperatures in the River Creedy and in nearby streams never reached 20°, and the responses of the fungi to temperature change within the range 3-15° are of most interest. The fact that L. curvula is capable of more rapid growth than Tricladium may tend to obscure the fact that when growth rate is expressed as a percentage of the maximum (Fig. 5), Tricladium shows relatively better growth than Lunulospora at temperatures below 15°. The relative performance of the two fungi when growing in competition with each other shows up differently, with maximum sporulation of T. chaetocladium near S°, compared with 10° for L. curoula. No evidence of antagonism between the two fungi was found when one fungus was grown over agar separated by a layer of cellophane, which was then stripped off and the area re-inoculated with the other fungus. The better performance of L. curvula at higher temperatures may be reflected in its world-wide distribution, with particular abundance in tropical countries (Ingold, 1975). The seasonal fluctuations in spore concentration of these two fungi may be controlled by other factors, such as availability of substrate. Iqbal (1972) has suggested that in the rivers he studied, L. curvula is most common on

495

leaves of Alnus glutinosa, and has claimed that since the tissues of this leaf are more susceptible to decomposition, and to breakdown by current action, the relatively early depletion of Alnus leaves may be an explanation of the early disappearance of L. curvula in detectable concentration from river water. Another possibility, which the experiments reported here do not explore, is that temperature influences the ability of the two fungi to compete with other organisms colonizing available substrata. REFERENCES

Goos, R. D. (1970). In vitro sporulation in Actinospora megalospora. Transactions of the British Mycological Society 55, 335-357· INGOLD, C. T. (1974). Tricladiumchaetocladium sp.nov., an aquatic Hyphomycete from Britain. Transactions of the British Mycological Society 63, 38-40. INGOLD, C. T. (1975). An illustrated guide to aquatic and water-borne Hyphomycetes (Fungi Imperfecti) with notes on their biology. Scientific Publication No. 30, Freshwater Biological Association, 96 pp. IQBAL, S. H. (1972). Some observations on aquatic Hyphomycetes. Ph.D. Thesis, University of Exeter. IQBAL, S. H. & WEBSTER, J. (1973). Aquatic Hyphomycete spora of the River Exe and its tributaries. Transactions of the British Mycological Society 61, 33 1-346.

WEBSTER, J. & TOWFIK, F. H. (1972). Sporulation of aquatic Hyphomycetes in relation to aeration. Transactions of the British Mycological Society 59, 353-364.

EFFECT OF INOCULUM SIZE ON CONIDIAL GERMINATION IN ASPERGILLUS FLAVUS P. M. MOORE* AND

J.

F. PEBERDY

Department of Botany, School of Biological Sciences, University of Nottingham, University Park, Nottingham NG7 2RD

The subject of autochemotrophic response in fungal spores has recently been reviewed (Robinson, 1973). The production of germination selfinhibitors was reported by Boyd (1952) and methyl ferulate was identified as such an agent in Puccinia graminis P. uredospores (Macko et al., 1971). Growth promotors were demonstrated in the culture filtrates during germination of Rhizopus stolonifer Ehrent. spores (Robinson, Dark & Graham, 1968). Other workers have cited in* Present address: Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7. Trans. Br, mycol. Soc. 67 (3), (1976).

sufficient supplies of oxygen or carbon dioxide as germination inhibitors (Doran, 1922; Trinci & Whittaker, 1968). During quantitative experiments involving the production and regeneration of fungal protoplasts, the presence of ungerminated spores is a critical factor. In view of the difficulties involved in separating such spores from protoplasts, it is essential to reduce their numbers as much as possible. We have found, during studies on protoplasts of Aspergillus fiavus Link (peberdy et al., 1976; Moore & Peberdy, 1976a), that we were

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496

Notes and brief articles

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samples removed after 24 h incubation were determined using a haemocytometer. Dry weight estimates were made by filtering entire cultures onto preweighed Whatman No . 1 filter disks, washing with distilled water, and drying at 90°. Over the range tested an apparently linear relationship was found to exist between the number of ungerminated spores at 24 h and initial spore concentration (Fig. 1). Mycelial yields are expressed as dry weight produced per germinated spore (Fig. 2). After 24 h incubation, the yield of material per spore remains fairly constant at concentrations of 2'5 x 106 ml and above. Below this concentration the yields per conidium rise quite sharply. The mechanism controlling spore germination is not fully understood. We have found, however, a linear relationship between the number of ungerminated conidia in a culture and inoculum concentration. Where the number of ungerminated conidia present in a culture is critical, as is the case with protoplast production, we feel that it is important to use low inoculum concentrations, and that the loss in growth yields are compensated for by a higher yield per spore.

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REFERENCES

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BoYD, A. E. W. (1952). Dry rot disease of potato. IV. Laboratory methods used in assessing variations in tuber susceptibility. Annals of Applied Biology 39,

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DORAN, W. L. (1922). Effect of external and internal factors on the germination of fungal spores. Bulletin

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of the Torrey Botanical Club 49,313-323.

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MACKO, V., STAPLES, R. C., ALLEN, P. J. & RENWICK, J. A. A. (1971). Identification of the germination self inhibitor from wheat stem uredospores, Science New York 173, 835-836. MOORE, P. M. & PEBERDY, J. F. (1976 a). The release and regeneration of protoplasts from the conidia of Aspergillus flaous , Transactions of the British Mycological Society 66,421-425. MOORE, P. M. & PEBERDY, J. F. (1976 b). A particulate chitin synthase from Aspergillus fla ous Link: The

properties, location and levelsof activity in mycelium and regenerating protoplast preparations. Canadian able, by controlling spore inoculum density, to reduce drastically the numbers of ungerminating spores in the medium. A . fiavus conidia were harvested from 3 day malt extract agar slants and germinated in 250 ml minimal salt medium N (Vogel, 1956) under conditions described elsewhere (Moore & Peberdy, 1975b). Fl asks containing 0 '1-12 '5 x 106 spores ml were incubated at 30° on a rotary shaker (200 rpm). The numbers of ungerminated spores in Trans. Br. my col. Soc. 67 (3), (1976).

Journal of Microbiology. 27,915-921.

PEBERDY, J. F., BUCKLEY, C. E., DALTREY, D. C. & MOORE, P. M. (1976). Factors affecting protoplast release in some filamentous fungi. Tran sactions of the British Mycological Society 67, 23-26.

ROBINSON, P. M. (1973). Autotrophism in fungal spores and hyphae. The Botanical R eviews 39, 367384 .

ROBINSON, P. M., DARK, D. & GRAHAM, T. A. (1968). Autotrophism in fungi. Journal of Experimental Botany 19, 125-134.

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Notes and brief articles 497 WHITTAKER, C. (1968). Self in- VOGEL, H. J. (1956). A convenientgrowth medium for

TRINCI, A. P. T. & hibition of spore germinationin Aspergillusnidalans. Transactions of the British Mycological Society 51, 594-596.

Neurospora (Medium N). Microbial GeneticsBulletin

13, 42-43.

EFFECTS OF TEMPERATURE, LIGHT AND AERATION ON THE PRODUCTION OF MICRO SCLEROTIA BY PYRENOCHAETA LYCOPERSICI J. G. WHITE*

University of Reading, Horticultural Research Laboratories, Shinfield Grange, Reading, Berks.

In the context of studies on Pyrenochaeta lycopersici Schneider & Gerlach, a reliable method was sought for the production of micro sclerotia. The fungus produces aerial and submerged microsclerotia (White & Scott, 1973), but while the conditions favouring their selective production were not clear, the limited projectin hand did not justify a complex investigation. In preliminary experiments P. lycopersici produced many of both aerial and submerged microsclerotia on 2 % malt agar (Oxoid L.39 malt extract and 2 % Davis Standard Agar), only aerial microsclerotia on potato dextrose agar (Oxoid CM.139) and small numbers of both types on Czapek Dox (Oxoid CM.97) or V8 Juice agar (10 % Campbell's V8 juice with 2 % Davis Standard Agar). No micro sclerotia were produced by cultures grown on agar containing a range of concentrations of peptone or sucrose, on sand/ maize meal mixtures or on malt extract agar in response to physical damage to the mycelium. There were, however, indications that microsclerotium production on malt extract agar could be affected by the conditions under which the the cultures were incubated. Aerial microsclerotia were germinated on plates of the 2 % malt extract agar and incubated at 25° in the dark for 14 days to provide mycelial inoculum for freshly poured plates of malt extract agar. Stacks of 10 plate cultures were incubated at 25° in a growth room in constant light (500 ft candles, approximately 19 watts 1m2 from warm white fluorescent tubes) or in blacked-out boxes (constant dark) in the same growth room. Similar cultures were incubated in diffuse daylight or in blacked-out boxes on the laboratory bench (range 8-22°). The polyethylene bags in which the plates were stored were either left unsealed, or sealed with adhesive tape to give conditions of reduced

* Now at National Vegetable Research Station, Wellesbourne, Warwickshire, CV35 9EF. Trans. Br, mycol, Soc. 67 (3), (1976).

Table 1. Microsclerotia produced under individual treatment conditions Submerged microConditions sclerotia 25°, light, unsealed 641 25°, light, sealed 178 25°, dark, unsealed 634 25°, dark, sealed 245 Room temp., light, unsealed 83 Room temp., light, sealed 14 Room temp., dark, unsealed 27 Room temp., dark, sealed 103

Aerial microsclerotia 264 152 25 17 651 281 433 209

* Each figurerepresents the mean of three replicates of three subcultures (80 individual em" readings). aeration. In this way, all the conditions under which microsclerotia had previously been produced could be compared in one experiment. After 28 days the numbers of aerial and submerged microsclerotia occurring in 8 x 1 em band transects across the top and bottom of each culture were counted and mycelium from representative cultures was used to inoculate a new series of plates. This process was repeated twice to give a series of three subcultures from the original microsclerotial cultures and the experiment was repeated three times. The mean numbers of microsclerotia produced under the eight individual treatment combinations are shown in Table 1. The most favourable conditions for the production of submerged microsclerotia were light or dark at 25° in unsealed polyethylene bags, whilst those for the production of aerial microsclerotia were room temperature with diffuse daylight and unsealed polyethylene bags. It would appear that the optimum temperature for production of aerial micro sclerotia is lower than that for production of submerged microsclerotia since the laboratory temperature was always somewhat lower than 25°. In all cases fewer

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