Autotropism in hyphae of Saprolegnia ferax

[257 ] Trans. Br . mycol. Soc. 83 (2), 257-263 (1984)

Printed in Great Britain

AUTOTROPISM IN HYPHAE OF SAPROLEGNIA FERAX By P. M . ROBINSON AND S . K . BOLTON Department of Botany, The Queen's University, Belfast BT7 INN Hyphae of Saprolegnia ferax displayed marked negative autotropism on cellophane-overlaid media. The closest distance that hyphae approached other hyphae during negative autotropism increased from 0 to a maximum of - 28 /lm as the concentration of either malt extract or a mixture of 21 amino acids decreased in the medium. The percentage of hyphae which displayed negative autotropism increased with increase in the number of amino acids in media containing the same total concentration of amino acids. The rate of hyphal extension did not alter significantly during the autotropic response. Negative autotropism in S. [erax is considered to be a positive chemotropism to a gradient in amino acid concentration which results from depletion of amino acids in the vicinity of actively-growing hyphae , Clark (1902) proposed that fungal chemotropism could possibly be explained by fungi exhibiting a negative tropism to their own metabolites, He concluded that this negative autotropism was greater than any positive chemotropism to nutrients or oxygen. This conclusion was supported by the work of Fulton (1906), Graves (1916) and Stadler (1952). The early work on chemotropism in fungi has been reviewed by Ziegler ( 1962), Carlile ( 1966), Robinson (1973) and Gooday ( 1975) but there have been few recent reports on autotropism in hyphae. Trinci (1970) described a tendency for lateral branches of Aspergillus nidulans to grow parallel to and close to the parent hyphae. Strands of three or four parallel hyphae were frequently observed. This could be interpreted as positive autotropism and a similar effect was noted with leader hyphae of Geotrichum candidum when colonies were grown on cellophane-overlaid media (R obin son, 1973 ). Trinci, Saunders, Gosrani & Campbell (1979) noted that some hypha I tips of A . nidulans, Mucor hiemalis and Neurospora crassa displayed negative responses on cellophane-overlaid media as they approached the sides of neighbouring hyphae. Hutchinson, Sharma, Clarke & MacDonald (1980) made an extensive study of the control of hyphal orientation in colonies of M. hiemalis. In a series of micromanipulation experiments they demonstrated negative autotropism between pairs of hyphal tips, each pair comprising a parent hypha and its branch. They analysed colony growth patterns by computer simulation and concluded that there was' no evidence that orientating interactions between hyphal tips have any biologically significant effect on colony shape ' . In the course of studies on fungal autotropism in this laboratory it was noted that hyphae of 9

Saprolegnia ferax (Gruith.) Thuret displayed marked negative autotropism. The results of the investigation are reported here, MATERIALS AND METHODS

Routine culture Stock cultures of S .ferax were maintained in 9 em diam Petri dishes on a medium with the following composition (g I-I): Oxoid peptone, 0 '01 ; Oxoid agar no. 1, 10. The fungus was subcultured by transferring 7 mm diam discs from a stock culture to fresh medium. Cultures were incubated at 25 °C in the dark. Preparation and inoculation of m edia Culture media were dispensed in 20 em" lots to 9 cm diam plastic Petri dishes. The medium used for the initial part of the investigation was malt agar (O xoid malt extract, as appropriate ; Oxoid agar no. 1, 10 g ; distilled water to 1 1). A defined medium buffered to pH 6,6 was developed for the later stages of the investigation and this had the following composition (g I-I) : L-glutamic acid, 0'05; t.-merhionine, 0'05; KH 2PO .. 5'2 ; Na 2HP0 4 • 12H 20, 7'4; MgS0 4 • 7H20, 0'5; Oxoid agar no. 1, 10. The r.-amino acids were obtained from Kit no. LAA-21 (Sigma Chemical Company). The medium in each Petri dish was overlaid with a 5 em square of cellophane (PT 300, British Cellophane Ltd). The cellophane squares were boiled for 5 min in distilled water and autoclaved at 121 ° for 15 min in fresh distilled water prior to use. Excess surface water was drained from the cellophane by inclining the plates at an angle of 45 0 for 18 hat 25 0 before inoculation. Dishes were inoculated centrally with 7 mm diam discs cut from just behind the M YC 83

Autotropism in Saprolegnia colony margin of a 3-day-old stock culture of S . ferax which was cultured on a weak medium, 0 '001 % peptone, to minimize contamination of the as say system with additional nutrients .

M easurement of th e autotropic response There were many instances of branch hyphae growing towards parent hyphae or other branch hyphae when S . ferax was grown on cellophane over various concentrations of malt agar. The hypha 1 t ip of a first order branch hypha generally showed a negative autotropic response to the side of a neighbouring h ypha and grew in such a wa y as to avoid contact with any part ofthe m ycelium. The most common type of avoidance reaction was a U-turn which resulted from a hypha making a more or less right-angled approach to the side of a neighbouring hypha (Fig. 1). A less common reaction was a sinuous growth pattern exhibited by the central member of three hyphae growing alongside, but not touching, each other. Sometimes a spiral pattern occurred when a hypha 1 tip became trapped within older regions enclosed by the same hypha . Four replicate cultures were prepared for each treatment and incubated at 25 0 for 48 h before measurements of 25 autotropic responses were recorded for each culture from a region just proximal to the colony perimeter. Only the U-shaped reactions were recorded and these were randomly selected and a calibrated eyepiece micrometer used to record the closest distance of approach (C D A) of each pair of hyphae during the autotropic response. An estimate for the frequency of the autotropic response was obtained from the same colony regions used for CDA determinations. For each treatment 100 pairs of hyphae, each in a tip-to-side formation, were randomly selected from each of the 4 replicate cultures and the percentage of pairs

which displayed negative autotropism wa s recorded. Whenever a pair of hyphae failed to respond in a negative autotropic fashion the approaching tip usually grew across the side of the neighbouring hypha and showe d n o deviation in its direction of growth. Occasionally an approaching hypha would touch a neighbouring hypha and grow alongside, in contact with it . Such instances were not score d as autotropic responses.

Tim e-lapse photography H yphal pairs were selected in which a hyphal tip was growing towards the side of a neighbouring hypha. The potential interactions were photographed at 2 m in intervals on 35 rom film with an Olympus OM t ime-lapse system (O lym p us Optical Co., Honduras Street, London) attached to a Leitz Dialux 20 EB microscope. The use of a long working distance objective (x 32) eliminated the need to open the Petri dish during the experiment. The rates of hypha 1 extension before, during and after each autotropic response were determined at 20 0 from photographic enlargements of the negatives. For these experiments S. ferax was cultured on malt extract at 0'5 g 1-1 and on defined media containing all 21 amino acids at total concentrations of 0 '05 and 0 '1 g I-I respectively. The extension rate of first order branches was determined for a series of defined media in which the total amino acid concentration ranged from 0 '025 to 0 '5 g 1-1. Areas of the colony normally used for the determination of CDA and frequency analysis were photographed at 0 '5 h intervals during a period of 1.5 h. The average extension rate for 25 lateral branches which continued extending throughout this period was determined for each medium from photographic enlargements. The percentage of branches which did not grow during this l ' 5 h period was also recorded.

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(b)

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T ypes of negative autotropism exhibited by lateral branch es of S . fe ra x , (a) U-sh aped re act ion , (b) sin uous reaction (c) spiral reaction.

P. M . Robinson and S. K. Bolton Statistical treatment of results The relationship between CDA and log of either the malt extract concentration or the total amino acid concentration in the culture media (F igs 2 , 3) was examined by regression analysis (P arker, 1979 ). The data for frequency in Table 1 were subjected to analysis of variance and a fixed range test (usin g the least significant range) applied to make comparisons between treatment means (P arker, 1979 ). In Figs 2 and 3 the confidence intervals (9 5 % ) for mean percentage values of frequency were determined from tables of confidence limits for percentages (R oh lf & Sokal, 1969). RESULTS

Effect of nutrient concentration on autotrop ism A series of malt agar media was prepared in which the malt extract concentration ranged from 0'02 to 2 g 1-1. The media were overlaid with cellophane, inoculated centrally with S. ferax, and the CDA and frequency of negative autotropism determined as already described. There was a significant (P < 0 '001 ) negative rectilinear relationship between CDA and the log of the malt extract concentration in the media (F ig. 2). The higher CDA values were correlated with the media containing the lower concentrations of malt extract. Results for media containing malt extract at less than 0 '02 g 1-1 are not recorded as colonies grew very poorly on media containing malt extract at 0 '01 g 1-1 and had a very low frequency of branch formation. This resulted in a decreased number of

259

potential hyphal interactions although negative autotropism did occur in some of these instances . Colonies exhib ited starvation growth on distilled water agar and no avoidance reaction occurred in the rare event of a potential hyphal interaction. Extrapolation of the regression line in Fig. 2 predicted a CDA of zero when S .ferax was grown on malt extract at 7.6 g 1-1 . This proved to be the case but many h yphal tips exhibited negative autotropism after first making contact with the side of the neighbouring hypha. On a more concentrated medium such as malt extract at 20 g 1-1 there were very few instances of negative autotropism, most hyphae grew across any others in their path without any deviation in their direction of growth before, during and after contact. The frequency of negative autotropic responses for hyphae in tip-to-side approaches reached maximum values of 68 and 66 % on media containing malt extract at 0'2 and 0'5 g 1-1 respectively (F ig . 2 ).

Autotropism on defined media A defined medium was developed to determine if the presence of one or more particular nutrients was critical for hyphal autotropism to occur. It was essential to have at least two L-amino acids present for growth, one of which had to contain su lp h u r, and it was necessary to buffer the medium as the addition of certain amino acid combinations produced pH changes beyond the limits for normal growth and development. When S. fera x was cultured on cellophane over this defined medium only 18 % of hyphal pairs displayed negative autotropism (T ab le 1). The frequency increased when the number of amino 32 , - - - - -- - - - - - - - - - - - . , lOa acids incorporated in the medium was increased. 28 The aromatic amino acids (P he, Tyr and Trp) played a significant role in promoting the frequency 24 75 of negative autotropic responses. The inclusion of Phe with Glu + Met, for example, raised the 20 ~ frequency from 18 to 53 %, a value not significantly • ;., different from that obtained with a combination of o ::: ~ 16 50 ., all 21 essential amino acids. In a mixture of 18 =' ? .,... amino acids which did not include Phe, Tyr and 12 • Trp the frequency was 31 % which, although "'" significantly greater than for mixtures (minus those 8 25 three amino acids) of from 2 to 4 amino acids, was ,, ,, significantly less than the value obtained for the 4 ,, combination of all 21 amino acids. , a a A significant (P < 0 '001 ) negative rectilinear 0·01 0·1 10 relationship was established between CDA and the Malt extract (g 1-1. log scale) log of the total concentration of amino acids in the Fig. 2 . Effect of malt extract concentration on CDA (. ) defined medium when all 21 amino acids were and fr eq uency ( 0 ) of negative autotropism in S. ferax. combined (F ig . 3). The CDA increased as the total Vertical lines represent 95 % confidence int ervals. concentration of amino acids decreased. Extra-

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260

Table

Autotropism in Saprolegnia 1.

Effect of different combinations of amino acids on frequency of negative autotropism in S . ferax Frequency of negative autotropism ( 00)

Amino acid combination Total (2 1) amino acids Glu, Met, Leu, Thr, L ys, C ys, Glyn, Val, Ala, Phe Glu, Met, Leu,Thr Glu, Met, Leu Glu, Met Total minus Phe Total minus Tyr Total minus Trp Total minus Phe, Tyr and Trp Glu, Met, Phe Peptone ( 0 -1 g 1-' )

588

53 8 19 c 11 c

18 C 518

56 8 528 31b

53 8 62 8

Each mean value for negative autotropism was based on 400 ( 100 from each of 4 replicate cultures) pairs of hyphae in tip-to-side approaches. Negative autotropisrn was considered to have occurred whenever the tip of the approaching hypha turned and grew away from the side of the neighbouring hypha. Each amino acid combination was assayed at a total concentration of 0 '1 g 1-1 and each amino acid was present at o-l /n g I-I, where n = the number of amino acids in a particular combination. The non-amino acid components of the medium were as in the defined medium (see Materials and Methods). Mean values with different letters differ significantly at P < 0'05.

100

• 75

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0·1 Total amino acids (g I-I , log scale) Fig. 3. Effect ofamino acid concentration on CDA (.) and frequency (0 ) ofnegative autotropism in S .fe rax , Vertical lines represent 95 % confidence intervals.

polation of the regression line predicted a CDA of zero when the total amino acid concentration was 0'47 g I-I. This was confirmed. The values for frequency of the negative autotropic response reached a plateau on media containing amino acids at 0'025, 0 '05 and 0'1 g I-I.

Time-lapse photomicroscopy Negative autotropism in S.ferax was studied using time-lapse photography as described in Materials and Methods . Photographs were taken of hyphaI tips at 2 min intervals from the time at which a hyphal tip was on course to collide with a neighbouring hypha to the time at which negative autotropism had been displayed. There was no significant change in the rate of hyphal extension during the tropism (Fig. 4). The tropism illustrated was typical of six similar tropisms photographed in separate colonies on malt extract at 0'5 g I-I. The rate of hyphaI extension did not change during the avoidance reaction when colonies were grown on the defined medium containing all 21 amino acids at total concentrations of 0'05 and 0 '1 g I-I . The extension rate of first order branches increased with increase in the total amino acid concentration in the defined medium (Fig. 5). The decreased extension rate at the lower amino acid concentrations was correlated with the tendency for a greater percentage of the lateral branches not to extend during the l' 5 h period of measurement. DISCUSSION

Several workers have recorded the distance at which negative autotropism occurs between two neighbouring hyphae. When M . hiemalis, A. nidulans and N. crassa were grown on media overlaid with cellophane the mean distances separating hyphae when negative autotropism was

P. M. Robinson and S. K. Bolton

261

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0·025 0-05 0·1 0·25 0-5 Total amino acids (g 1-1. log scale) Fig. 5. Mean extension rates (.) and frequency of growth cessation CO) of lateral branch hyphae of S. ferax on media containing different total concentrations of amino acids. Vertical lines represent 95 % confidence intervals.

observed for tip-to-side approaches were 24, 27 and 30 pm respectively (Trinci et al., 1979). Hutchinson et al. (1980) reported that hyphal tips ofM. hiemalis repelled each other when 10-20 pm apart on media not overlaid with cellophane. In the present investigation the closest distance of approach

(CDA) of neighbouring hyphae of S. ferax was consistent for a particular growth medium but increased from 0 to 29 pm as the malt extract concentration decreased from 7'6 to 0'02 g 1-1. Positive and negative autotropic effects in fungi have often been attributed to self-produced metabolites (Stadler, 1952; Muller & Jaffe, 1965; Jaffe, 1966; Robinson, Park & Graham, 1968; Robinson, 1980) but the existence of such factors has not been unequivocally demonstrated. With respect to negative autotropism of hyphae it is important to consider an alternative hypothesis that the avoidance may be a positive chemotropism resulting from depletion of one or more nutrients in the immediate vicinity of each hypha. Hutchinson et al. (1980) considered both hypotheses in relation to negative autotropism of hyphae of M. hiemalis but were unable to eliminate either possibility completely. It seemed relevant to examine the nutrient depletion hypothesis in relation to the present investigation with S.ferax as positive chemotropism to sources of amino acids has been recorded for hyphae of Saprolegnia (Wortmann, 1887), Saprolegnia mixta, S .ferax and Achlya polyandra (Fischer & Werner, 1955) and Achlya bisexualis (Musgrave, Ero, Scheffer & Oehlers, 1977). The linear relationship between CDA and log malt extract concentration (Fig. 2) pointed to a diffusion phenomenon, either to production of a metabolite

262

Autotropism in Saprolegnia

which induced negative autotropism or to depletion of nutrients in the vicinity of hyphae . The results of further experiments tended to support the latter hypothesis, more specifically, that negative autotrop ism re sulted from a reduction in amino acid concentration in the neighbourhood of growing hyphae . The main evidence was the correlation of CDA with the concentration of amino acids in a defined medium (F ig . 3). The increase in CDA as the total amino acid concentration was reduced was consistent with the results for CDA and different malt extract concentrations . The autotropic resp on se occurred when the medium consisted of a mixture of Z1 amino acids and agar without any of the other constituents of the defined medium. Additional support for this hypothesis came from an experiment, not described in this paper, in which agar discs containing Z1 amino acids at a total concentration of 0 'Z5 g 1-1 were placed behind the margin of colonies of S. fera x growing on the defined medium (minus cellophane) plus Zl amino acids at a total concentration of 0 '05 g 1-1. The leader hyphae and lateral branches in the neighbourhood of the discs turned and grew backwards, i.e. in a proximal direction, towards the discs. Musgrave et al, (1977) reported that germlings of A. bisexualis exhibited positive chemotropism to a wide range of concentration of casein hydrolysate ; the response to a mixture of equal quantities of all the essential amino acids occurred over a narrower concentration range. In the present in vestigat ion the full range of the autotropic response from 0 to - 28 fLm, was realised over a wider range of medium dilution for a medium containing malt extract (F ig . z) than for a defined medium containing equal quantities of 21 amino acids (F ig . 3). Both casein hydrolysate and malt extract would reflect a more natural qualitative and quantitative balance of amino acids and this could partly account for these effects. The chemotropic response could also be sensitized by som e of the non-amino acid components in these mixtures . The frequency of negative autotropic reactions varied not only with the total concentration of amino acids (F ig . 3) but also with the number of amino acids in the medium (Table 1). Frequency would be expected to fall at amino acid concentrations above and below the critical levels beyond which a gradient in concentration of one or more amino acids would not be perceived across the apex of a hypha of S . [erax , The decline in frequency observed at amino acid concentrations above 0 '10 g 1-1 and below 0 '025 g 1-1 fits this supposition and correlates with the attainment of the near m in imum and maximum values for CDA at these concentrations (F ig . 3). Musgrave et al, ( 1977) determined the upper and lower limits of con-

centration ofamino acid combinations which would elicit a positive chemotropic response in germlings of A . bisexualis, The greater the number of amino acids in the m ixtures (t h e total concentration of amino acids was constant) the greater the con centration range over which chemotropism wa s detectable . If these results can be applied to Si fera x then frequency would be expected to fall as the number of amino acids decreased. The en hancement of frequency when one of the aromatic amino acids was included in defined media also indicated the importance of the qualitative balance of amino acids in relation to ch em ot rop ic effects (T ab le 1). Do the re sults have any relevance to the pattern of colony development in S. f erax? On cellophane the hyphae are over a large sink of nutrients but growth is restricted to one plane and limited by the rate of diffusion of nutrients from below in relation to demand. Without cellophane the hyphae radiate in three dimensions and the concentration of the growth-limiting component(s) of the medium would be very low just proximal to the apices of the leader hyphae . Under these conditions the CDA might be expected to be high but pronounced negative autotropism would not be apparent for two reasons. Firstly, the hyphae can manoeuvre in three dimensions and so the pronounced curvatures seen on cellophane do not occur. Secondly, the steep gradient in amino acid concentration from in front of to behind the colony margin would tend to direct branch hyphae in a radial direction and override tangential movement of branch tips . The ob servation of Fischer & Werner ( 19 55) that the leader hyphae of colonies of S. ferax can be induced to turn through 180 0 to a source of amino acids placed proximal to the tips lends support to the idea that concentration gradients of particular nutrients may contribute, at least in some species, to the observed centrifugal growth pattern. We wish to thank Miss J. C . Walker of the Commonwealth Mycological Institute for the identification of the Saprolegnia used in this investigation and also the Department of Education for Northern Ireland for the award of a research scholarship to S. K. Bolton.

REFERENCES CARLILE, M. J. ( 19 66) . The orientation of zoo sp ores and germ-tubes. In The Fungu s Spore (ed. M . F . M adelin), pp. 175-186. London : Butterworths. CLARK, J. F . ( 190 2). On the toxic properties of some copper compounds w ith sp ecial reference to Bordeaux m ixture. B otanical G azette 33, 26--48 . FISCHER, F. G . & W ERNER, G . (19 55). Eine Analyse des Chemotropismus einige Pilze, ins be sondere der Sapro-

P. M . Robinson and S. K. Bolton legniaceae. Hoppe-Seyler's Z eitschrift fur physiologis che Chem ie 300, 211-236. FULTON,H . R . (1906) . Chemotropism of fungi . Botanical Ga zette 62, 337-369. GOODAY, G. W. (1975). Chemotaxis and chemotropism in fungi and algae. In Primary Sensory and Communication Systems (ed . M. J . Carlile), pp. 155-204. London : Academic Press. GRAVES, A . H. ( 1916) . Chemotropism in Rhizopus nigri cans. Botanical Gazette 62 , 337-369. HUTCHINSON, S. A ., SHARMA, P ., CLARKE, K . R. & MACDONALD, I. ( 1980) . Control of hyphal orientation in colonies of Mucor hiemalis. Transactions of the Brit ish Mycological Society 75, 177-191. JAFFE, L. F. (1966). Onautotropism in Botrytis: measurement technique and control by CO 2 • Plant Physiology 4 1, 303-306. MiiLLER, D . & JAFFE, L. F. ( 1965) . A quantitative study of cellular rheotropism. Biophy sicalJournal 5,317-335 . MUSGRAVE, A., LOES ERO, SCHEFFER, R. & OEHLERS, E. (1977). Chemotropism of A chlya bisexualis germ hyphae to casein hydrolysate and amino acids. J ournal of G eneral Microbiology 101 ,65-70. PARKER, R. E. (1979). Introductory Statistics for Biology. Studies in Biology no. 43 . London: Edward Arnold.

ROBINSON, P . M . (1973). Autotropism in fungal spores and hyphae. Th e Botanical Review 39, 367-384. ROBINSON , P . M . ( 1980) . Autotropism in germinating arthrospores of Geotrichum candidum. Transactions of the British M y cological So ciety 75, 151-153. ROBINSON, P. M ., PARK, D. & GRAHAM, T. A. ( 1968). Autotropism in fungal spores. Journal of Experimental Botany 58, 125-134. ROHLF, F. J. & SOKAL, R . R. (1969). Statistical Tables. London : Freeman. STADLER, D. R . ( 1952) Chemotropism in Rhizopus nigricans: the staling reaction. Journal of Cellular and Comparativ e Physiology 39, 449-474· TRINCI, A. P. J . (1970). Kinetics of apical and lateral branching in Aspergillus nidulans and Geotrichum lactis. Tran sactions of the British Mycological Society 55, 17-28. TRINCI, A. P. J ., SAUNDERS, P . T., REKHA GOSRANI & CAMPBELL, K. A. S . (1979). Spiral growth of mycelial and reproductive h yphae. Transactions of the British M y cological Soc iety 73 , 283-292 . WORTMAN, J. (1887).ZurKennmissderReizbewegungen. Botanische Z eitung 45, 801-812. ZIEGLER, H. (1962). Chemotropismus der Pilze. In Handbuch der Pjlanzenphysiologie, XVII /2 (ed . W. Ruhland), pp. 398-411 . Berlin : Springer-Verlag.

(R eceiv ed for publication 19 January 1984)