Turgor and fungal growth: Studies on water relations of mycelia of Serpula lacrimans and Phallus impudicus

Turgor and fungal growth: Studies on water relations of mycelia of Serpula lacrimans and Phallus impudicus

[ 527 ] Trans. Br. mycol. Soc. 86 (4), 527-535 (1986 ) Printed in Great Britain TURGOR AND FUNGAL GROWTH: STUDIES ON WATER RELATIONS OF MYCELIA OF S...

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[ 527 ] Trans. Br. mycol. Soc. 86 (4), 527-535 (1986 )

Printed in Great Britain

TURGOR AND FUNGAL GROWTH: STUDIES ON WATER RELATIONS OF MYCELIA OF SERPULA LACRIMANS AND PHALLUS IMPUDICUS By D. EAMUS AND D. H. JENNINGS Botany Department, The University, Liverpool L69 3BX, U.K. The influence of substratum water potential on the growth of Serpula lacrimans and Phallus impudicus was investigated. Two methods of adjusting the water potential of the substratum were used; both gave qualitatively similar results. Linear growth rate decreased with substratum water potential. The turgor potential at the mycelial front and linear growth rate were positively correlated in both species on both media. An experiment concerned with growth from a medium of high water potential to one oflower water potential and vice versa produced results consistent with those obtained for growth on a single medium. The influence of water potential of the substratum on fungal growth has received increasing attention in recent years (Boddy, 1983; Clarke, Jennings & Coggins, 1980; Dube, Dodman & Flentie, 1971; Griffin, 1977; Hocking & Pitt, 1979; Magan & Lacey, 1984; Pitt & Hocking, 1977; Wilson & Griffin, 1979). These studies have shown that although there may be a stimulation of linear growth rate on those media whose water potential has been made slightly more negative than the control, further decrease in the water potential of the medium brings about a reduction of growth, the extent of the reduction in growth depending upon the amount of reduction of the water potential of the substratum. Wood-decaying basidiomycetes appear to be particularly sensitive to decreased water potential of the medium (Boddy, 1983; Clarke et al., 1980; Griffin, 1977), being able to grow in most instances only above - 10 MPa. On the other hand, xerophilic fungi can often grow relatively well at a water potential around -30 MPa (Hocking & Pitt, 1979; Pitt & Hocking, 1977)·

The most appropriate hypothesis for the mechanism by which growth is reduced with reduced water potential places a central role on turgor. It may be proposed that, as in higher plant cells, turgor provides the driving force for growth. A reduction in this driving force will result in reduced growth. Adebayo, Harris & Gardner (1971), studying Aspergillus wentii and Mucor hiemalis, were not able to find any correlation between mycelial turgor and growth rate, and concluded that a low internal solute potential inhibited growth probably via inhibition of enzymes. Similarly, Luard & Griffin (1981) observed no relation between growth and turgor for a range of fungi.

However, in both studies it was assumed that the mycelial water potential was the same as the water potential of the medium. Consequently, only the solute potential of the mycelium was determined directly and turgor potential deduced by difference between the assumed water potential and the measured solute potential. The rationale for these assumptions lay in the belief that in the enclosed system of the Petri dish there is equilibrium between the mycelium, agar and vapour phase with respect to water movement. Hoch & Mitchell (1973) transferred mycelia of Aphanomyces euteiches from a solution of low to higher water potential in an attempt to increase growth by increasing mycelial turgor. They believed that there would be water influx into the hyphae from the surrounding medium, on the basis of water potential gradient estimated on the same assumptions as those above. They did not observe an increased growth, which was contrary to these assumptions. The water potential of the growth medium may be adjusted by the addition of solutes. A variety of solutes has been used in previous studies: glucose, fructose (Pitt & Hocking, 1977), sucrose (Clarke et al., 1980), polyethylene glycol 6000 (PEG 6000) (Sterne & McCarver, 1978), glycerol and NaCl (Hocking & Pitt, 1979), and KCl (Wilson & Griffin, 1979). Ideally one should use a solute which does not enter the cytoplasm. However, as yet, there is no evidence for a compound of suitable molecular weight which neither enters fungal hyphae nor is degraded externally by them. Some compounds present other problems. Thus PEG 6000 may restrict oxygen diffusion through the medium (Luard, 1980). Salts, particularly of monovalent ions, are the least preferable, since they are rapidly absorbed by mycelia to achieve high concentrations

528

Turgor and fungal growth

within the cytoplasm (Wethered, Metcalf & Jennings, 1985), such that enzyme activity per se is influenced and thus growth rate could be affected directly rather than via changes in the water relations of the hyphae. Given that a penetrating solute must be used to adjust the medium solute potential, it is probably best to use one which is a normal substrate for metabolism. There is good evidence that non-metabolized compounds can affect indirectly metabolism and can alter the pattern of growth (Jennings & Austin, 1973; Moore, 1981). In all studies of the effect of water potential upon growth it is necessary to show that the observed effect is a function of the growth medium water potential, and not a function of the solute used to adjust the media. Consequently at least two osmotica must be used for comparison. In this study, sucrose was chosen as an appropriate solute, but in addition the water potential of the growth media was adjusted by increasing the overall concentration of the medium. Throughout the above, the term' water potential' has been used instead of ' water activity', the term most often used in other studies on fungal water relations. Water activity decreases from a maximum of one and is a dimensionless number, while water potential decreases negatively from a maximum of zero with the units MPa . Howe ver, throughout the rest of this paper, water potential is used exclusively, since this can be used to describe both the growth media and the fungus itself for which the water potential can be separated into solute and turgor potentials. This paper reports studies on the relationship between growth and the water potential of the med ium for two members of the Basidiomycotina, Serpula lacrimans (Wulf.) Schrot. and Phallus impudicus L. Both mycelial water and solute potentials were measured, as was the water potential of the growth medium, in all instances using the same method. MATERIALS AND METHODS

Cultures S erpula lacrimans and Phallus impudicus were cultured as described by Eamus & Jennings (1984). A range of substratum water potentials was generated by two methods. First, sucrose was used to adjust to an appropriate value the water potential of 2 % (wIv ) malt extract (Oxoid) 1'5 % (wI v) agar (Oxoid NO.3) medium, that which was used to maintain cultures. Second, the amounts of malt extract and agar in the medium were increased to give media of differing water potentials in which the ratio (by weight) of malt extract : agar was always

2'0 : 1'5. No other substances were added. Ten 9 ern diam Petri dishes were filled to a depth of 5 mm with either the sucrose- or malt/agar-adjusted media. Each plate was inoculated with an 8 mm diam plug from the mycelial front of a colony growing on agar used to maintain cultures. Five plates per species per water potential per adjustment method (sucrose or increasing malt/agar) were used. The procedure of Coggins et al. (1980) was used for experiments involving growth from agar medium of one water potential to agar medium of another, diffusion from one medium to another being prevented by a glass barrier. Unless stated otherwise all results are given as means of five replicates ± standard error. Thermocouple psychrometry The water potential of each medium was determined by thermocouple psychrometry using a Wescor HR-33T microvoltmeter. Sufficient agar was taken from the poured plates to fill the sample chamber, the use of which has been described previously (T hompson et al. , 1984). It should be noted that, while the initial composition of one batch of agar might be nominally the same as another, the water potentials of the two could differ quite significantly because of the difficultie s of standardizing the preparation of a medium in the sterile state. Mycelial water, solute and turgor potentials were determined as previously described (T hompson et al ., 1984). Growth measurement Colony radial growth was measured by measuring colony diameter. Due to deviations from absolute circularity, both the largest and smallest diameters of the colonies were measured, and the mean taken. RESUL TS

From Fig. 1 it can be seen that on sucrose-adjusted media the rate of growth and mycelial turgor potential of S. lacrimans both decreased linearly as a function of decreasing substratum water potential. Similar relationships were observed on malt/agaradju sted media. The decrease in colony radial growth rate per unit decrease of substratum water potential was greater on malt /agar-adjusted media than on sucrose-adjusted med ia (Fig. 2). Figs 3 and 4 show that for S . lacriman s on both sucroseadjusted and malt/agar-adjusted med ia the decline in turgor potential at the mycelial front and in radial growth rate were linearly related for the range of radial growth rates for which measurements were made . The observation that the line of best fit does not pass through the origin will be discussed later. Figures 5 and 6 show that for P. impudicus, as substratum water potential decreased with addition

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Fig. 1. Colony radial growth rate C.A.) and turgor potential at the mycelial front Ce) for Serpula lacrimans growing on sucrose-adjusted media of different water potentials. Results are means of five replicates ± S.E. Equations for the lines are as follows: growth rate, y = o'0395-0'oo42X, correlation coefficient 0 '97, significant at 0 '1 % level ; turgor potentialy = 0 '397 - 0'049 X, correlation coefficient 0 '93 , significant at 0 '1 % level.

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F ig. 2 . As for Fig. 1 but for malt/agar-adjusted media . Results are means of five replicates ± S.E. Equations for the lines are as follows: growth rate, y = 0'0284-0'oo66x, correlation coefficient 0 '925, sig0 '01 % level; turgor potential nificant at y = 0 '358-O'14X, significant at 0 '1 % level.

Fig . 3. Relat ion between colony radial growth rate and turgor potential at the mycelial front for S erpula lacrimans growing on sucrose-adjusted media of different water potentials (data from Fig. 1). Equation for the line, y = - 0 '08 + 12'6x, correlation coefficient 0 '91 , significant at 0 '1 % level.

of sucrose or increased malt /agar concentration, radial growth rate and mycelial water turgor potential both decreased linearly. As with S. lacrimans, the decline in colony radial growth rate per unit decrease of substratum water potential was

more pronounced on malt/agar-adjusted media than on sucrose-adjusted media. Figure 7 shows that on sucrose-adjusted media, the decline in turgor potential at the mycelial front and in colony radial growth rate were linearly

Turgor and fungal growth

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Fig. 5. Colony radial growth rate (A ) and turgor potential at the mycelial front (.) for Phallus impudicus growing on sucrose-adjusted media of different water potentials. Results are means of five replicates j s.s. Equations for the lines are as follows : growth rate, y = 0 '01120'0022X, correlation coefficient 0'96, significant at 0 '1 % level; turgor potential y = 0'415 -0'088x, correlation coefficient 0'92, significant at 0 '1 % level.

Fig. 7. Relation between colony radial growth rate and turgor potential at the mycelial front for Phallusimpudicus growing on sucrose-adjusted media of different water potentials (data from Fig. 5). Equation for the line y = 0'035 +40x, correlation coefficient 0'83, significant at 2% level.

related. The same relationship was observed for data obtained from mycelium growing on malt/ agar-adjusted media (Fig. 8). Again the lines of best fit do not go through the origin. Table 1 gives data for the solute potentials within

the mycelium of both S .lacrimans and P . impudicus in relation to the water potentials of the media. On both media S.lacrimans seemed to change its solute potential more than P. impudicus in response to a reduced external water potential.

D . Eamus and D . H. Jennings

53 1

When growth was from - 2 ' 2 4 to - 0'4 MPa, there appeared to be no effect on the extension rate of the mycelium as it crossed from one agar to the other, On the other hand, when growth was from agar at 0,3 -0'4 MPa to agar at - 2'24 MPa, there was a check on growth for at least 24 h. However, by 96 h, the /cO radial growth rate had returned to that on the agar ' 0.. 0,2 with the higher water potential. Table 2 gives ~ values for the water, solute and turgor potential of ~~ the mycelial front when it was on the first agar and 0, ) then on the second. It can be seen that in all four of cases, a positive turgor potential was generated. As above, when the turgor potential of the mycelium for the two agars from which the colonies expanded o 0,01 0,02 0,030,04 0'050'060'070'080 ,090'10 is compared, there was a higher value for that agar Colony radial growth rate (mm h - I ) with the higher water potential. The same is true \.,VI U UY IdUldl gIvvy ll1 \ Jl IU I J when a comparison is made with respect to the two Fig. 8. As for Fig, 7 but for malt/agar-adjusted media outer agars, (data from Fig. 6), Equation for the liney = -0'0057+ Figure 10 shows similar information for P, 44X, correlation coefficient 0'99, significant at 0'1 % level. impudicus . Hyphal extension was not affected when the mycelium crossed from agar at - 2'24 MPa bars to agar at - 0 ' 4 MPa. When growth was in the other The relationship between turgor potential at the direction, it became severely reduced when the mycelial front and colony radial growth rate was mycelium reached the medium of -2'24 MPa. examined further by a study of mycelial extension Table 2 shows that while a positive turgor is from an agar of one water potential to that with a generated at the mycelial front on the agar at different potential. Figure 9 shows radial growth of -0'4 MPa, almost no turgor was generated when S. lacrimans from sucrose-adjusted agar of water the mycelium was on the agar at - 2 ' 2 4 MPa. In potential of -0'4 MPa on to sucrose-adjusted agar keeping with data presented above, the value for the of water potential of -2'24 MPa and vice versa, turgor potential of mycelium growing on the agar 0-4

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Table 1. Solute potential ( - M Pa) in mycelium of Serpula lacrirnans and of Phallus impudicus grown either on sucrose-adjusted or on malt/agar-adjusted media S. lacrimans

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initially inoculated at -0 '4 MPa was greater than the value for mycelium on the equivalent agar at -2 '24 MPa. DISCUSSION

It is clear from the results that the colony radial growth rate of both S. lacrimans and P. impudicus was reduced when water potential of the medium was reduced, irrespective of whether or not the water potential was lowered by increasing the sucrose concentration of the medium or by increasing the concentration of malt/agar, As indicated above, these observations are in keeping

with what has been observed previously for other fungi. The data for S. lacrimans are very similar to those obtained by Clarke, Jennings & Coggins (1980). From extrapolation of the data, growth of S. lacrimans would be expected to be inhibited completely at an external water potential of -7'4 MPa on sucrose-adjusted media but at -4'4 MPa on malt/agar-adjusted media. For P. impudicus the figures are - 5'1 MPa and - 2 '7 MPa respectively. This matter of the better growth on sucrose requires further investigation. There is no doubt, from the results that over the whole range of medium water potentials used, the turgor potential at the mycelial front was related in a relatively simple manner to colony radial growth rate. High growth rate was associated with a high turgor potential, low growth rate with a low turgor potential. For both fungi on both media, there was a significant linear relation between turgor potential and growth rate, It could be argued that the use of a solute, which is absorbed by the hyphae to change the water potential of the medium will lead to local changes in the external water potential and thus

Table 2. Water ('I' w), solute ('I' s) and turgor potentials ('I' p) at the mycelial front for Serpula lacrimans and Phallus impudicus during growth from a medium of one wat er potential to a medium of another. Results are means of a number of replicates, for which details are given below. See Figs 9 & 10 for other details. P. impudicus S. lacrimans Region of Petri dish.. . Position on graph.. , Time '¥ IV medium ( - MPa) Potential (MPa)

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-2'77 -2 '9 +0'13 3

- 2'79 -2'97 +0'18 6

D. Eamus and D. H. Jennings invalidate this linear relationship. However, the area of mycelium sampled was of a size that made it unlikely that there was much heterogeneity in the water potential of the agar beneath. In any case, the determination of the mycelium turgor potential does not demand knowledge of the water potential of the medium. The requirement of positive turgor for growth was confirmed by the studies on mycelial spread from a medium of one potential to a medium of another. As with the data for growth in Petri dishes containing a single medium, the lower the external water potential, the lower the turgor of the apical region of the mycelium. It was particularly striking to observe that when mycelium of P. impudicus crossed from a medium with a water potential of - 0'4 MPa to one with a water potential of - 2' 24 MPa the rate of growth was reduced to a very low level with turgor very close to zero. It is not clear why mycelium of S . lacrimans could continue to extend from a medium at -0'4 MPa water potential over the medium at -2 '24 MPa, whereas P. impudicus showed little ability for such extension, though the same species grows readily when inoculated directly onto a medium of - 2 '24 MPa water potential. Mycelium of S. lacrimans appeared to be better able than that of P. impudicus to produce a lower solute potential in response to a lower medium water potential. However, whether this observation is of any significance with respect to spread from a medium of high water potential to that of a lower potential remains to be established. While a relatively simple relationship has been observed between the turgor potential at the mycelial front and colony radial growth rate , the interpretation of the observation is not necessarily the simple one that turgor potential controls hyphal extension. In order to fully understand the way in which an environmental factor influences hyphal extension, it is also necessary to know how the factor influences specific growth rate (IX), hyphal branching and the width of the peripheral growth zone (Trinci, 1971). It is clear that a positive turgor is needed for hyphal extension but, provided that can be achieved, it is just as possible that the requirements for hyphal extension control turgor potential. This latter interpretation must be considered particularly apposite for the data obtained using malt/agar-adjusted media in which the concentrations ofall nutrients were increased to provide the appropriate lower water potential. On the other hand, using a significantly different method of changing the water potential of the medium, that is by increasing only the concentration of sucrose, there was a very similar relationship between turgor potential and linear growth rate.

533

For thi s reason, we favour the interpretation that turgor potential controls hyphal extension. As indicated above, for the range of growth rates studied, there was a significant linear relationship between colony radial growth rate and turgor potential at the mycelial front. But, as has been pointed out, the lines of best fit do not go through the origin. Indeed if extrapolated, they give negative values of turgor at zero growth rate . Theoretically, one would anticipate that, even at zero growth rate there ought to be positive turgor generated at the mycelial front as a consequence of the cell wall due to its elastic properties inhibiting hyphaI elongation until a threshold turgor has been achieved. Above this threshold growth occurs, below this the turgor is insufficient to overcome the elasticity of the wall. Since the data do not appear to support directly the theoretical view of the relationship between turgor and growth, another explanation must be sought. One possibility is that the turgor potential measured here underestimates the hydrostatic pressure generated in growing hyphae at the mycelial front. This underestimate might follow if turgor potential at the apices is maintained via connexions with the remainder of the mycelium. Some of those connexions might be channels by which solution is driven by hydrostatic pressure from the older parts of the mycelium to the advancing front. Visible manifestation of such movement of solution through mycelium is the presence of droplets at the apices of hyphae of S. lacrimans , both the volume of the droplets and the extension of the hyphae being under the influence of the water potential of the medium some distance away from the mycelial front (Brownlee & Jennings, 1981 ). Nevertheless, the inability of P. impudicus to grow from a medium of higher water potential onto one oflower water potential suggests that a hydrostatic pressure transmitted over a distance may not necessarily be effective in this case. Another possibility for the divergence of the observed results from theory might be that the values for turgor potential presented here while representing the bulk of the sample used nevertheless underestimate that potential in the apical compartments of the extending hyphae. The third possibility is that the solute potential determined is an over-estimate due to the presence of a significant volume of solution external to the plasma membrane. If there were to be such a volume of solution, the potential of its solutes , given that there is no binding to the wall and this would certainly be so for sucrose, would be higher than that of the osmotically active material within the plasma membrane. On freezing and thawing, the protoplasmic solute potential would be increased

534

Turgor and fungal growth

due to dilution by the solution external to the plasma membrane. With any over-estimate of the solute potential, there will be an under-estimate of the turgor potential. The biological basis to the observed relationship between turgor potential at the mycelial front and growth rate requires further investigation. Nevertheless, the results presented in this paper indicate that Adebayo et aI. (1971) and Luard & Griffin (1981) were wrong to suggest that turgor and hyphal extension might not be related. We would argue that these latter studies are based on the mistaken assumption that mycelial water potential is at equilibrium with that of the medium. The data presented here shows that this assumption is not correct. For it to have any validity the hydraulic conductivity of the plasma membranes must be such as to allow a flow of water of great rapidity to ensure the establishment of an equilibrium between the internal protoplasmic environment and that external to the membrane. The flux of water will of course be a consequence of the accumulation of solutes within the mycelium, the accumulation being ultimately dependent upon energy-dependent fluxes at the plasma membrane (Jennings, 1976). Thus the maintenance of a water potential gradient between the internal and external solution is of major importance in the maintenance of water influx and as such has been postulated to be crucial to the generation of turgor gradients within mycelium of basidiomycetes including S.lacrimans and P. impudicus, by Eamus & Jennings (1984) and Thompson et al. (1984). These studies demonstrated the existence of turgor gradients between that part of the mycelium which is extending and the more distal older portions. Such gradients have been implicated in longdistance translocation of solutes through mycelium (Jennings, 1984). Further studies of water movement at the food source into translocating mycelium (Thompson et aI., 1984) have shown that under certain conditions the water movement can be reversed without growth being affected. Under these conditions, a positive turgor at the mycelial front was still demonstrated. Finally, it is appropriate to make a briefcomment on the extent to which turgor is regulated in the two fungi. Gutknecht, Hastings & Bisson (1978) in their consideration of turgor pressure regulation in giant algal cells give a hypothetical example of turgor regulation. Perfect regulation would be manifested by a turgor potential which does not change with change of external water potential. Conversely, the more the turgor potential changes with change of external water potential, the less the degree of regulation of turgor. Thus it can be seen from the slope of plots of turgor potential against medium

water potential that P. impudicus was less able to regulate its turgor than S. lacrimans on both sucrose and malt/agar media. In addition, both species were better able to regulate their turgor on sucrose media than on malt/agar media. The significance of these observations is not clear. We believe that the simplest hypothesis to explain the data presented here is that hyphal turgor and colony radial growth rate are directly related and that the cause of the decreased growth rate observed on media of reduced water potential is due to the reduction in the driving force for growth at the hyphal apices. We hope that these observations will help to re-awaken an interest in the role of turgor in the development of the hyphal apex first highlighted so strikingly in the seminal observations of Park & Robinson (1966) and Robertson & Rizvi (1968). We wish to thank NERC for the support of this investigation. REFERENCES

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(Received for publication 28 January 1985)