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Fungal turgor pressure is directly involved in the hyphal growth rate
Microbiol. Res. (1999) 154, 81- 87 http://www.urbanfischer.de/journals/microbio1res ©
Urban & Fischer Verlag
Fungal turgor pressure is directly involved in the hyphal growth rate Patrick Gervais, Christophe Abadie, Paul Molin Laboratoire de Genie des Procedes A1imentaires et Biotechno10giques, ENSBANA - 1, Esplanade Erasme, 21000 Dijon, France Accepted: February 1, 1999
Abstract From a rigorous momentum balance it was shown that fungal turgor pressure can be expressed as a sum of two components: a) a static contribution, related to the membrane elastic properties and resolved by capillary probe measurements; b) a dynamic part (M), responsible for the hyphal extension rate and proportional to it. Both contributions add up and explain why some researchers have found hyphae growing at finite rates when the capillary probe measures no pressure difference at all. This approach was applied to Aspergillus oryzae and we have found an increasing relation between the dynamic part of turgor pressure eM) and hyphal extension rate.
Key words: hyphal extension - water potential - image analysis - Aspergillus oryzae
Introduction Growth of microorganisms is very sensitive to environmental parameters such as temperature (Suutari et at. 1990), water potential or water activity (Scott 1957). In the cell, there is no active water transport and water moves according to the water potential gradient between the intra- and extracellular medium. In heterogeneous media, water potential or water activity is related to the solute concentration, capillary forces and absorption properties of the insoluble solid substrate. Numerous workers have demonstrated the sensitivity of hyphal extension (Robertson 1958, Robertson and
Corresponding author: P. Gervais (email: [email protected]) 0944-5013/99/154/01-081
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Rizvi 1968; Wilson and Griffin 1979) fungal spores germination and aroma production (Gervais et al. 1988a; 1988b) to water stress. A variety of experiments have shown that a turgor pressure exists in hyphal tips. This work is intended to provide a theoretical approach in order to define turgor pressure and to validate a method of determination of pressure balance in growing fungi.
Materials and methods Microorganism and culture media. Aspergillus oryzae CBS 81772 var. bruneus was furnished by the Centraal Bureau voor Schimme1cultures (The Netherlands). The fungus was cultivated on potato-dextrose agar (PDA, Institut Pasteur Production, Paris) containing 20 gil glucose, 15 gil agar and 4 gil potato extract. Xylitol, a compatible solute, was added to the PDA medium to obtain different water activity values. Media were sterilized at 121 DC for 20 min. Water activity values indicating the water potential were checked before and after cultivation, using a dew point osmometer Aqualab CX2 (Decagon Devices Inc., USA). Four different water potential (\jf) levels of the medium were tested in order to measure the evolution of the turgor pressure as a function of the external water potential (-0.3 MPa; -2.8 MPa; -4.2 MPa; -7.0 MPa). Petri dishes were partially filled with 4.5 ml of PDA along a band and inoculated with 24-hours-old mycelia. Dishes were stored at 27 DC in boxes containing 200 ml of water-xylitol solution in order to control the relative humidity of the atmosphere. The water potential of the water-xylitol solution was the same as that of the dishes, so the surrounding Microbiol. Res. 154 (1999) 1
81
environment was in equilibrium with the solid media. Measurements were then made after 24 hours of cultivation on the band. Microscopic measurement. The hyphal growth rate of Aspergillus oryzae was measured after 24 h of cultivation when the radial extension rate had reached a constant value for a given aw ' The hyphal growth rate (Vh) was measured at the periphery of the colony by analyzing images of individual hyphae; three hyphae, randomly chosen, were tracked for 15 min taking images every three minutes (Molin et al. 1992). At the colony front, the observed hyphae were always in contact with the medium (Nielsen 1992; Oh et al. 1993). A CCD camera (Cohu, model 6710, Japan) was set on an inverted light microscope and this sent a numerical signal to the image analyser (serie 151, Imaging Technology Inc., USA). The image produced consisted of pixels which represented different gray intensity levels varying from 0 to 255. Experimental procedure allowing the iJP determination. To approach the iJP value of the fungus, different plates were subjected to slightly decreasing stepwise changes of water potential (i.e. increasing stepwise changes of osmotic pressure levels) until a change stopped hyphal growth, but without any volume decrease. Different levels of changes for water potential were then used in order to estimate iJP for different water potential levels (see Table 1). Practical realization of stepwise changes was made by the instantaneous injection ofaxylitol water solution at the defined water potential in the plate in order to totally and instantaneously submerge the fungus culture. Microscopical observation of the hyphal growth rate was going on during and after this immersion. It has been verified that immersion of filamentous fungi (with a solution at the same water potential as the cultivation medium for periods of up to 45 minutes) does not modify the extension rate.
Results and discussion
gation, division and hence the increase in biomass:
(0) with: PI the turgor pressure, IIi the intracellular osmotic pressure, IIe the external osmotic pressure. The turgor pressure value is balanced by the cell wall resistance of the organisms, the higher the wall resistance the higher is the turgor pressure (Trinci 1978). This can be explained as follows: since the cell wall must be in equilibrium, it has to be under tension so that each surface element can balance the intra and extracellular osmotic pressures. This translates mechanically into a compensating pressure, given by the ratio of a surface tension over a curvature radius, that is exactly as in the case of a soap bubble. It follows that for a given organism:
Pt = Pr
(1)
where Pr will be a constant value unless wall geometry or properties are modified. The growth of individual cells is discontinuous and can be separated into two phases: a slow evolution phase and a rapid division phase corresponding to mitosis or budding. The turgor pressure was found to be constant for a cell at a specific physiological stage and the budding phenomenon in yeasts was found to be related to a specific and localized hydrolysis of the cell wall which induces an important decrease in wall resistance and leads to a volumetric expansion of the cell wall (a passive flux of water into the cell). Turgor pressure can be measured in three ways (Money 1990). First, osmometry can be used to determine the difference in osmotic potential between cytoplasm and growth medium, i.e. turgor pressure. Internal osmotic potential is difficult to determine accurately since the cytoplasm must be extracted by freezing and thawing (Luard and Griffin 1981; Luard 1982; Woods and Duniway 1986). Second, by exposing the cell to varying solute concentrations, the onset of plasmolysis can be determined. Third, pressure probe micropipettes can be inserted into a cell and the pressure that must be applied to prevent a low-viscosity oil droplet from being forced back up the pipette can be measured (Money 1990; Money and Harold 1992).
1) Theoretical approach
Non equilibrium states: fungal cell growth Equilibrium states and cell wall tension
Cell turgor pressure is defined as the hydrostatic pressure due to the difference between the intracellular and extracellular osmotic pressures at a steady state characterized by a steady cell volume. Turgor pressure exists in all types of cells: microorganism, plant and animal cells. In all theses cases, cell turgor pressure corresponds to an excess pressure which supports cell shape, elon82
Microbiol. Res. 154 (1999) 1
For fungal species, it is largely admitted that the growth occurs continuously at the apex of numerous hyphae of a colony and at constant growth rate under defined conditions of the medium (i.e. pH, temperature and water activity) (Scott 1957; Gervais et al. 1988c), and the maintenance of the turgor pressure could not be sufficient to explain the growth phenomena (Zhu and Boyer 1991; Kaminsky et al. 1992; Money and Harold
1993). Nevertheless, Trinci (1974) has observed oscillations of the mycelium growth rate just after spore germination. During hyphal elongation, synthesis of cell materials such as cytoplasmic organelles or membrane components must continuously occur in order to keep the cell wall constant in mechanical resistance in spite of the continuous deformation induced by the turgor pressure. The most numerous components in the apical zone are the apical vesicles (Howard 1981) that originate in the Spitzenkorper (or Vesicle Supply Centre): a central and dense component of the apex consisting of accumulated vesicles and presumably anchored to the apex walls (Howard 1981). It appears there are two classes of size classes of vesicles: microvesicles with a diameter of about 40-70 nm and macrovesicles with a diameter of more than 100 nm (Gow 1989). The macrovesicles are more numerous and probably contribute most to the new membrane for tip expansion. The microvesicles of hyphae or chitosomes have been described in fungi representative for all taxonomic groups that contain fungi with chitin in their walls (Gow 1989). The chitosomes were described to be zymogenic multienzyme structures which deliver chitin synthase units to specific sites in the cell wall membranes. Bartnicki-Garcia et al. (1979) gave a detailed description of the morphology and structure of chitin microfibrils and of the particles associated with their formation in Mucor rouxii. Hence, cell growth is the result of a dynamic balance between the maintenance of an intracellular turgor pressure via solute synthesis and water input, and a synthesis of macromolecules which causes the membrane to provide a constant resistance despite the elongation phenomenon (Wessels 1993). We give further details below. In the case of continuously growing cells such as filamentous fungal tips, the steady-state conditions mentioned in the preceding section (see eq. (1») can no longer be applied. So the evaluation of intracellular turgor pressure must take into account the type of system under consideration: there will be differences between non-extending cells ( such as bacteria, yeasts, or plant cells) for which both cell volume V and intracellular osmotic pressure Jr; could be considered to be constant, and extending non-equilibrium cells (such as fungi). In order to study more precisely the case of fungi, let us consider the example of fungal hyphae growth during a time interval dt. During this interval dt, dns moles of a solute j are synthesized or incorporated in the internal medium which induces a dII; increase in osmotic pressure, as for diluted media II; could be considered to be equal to: n
II; = RTICj j=i
(2)
with R =ideal gas constant; Cj =molar concentration of intracellular solute j; and T =temperature. Such an increase in osmotic pressure, in addition to a continuous apex wall elongation (through vesicle incorporation), will simultaneously provoke a thermodynamic flow of water into the cell which will decrease the Cj concentration and will thus simultaneously reduce the osmotic pressure level of -dII; as shown in the previous sentence. All these previous phenomena occur simultaneously, which leads to a continuous hyphal extension. Cell growth could then be attributed to a constant increase in the osmolytes concentration near the apical part which is continuously balanced by a water flow input. So in the case of filamentous fungi, growth, regarded as volume variation (essentially due to water input), should occur under a constant intracellular osmotic pressure (II). The increase in cell volume is in tum limited by the cell wall's mechanical properties, which were previously discussed in this section. Assuming that the tension developed by the cell wall is constant in the apical part during hyphal growth (i.e. the continuously synthesized wall exhibits steady mechanical properties), it is possible to calculate the cell pressure balance for extending fungi. In this case the sum of the exerted pressures is no longer equal to zero (as previously shown for noncontinuously extending cells in equation (1» but equal to a pressure loss value (LiP) which is the origin of the continuous apical extension, (that is, water inflow to the cell). At a time t it could be written:
(II;-IIe}-Pr=LiP
or
Pt-Pr=,jP
(3)
This very simple equation allows to describe fungal growth; thus turgor pressure in extending cells is no longer counterbalanced only by the membrane pressure resistance (Pr) but also by the apical deformation (due to LiP). Previous works (Lockhart 1965; Money and Harold 1993) have not considered the parameter LiP in the turgor pressure evaluation or measurement in continuously extending cells. Now it is possible to relate the volume variation dV during the time dt to this constant pressure loss LiP over time (as II;, IIe and Pr are constant) and to physical characteristics of the fungal apex through the application of an equation (Kedem and Katchalsky 1958) describing laminar water flow driven by pressure gradients between intra- and extracellular media.
(4) with: A
= exchange surface where the water flow takes
place (m 2), dV
dt
= water flow between intra and extra-
cellular media (m3 .
S-I),
Lp = hydraulic permeability of
Microbiol. Res. 154 (1999) 1
83
the apex wall (m . S-I . Pa- I ), M =(lli - lle) - P r (Pa). Integration of previous equation (4) yields: (5)
In obtaining (5) it is assumed that the surface A is constant versus time. Indeed the parameter A is related to the surface of the apex where the water flow takes place, i.e. where the pressure gradient occurs. This apex wall surface corresponds to the cell's active volume (Va) which can be defined as the hyphal apex volume which is osmotically balanced or equilibrated by both, continuous incorporation or synthesis of solutes and water input. Indeed it was shown by 14C glucose labeling (Trinci 1971 ; Fevre 1979; Fevre and Rougier 1982), that glucose incorporation was about ten times higher at the apex than in the subapical zone of Saprolegnia monoi'ca. Such a local increase in solute incorporation (mainly sugars and ions) would immediately trigger a fast water input to the fungi, this is known to be many times faster than solute diffusion towards the rear of the apex (Mauro 1957). This confirms the view that the rate of hyphal growth will be only controlled by the water input in the apical part, as estimated in equation (5). As the apex is extending during the time dt, the active volume Va is increased by dV, but is also decreased by dV due to the advance of the apex which is supposed to be the only place of osmotic perturbation (i.e. solute incorporation or synthesis) as proposed in Fig. 1. The surface A could then be considered constant during fungal extension. Two hypotheses could be proposed to explain this behaviour, i) cell walls corresponding to the
Va timet
lime I + dl
or
dV
7
/"
\\
(6)
These equations imply that the hyphal growth rate dx
dt is constant for defined medium conditions (A, Lp ' M, s
are constant), a well known fact since 1957 (Scott). The expression of the turgor pressure could be easily obtained from the previous equation (6). Substituting M by its value (Pt - Pr ) given by equation (3) in equation (6) gives:
~ = Pr + K( dX) dt
with
K= ~ ALp
(7)
Comparison of this equation with equation (1) allows us to conclude that in the case of growing tips the turgor pressure, considered to be a potential pressure, is greater than the equilibrium value Pt = Pr (case of nonextending cells). Equation (7) trivially reduces to (1) when cell extension stops. This difference between non extending cells and growing tips is easily understandable, because in the
Major consequences
)
1/
Va
Fig. 1. Representation of the apex volume variation during the time dt (the cell active volume Va remains constant). 84
or
the turgor pressure continuously drives the volume expansion and thus cannot be measured just through the membrane pressure resistance P,. We discuss experimental details below.
------rl~
~~)
dx = ALp t1.P dt S
case of continuous hyphal elongation, a part [ K : ] of
/
,
dV volume in Fig. 1 have acquired a weaker permeability to external solutes, and ii) the cytoplasmic volume corresponding to dV has lost its ability to synthesize compatible solutes (glycerol, mannitol, etc.). With these assumptions, the fungal growth expansion is represented by equation (5). As the hyphal growth rate is unidirectional (x axis), the volume variation dV during the time dt could be written dV =dx . S where dx is the axial hyphal extension and S is the section of the hyphal tube. Consequently, equation (4) becomes:
Microbiol. Res. 154 (1999) 1
Recent works using the capillary probe technique have measured such a low turgor pressure level in growing tips that the question of how growth is possible without turgor pressure has been put forward (Money and Harold 1993). This controversy is clarified by our theory. In fact, such a probe incorporated as the hyphae are still growing will measure a hydrostatic pressure gradient between intra and extracellular medium, but without taking into account the pressure which continuously drives the volume expansion i.e. the M value in equation (3).
Hyphal extension rate
Whithin the framework developed here, such measurements will only give the value P't = II'i - IIe = P, and not the total value which is IIi - IIe = Pr + !1P, i.e. Pt =P r +!1P· A main analytical conclusion of this theoretical development is that internal fungal osmotic pressure as well as fungal turgor pressure were underestimated when the capillary probe technique was used or when any other static measurement is performed.
Table I gives the different hyphal extension rates for different water potential values of the culture medium. These results confirm previous findings (Scott 1957) and show that hyphal extension rate is constant for a given water activity in the culture medium. Nevertheless there are significant variations as the water potential of the medium is modified. The hyphal extension rate corresponding to the optimal water potential value (about -4 MPa) was about twice the values found for higher water potential values (-OJ MPa) or for lower water potential values (-7 MPa).
2) Experimental approach Previous theoretical considerations allowed us to understand the cell turgor pressure, particularly in mobile microorganisms. For such cells, an important part of the turgor pressure was continuously converted into volume expansion and thus cannot be measured by an intracellular pressure measurement. Equation (7) also evidences a linear relation between the turgor pressure and the hyphal extension rate. Nevertheless, such a linear relationship will only be valid for standard culture conditions (temperature, pH, osmotic pressure) which correspond to constant membrane characteristics. In order to relate experimental methods to the previous considerations about the underestimation of turgor pressure in growing tips, we propose a method intended to evaluate only that part of turgor pressure in growing filamentous fungi continuously used for volume expansion. This method is based on the application of different osmotic stepwise increases to the external culture medium in order to evaluate the intensity of the specific osmotic shift which would stop the hyphal extension rate without any plasmolysis, i.e. without decrease of the hyphal volume. The osmotic shift must be as fast as possible in order to prevent active osmoregulation mechanism from the fungi. The perturbation corresponds to a sudden increase of the IIe value from IIer dx
(growing fungus at a constant hyphal growth rate,' dt)
to IIe?_ (stopped hyphae i.e.
dx = 0). It follows from
dt
L1P measurements Previous considerations have allowed to propose a method for L1P evaluation ( !1P = K ~~ with K A~p ). As an example of LJp determination, Fig. 2 shows the hyphal extension rate for an initial water potential value of -2.8 MPa and for three different stepwise decreases in water potential: - 3.1 MPa, - 3.5 MPa, and - 3.9 MPa, respectively. From the plots, the minimum level of water potential required in order to stop growth without modification of the hyphal volume would be between the decrease values -3.5 MPa and -3.9 MPa. So LJp will be (+3.5 -2.8) < Pt < (+3.9 -2.8) i.e. 0.7 < Pt < 1.1 (in MPa). The procedure has been repeated for three other initial water potentials and the results are presented in Table 1. Analysis of the fungal !1P values proposed in Table 1 shows that Aspergillus oryzae exhibits relatively high 3.0
c
'E
2.5
(jj a. E 2; 2.0
2
~
equation (7) that:
c 1.5 0
'iii
(8)
Hence the difference between the two external levels in osmotic pressure gives the part of the turgor pressure which is continuously used for volume expansion i.e. the !1P value of equation (3). The total turgor pressure value could then be experimentally determined by the sum of the experimental !1P value and of the Pr value given by the capillary pro~e techniques or by a direct measurement of the osmotIc step increase leading to the incipient plasmolysis (Money 1990).
c
Q)
xQ) '(ii
1.0
,.,a.
J::
I
0.5
0.0 0
5
10
15
Time after an osmotic shock (min)
Fig. 2. Evolution of the hyphal extension rate for different stepwise decreases in water potential: . : - 3.1 MPa, 0 : -3.5 MPa, + : -3.9 MPa, (initial water potential value was -2.8 MPa). Microbiol. Res. 154 (1999) 1
85
Table 1. Evaluation of LJP values for Aspergillus orizae cultivated at different levels of water potential. Water potential of the culture medium (MPa)
-0.3 1.60 (0.34)*
Hyphal extension rate (f.unlmin) Water potential of the solutions used to stop growth (MPa)
LJp values (MPa) K =LJP/Vh
*
1.94 (0.30)
-4.2 3.08 (0.60)
-0.5
-3.1
-4.9
-1.0
-3.5 -3.9
-5.1 -5.6
0.2
0.7 < Pt < 1.1 0.36 - 0.57
-7.0 1.43 (0.41) -7.3
0.9
values into brackets represent confidence intervals at 95%.
3.5.,----------------------,
0.0 + - - - - + - - - t - - - - + - - - + - - - i - - - - - t - - - - - j - - ' 0.8 1.0 1.2 1.4 0.6 0.2 0.4 0.0
AP (Pa)
Fig. 3. Representation of the hyphal extension rate as a function of the pressure LJP.
AP values, which depend on the water potential value of the media. Such high values for AP are in contradiction with previous theoretical considerations which have concluded that a minuscule difference in water potential suffices to account for water inflow during hyphal growth and that this does not significantly influence turgor pressures as measured by a pressure probe (Money 1994). Similar values have been previously reported (Griffin 1981; Luard and Griffin 1981). Maximal AP values (about 1.3 MPa) were found for water potential values of the external medium of -4 MPa (the one having the highest Vh ). For higher as well as for lower values of external water potential (-0.3 or -7 MPa) the AP was found to decrease drastically to 0.5 MPa and 0.1 MPa, respectively (like behaviour of Vh ). The measured AP part of the turgor pressure is plotted in Fig. 3 against hyphal extension rate. It can be seen that the higher the AP, the higher the apical rate. The variability of the jjp measurements has not allowed to clearly show a linear relation between the two parame86
-2.8
Microbiol. Res. 154 (1999)
1
ters as expected from eq. (6). Nevertheless the relationship between the two parameters is clearly an increasing function. Further experiments will allow us to explore this relation. The theory developed here allows a better understanding of turgor pressure in fungi and mobile microorganisms. For such organisms a main part of the turgor pressure (AP) was shown to be continuously used for cell volume expansion. On the basis of this theoretical approach, a method allowing the evaluation of the jjp of mobile microorganisms has been proposed. This method does not give a precise value but defines a range for the AP values. Nevertheless, the use of osmotic stepwise changes of weaker intensity would certainly improve the resolution of the method. In order to measure the whole turgor pressure of mobile microorganisms, it would be necessary to combine the capillary probe technique in order to evaluate Pr' and the osmotic step technique in order to evaluate .1P. The turgor pressure is the sum of both values. Another method to evaluate the whole turgor pressure is to use the osmotic step technique until the cell shows incipient plasmolysis as proposed previously (Money 1990).
Acknowledgments This work was supported by funds from the Burgundy district. We are indebted to Pr. Hector Jorquera from the Catholic University of Santiago (Chile) for helpful advices and discussions.
References Bartnicki-Garcia, S., Ruiz-Herrera, 1., Bracker, C. E. (1979): Chitosomes and chitin synthesis. In: 1. H. Burnett, A. P. 1. Trinci (eds.): Fungal walls and hyphal growth. p. 149. Cambridge University Press. Cambridge 1979.
Fevre, M. (1979): Digitonin solubilization and protease stimulation of ~-glucan synthetases of Saprolegnia . Z. Pflanzenphysiol. 95, 129-140. Fevre, M., Rougier, M. (1982): Autoradiographic study of hyphal cell wall synthesis of Saprolegnia. Arch. Microbiol. 131,212-215. Gervais, P. , Fasquel, J. P., Molin, P. (1988a): Water relation of fungal spore germination. Appl. Microbiol. Biotechnol. 323, 72-75. Gervais, P., Belin, J. M., Grajek, w., Sarrette, M. (1988b): Influence of water activity on the aroma production by Trichoderma viride growing on solid substrate. J. Ferment. Technol. 66,403-407. Gervais, P., Grajek, w., Bensoussan, M., Molin, P. (l988 c): Influence of the water activity of a solid substrate on the growth rate and sporogenesis of filamentous fungi. Biotechnol. Bioeng. 31,457-463. Griffin, D. M. (1981): Water and microbial stress. Adv. Microb. Ecol. 5, 91-136. Gow, N. A. R. (1989) : Control of extension of the hyphal apex. In: Current topics in medical mycology. Vol. 3, 109-152. Howard, R. J. (1981): Ultrastructural analysis of hyphal tip cell growth in fungi Spitzenkorper, cytoskeleton and endomembranes after freeze-substitution. J. Cell Sci. 48, 89-103. Kaminsky, S. G. w., Garril, J. A. , Heath, B. I. (1992): The relation between turgor and tip growth in Saprolegniaferax: turgor is necessary but not sufficient to explain apical extension rates. Exp. Mycol. 16,64-75. Kedem, 0., Katchalsky, A. (1958): Thermodynamic analysis of the permeability of biological membranes to non-electrolytes. Biochim. Biophys. Acta 27, 229-246. Lockhart, J. A. (1965): An analysis of irreversible plant cell elongation. J. Theoret. BioI. 8, 264-275 . Luard, E., Griffin, D. M. (1981): Effect of water potential on fungal growth and turgor. Trans. Br. Mycol. Soc. 76, 33-40. Luard, E. (1982): Accumulation of intracellular solutes by two filamentous fungi in response to growth at low steady state osmotic potential. 1. Gen. Microbiol. 128, 2563-2574. Mauro, A. (1957): Nature of solvent transfer in osmosis. Science 26, 252-253. Molin, P. , Gervais, P., Lemiere, J. P. , Davet, T. (1992): Direction of hyphal growth : a comprehensive parameter of the development of filamentous fungi . Res. Microbiol. 143, 777-784. Money, N. P., Harold, F M. (1992): Extension growth of the
water mold Achyla: interpl ay of turgor and wall strength. Proc. Natl. Acad. Sci . USA 89, 4245-4249. Money, N. P., Harold, F M. (1993): Two water molds can grow without measurable turgor pressure. Planta 190,426-430. Money, N. P. (1990): Measurement of hyphal turgor. Exp. Mycol. 14,416-425. Money, N. P. (1994): Osmotic adjustment and the role of turgor in mycelial fungi, In: The Mycota I: Growth, Differentiation and Sexuality, Wessels and Meinhard (Eds.), Springer-Verlag Berlin Heidelberg. Nielsen, J. (1992): A simple morphologically structured model describing the growth of filamentous microorganisms. Biotechnol. Bioeng. 41, 715-727. Oh, K., Matsuoka, H., Nemoto, Y, Sumita, 0 ., Takatori, K. , Kurata, H. (1993) : Determination of anti-aspergillus activity of antifungal agents based on the dynamic growth rate of a single hypha. App\. Microbiol. Biotechnol. 39, 363-367. Robertson, N. F (1958): Observation of the effect of water on the hyphal apices of Fusarium oxysporum. Ann. Bot. London. 22, 159-173. Robertson, N. F , Rizvi, S. R. H. (1968): Some observations on the water relations of the hyphae of Neurospora crassa. Ann. Bot.-London. 32, 279-291. Scott, W. J. (1957): Water relation of food spoilage microorganisms. Adv. Food Res. 7, 83-127. Suutari, M. , Liukkonen, K., Laakso, S. (1990): Temperature adaptation in yeasts: the role of fatty acids. J. Gen. MicrobioI. 136, 1469-1474. Trinci, A. P. J. (1971): Influence of the width of the peripheral growth zone on the radial growth rate of fungal colonies on solid media. J. Gen. Microbiol. 67,320-344. Trinci, A. P. J. (1974): A study of the kinetics of hyphaI extension and branch initiation of fungal mycelia. J. Gen. MicrobioI. 81,225-236. Trinci, A. P. J. (1978): Wall and hyphal growth. Sci. Prog.-Oxf. 65,75-99. Wessels, J. G. H. (1993): Wall growth, protein excretion and morphogenesis in fungi. New Phytol. 123,397-413. Wilson, 1. M., Griffin, D. M. (1979): The effect of water potential on the growth of some soil basidiomycetes. Soil BioI. Biochem. 11,211-212. Woods, Duniway, 1. M. (1986): Some effects of water potential on turgor and respiration of Phytophthora cryptogea and Fusarium moniliformis. Phytopathology 76, 1248-1254. Zhu, G. L. , Boyer, J. S. (1991 ) : Turgor and elongation growth in Chara cells. Plant Physiol. 96, 38.
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Urban & Fischer Verlag
Fungal turgor pressure is directly involved in the hyphal growth rate Patrick Gervais, Christophe Abadie, Paul Molin Laboratoire de Genie des Procedes A1imentaires et Biotechno10giques, ENSBANA - 1, Esplanade Erasme, 21000 Dijon, France Accepted: February 1, 1999
Abstract From a rigorous momentum balance it was shown that fungal turgor pressure can be expressed as a sum of two components: a) a static contribution, related to the membrane elastic properties and resolved by capillary probe measurements; b) a dynamic part (M), responsible for the hyphal extension rate and proportional to it. Both contributions add up and explain why some researchers have found hyphae growing at finite rates when the capillary probe measures no pressure difference at all. This approach was applied to Aspergillus oryzae and we have found an increasing relation between the dynamic part of turgor pressure eM) and hyphal extension rate.
Key words: hyphal extension - water potential - image analysis - Aspergillus oryzae
Introduction Growth of microorganisms is very sensitive to environmental parameters such as temperature (Suutari et at. 1990), water potential or water activity (Scott 1957). In the cell, there is no active water transport and water moves according to the water potential gradient between the intra- and extracellular medium. In heterogeneous media, water potential or water activity is related to the solute concentration, capillary forces and absorption properties of the insoluble solid substrate. Numerous workers have demonstrated the sensitivity of hyphal extension (Robertson 1958, Robertson and
Corresponding author: P. Gervais (email: [email protected]) 0944-5013/99/154/01-081
$12.00/0
Rizvi 1968; Wilson and Griffin 1979) fungal spores germination and aroma production (Gervais et al. 1988a; 1988b) to water stress. A variety of experiments have shown that a turgor pressure exists in hyphal tips. This work is intended to provide a theoretical approach in order to define turgor pressure and to validate a method of determination of pressure balance in growing fungi.
Materials and methods Microorganism and culture media. Aspergillus oryzae CBS 81772 var. bruneus was furnished by the Centraal Bureau voor Schimme1cultures (The Netherlands). The fungus was cultivated on potato-dextrose agar (PDA, Institut Pasteur Production, Paris) containing 20 gil glucose, 15 gil agar and 4 gil potato extract. Xylitol, a compatible solute, was added to the PDA medium to obtain different water activity values. Media were sterilized at 121 DC for 20 min. Water activity values indicating the water potential were checked before and after cultivation, using a dew point osmometer Aqualab CX2 (Decagon Devices Inc., USA). Four different water potential (\jf) levels of the medium were tested in order to measure the evolution of the turgor pressure as a function of the external water potential (-0.3 MPa; -2.8 MPa; -4.2 MPa; -7.0 MPa). Petri dishes were partially filled with 4.5 ml of PDA along a band and inoculated with 24-hours-old mycelia. Dishes were stored at 27 DC in boxes containing 200 ml of water-xylitol solution in order to control the relative humidity of the atmosphere. The water potential of the water-xylitol solution was the same as that of the dishes, so the surrounding Microbiol. Res. 154 (1999) 1
81
environment was in equilibrium with the solid media. Measurements were then made after 24 hours of cultivation on the band. Microscopic measurement. The hyphal growth rate of Aspergillus oryzae was measured after 24 h of cultivation when the radial extension rate had reached a constant value for a given aw ' The hyphal growth rate (Vh) was measured at the periphery of the colony by analyzing images of individual hyphae; three hyphae, randomly chosen, were tracked for 15 min taking images every three minutes (Molin et al. 1992). At the colony front, the observed hyphae were always in contact with the medium (Nielsen 1992; Oh et al. 1993). A CCD camera (Cohu, model 6710, Japan) was set on an inverted light microscope and this sent a numerical signal to the image analyser (serie 151, Imaging Technology Inc., USA). The image produced consisted of pixels which represented different gray intensity levels varying from 0 to 255. Experimental procedure allowing the iJP determination. To approach the iJP value of the fungus, different plates were subjected to slightly decreasing stepwise changes of water potential (i.e. increasing stepwise changes of osmotic pressure levels) until a change stopped hyphal growth, but without any volume decrease. Different levels of changes for water potential were then used in order to estimate iJP for different water potential levels (see Table 1). Practical realization of stepwise changes was made by the instantaneous injection ofaxylitol water solution at the defined water potential in the plate in order to totally and instantaneously submerge the fungus culture. Microscopical observation of the hyphal growth rate was going on during and after this immersion. It has been verified that immersion of filamentous fungi (with a solution at the same water potential as the cultivation medium for periods of up to 45 minutes) does not modify the extension rate.
Results and discussion
gation, division and hence the increase in biomass:
(0) with: PI the turgor pressure, IIi the intracellular osmotic pressure, IIe the external osmotic pressure. The turgor pressure value is balanced by the cell wall resistance of the organisms, the higher the wall resistance the higher is the turgor pressure (Trinci 1978). This can be explained as follows: since the cell wall must be in equilibrium, it has to be under tension so that each surface element can balance the intra and extracellular osmotic pressures. This translates mechanically into a compensating pressure, given by the ratio of a surface tension over a curvature radius, that is exactly as in the case of a soap bubble. It follows that for a given organism:
Pt = Pr
(1)
where Pr will be a constant value unless wall geometry or properties are modified. The growth of individual cells is discontinuous and can be separated into two phases: a slow evolution phase and a rapid division phase corresponding to mitosis or budding. The turgor pressure was found to be constant for a cell at a specific physiological stage and the budding phenomenon in yeasts was found to be related to a specific and localized hydrolysis of the cell wall which induces an important decrease in wall resistance and leads to a volumetric expansion of the cell wall (a passive flux of water into the cell). Turgor pressure can be measured in three ways (Money 1990). First, osmometry can be used to determine the difference in osmotic potential between cytoplasm and growth medium, i.e. turgor pressure. Internal osmotic potential is difficult to determine accurately since the cytoplasm must be extracted by freezing and thawing (Luard and Griffin 1981; Luard 1982; Woods and Duniway 1986). Second, by exposing the cell to varying solute concentrations, the onset of plasmolysis can be determined. Third, pressure probe micropipettes can be inserted into a cell and the pressure that must be applied to prevent a low-viscosity oil droplet from being forced back up the pipette can be measured (Money 1990; Money and Harold 1992).
1) Theoretical approach
Non equilibrium states: fungal cell growth Equilibrium states and cell wall tension
Cell turgor pressure is defined as the hydrostatic pressure due to the difference between the intracellular and extracellular osmotic pressures at a steady state characterized by a steady cell volume. Turgor pressure exists in all types of cells: microorganism, plant and animal cells. In all theses cases, cell turgor pressure corresponds to an excess pressure which supports cell shape, elon82
Microbiol. Res. 154 (1999) 1
For fungal species, it is largely admitted that the growth occurs continuously at the apex of numerous hyphae of a colony and at constant growth rate under defined conditions of the medium (i.e. pH, temperature and water activity) (Scott 1957; Gervais et al. 1988c), and the maintenance of the turgor pressure could not be sufficient to explain the growth phenomena (Zhu and Boyer 1991; Kaminsky et al. 1992; Money and Harold
1993). Nevertheless, Trinci (1974) has observed oscillations of the mycelium growth rate just after spore germination. During hyphal elongation, synthesis of cell materials such as cytoplasmic organelles or membrane components must continuously occur in order to keep the cell wall constant in mechanical resistance in spite of the continuous deformation induced by the turgor pressure. The most numerous components in the apical zone are the apical vesicles (Howard 1981) that originate in the Spitzenkorper (or Vesicle Supply Centre): a central and dense component of the apex consisting of accumulated vesicles and presumably anchored to the apex walls (Howard 1981). It appears there are two classes of size classes of vesicles: microvesicles with a diameter of about 40-70 nm and macrovesicles with a diameter of more than 100 nm (Gow 1989). The macrovesicles are more numerous and probably contribute most to the new membrane for tip expansion. The microvesicles of hyphae or chitosomes have been described in fungi representative for all taxonomic groups that contain fungi with chitin in their walls (Gow 1989). The chitosomes were described to be zymogenic multienzyme structures which deliver chitin synthase units to specific sites in the cell wall membranes. Bartnicki-Garcia et al. (1979) gave a detailed description of the morphology and structure of chitin microfibrils and of the particles associated with their formation in Mucor rouxii. Hence, cell growth is the result of a dynamic balance between the maintenance of an intracellular turgor pressure via solute synthesis and water input, and a synthesis of macromolecules which causes the membrane to provide a constant resistance despite the elongation phenomenon (Wessels 1993). We give further details below. In the case of continuously growing cells such as filamentous fungal tips, the steady-state conditions mentioned in the preceding section (see eq. (1») can no longer be applied. So the evaluation of intracellular turgor pressure must take into account the type of system under consideration: there will be differences between non-extending cells ( such as bacteria, yeasts, or plant cells) for which both cell volume V and intracellular osmotic pressure Jr; could be considered to be constant, and extending non-equilibrium cells (such as fungi). In order to study more precisely the case of fungi, let us consider the example of fungal hyphae growth during a time interval dt. During this interval dt, dns moles of a solute j are synthesized or incorporated in the internal medium which induces a dII; increase in osmotic pressure, as for diluted media II; could be considered to be equal to: n
II; = RTICj j=i
(2)
with R =ideal gas constant; Cj =molar concentration of intracellular solute j; and T =temperature. Such an increase in osmotic pressure, in addition to a continuous apex wall elongation (through vesicle incorporation), will simultaneously provoke a thermodynamic flow of water into the cell which will decrease the Cj concentration and will thus simultaneously reduce the osmotic pressure level of -dII; as shown in the previous sentence. All these previous phenomena occur simultaneously, which leads to a continuous hyphal extension. Cell growth could then be attributed to a constant increase in the osmolytes concentration near the apical part which is continuously balanced by a water flow input. So in the case of filamentous fungi, growth, regarded as volume variation (essentially due to water input), should occur under a constant intracellular osmotic pressure (II). The increase in cell volume is in tum limited by the cell wall's mechanical properties, which were previously discussed in this section. Assuming that the tension developed by the cell wall is constant in the apical part during hyphal growth (i.e. the continuously synthesized wall exhibits steady mechanical properties), it is possible to calculate the cell pressure balance for extending fungi. In this case the sum of the exerted pressures is no longer equal to zero (as previously shown for noncontinuously extending cells in equation (1» but equal to a pressure loss value (LiP) which is the origin of the continuous apical extension, (that is, water inflow to the cell). At a time t it could be written:
(II;-IIe}-Pr=LiP
or
Pt-Pr=,jP
(3)
This very simple equation allows to describe fungal growth; thus turgor pressure in extending cells is no longer counterbalanced only by the membrane pressure resistance (Pr) but also by the apical deformation (due to LiP). Previous works (Lockhart 1965; Money and Harold 1993) have not considered the parameter LiP in the turgor pressure evaluation or measurement in continuously extending cells. Now it is possible to relate the volume variation dV during the time dt to this constant pressure loss LiP over time (as II;, IIe and Pr are constant) and to physical characteristics of the fungal apex through the application of an equation (Kedem and Katchalsky 1958) describing laminar water flow driven by pressure gradients between intra- and extracellular media.
(4) with: A
= exchange surface where the water flow takes
place (m 2), dV
dt
= water flow between intra and extra-
cellular media (m3 .
S-I),
Lp = hydraulic permeability of
Microbiol. Res. 154 (1999) 1
83
the apex wall (m . S-I . Pa- I ), M =(lli - lle) - P r (Pa). Integration of previous equation (4) yields: (5)
In obtaining (5) it is assumed that the surface A is constant versus time. Indeed the parameter A is related to the surface of the apex where the water flow takes place, i.e. where the pressure gradient occurs. This apex wall surface corresponds to the cell's active volume (Va) which can be defined as the hyphal apex volume which is osmotically balanced or equilibrated by both, continuous incorporation or synthesis of solutes and water input. Indeed it was shown by 14C glucose labeling (Trinci 1971 ; Fevre 1979; Fevre and Rougier 1982), that glucose incorporation was about ten times higher at the apex than in the subapical zone of Saprolegnia monoi'ca. Such a local increase in solute incorporation (mainly sugars and ions) would immediately trigger a fast water input to the fungi, this is known to be many times faster than solute diffusion towards the rear of the apex (Mauro 1957). This confirms the view that the rate of hyphal growth will be only controlled by the water input in the apical part, as estimated in equation (5). As the apex is extending during the time dt, the active volume Va is increased by dV, but is also decreased by dV due to the advance of the apex which is supposed to be the only place of osmotic perturbation (i.e. solute incorporation or synthesis) as proposed in Fig. 1. The surface A could then be considered constant during fungal extension. Two hypotheses could be proposed to explain this behaviour, i) cell walls corresponding to the
Va timet
lime I + dl
or
dV
7
/"
\\
(6)
These equations imply that the hyphal growth rate dx
dt is constant for defined medium conditions (A, Lp ' M, s
are constant), a well known fact since 1957 (Scott). The expression of the turgor pressure could be easily obtained from the previous equation (6). Substituting M by its value (Pt - Pr ) given by equation (3) in equation (6) gives:
~ = Pr + K( dX) dt
with
K= ~ ALp
(7)
Comparison of this equation with equation (1) allows us to conclude that in the case of growing tips the turgor pressure, considered to be a potential pressure, is greater than the equilibrium value Pt = Pr (case of nonextending cells). Equation (7) trivially reduces to (1) when cell extension stops. This difference between non extending cells and growing tips is easily understandable, because in the
Major consequences
)
1/
Va
Fig. 1. Representation of the apex volume variation during the time dt (the cell active volume Va remains constant). 84
or
the turgor pressure continuously drives the volume expansion and thus cannot be measured just through the membrane pressure resistance P,. We discuss experimental details below.
------rl~
~~)
dx = ALp t1.P dt S
case of continuous hyphal elongation, a part [ K : ] of
/
,
dV volume in Fig. 1 have acquired a weaker permeability to external solutes, and ii) the cytoplasmic volume corresponding to dV has lost its ability to synthesize compatible solutes (glycerol, mannitol, etc.). With these assumptions, the fungal growth expansion is represented by equation (5). As the hyphal growth rate is unidirectional (x axis), the volume variation dV during the time dt could be written dV =dx . S where dx is the axial hyphal extension and S is the section of the hyphal tube. Consequently, equation (4) becomes:
Microbiol. Res. 154 (1999) 1
Recent works using the capillary probe technique have measured such a low turgor pressure level in growing tips that the question of how growth is possible without turgor pressure has been put forward (Money and Harold 1993). This controversy is clarified by our theory. In fact, such a probe incorporated as the hyphae are still growing will measure a hydrostatic pressure gradient between intra and extracellular medium, but without taking into account the pressure which continuously drives the volume expansion i.e. the M value in equation (3).
Hyphal extension rate
Whithin the framework developed here, such measurements will only give the value P't = II'i - IIe = P, and not the total value which is IIi - IIe = Pr + !1P, i.e. Pt =P r +!1P· A main analytical conclusion of this theoretical development is that internal fungal osmotic pressure as well as fungal turgor pressure were underestimated when the capillary probe technique was used or when any other static measurement is performed.
Table I gives the different hyphal extension rates for different water potential values of the culture medium. These results confirm previous findings (Scott 1957) and show that hyphal extension rate is constant for a given water activity in the culture medium. Nevertheless there are significant variations as the water potential of the medium is modified. The hyphal extension rate corresponding to the optimal water potential value (about -4 MPa) was about twice the values found for higher water potential values (-OJ MPa) or for lower water potential values (-7 MPa).
2) Experimental approach Previous theoretical considerations allowed us to understand the cell turgor pressure, particularly in mobile microorganisms. For such cells, an important part of the turgor pressure was continuously converted into volume expansion and thus cannot be measured by an intracellular pressure measurement. Equation (7) also evidences a linear relation between the turgor pressure and the hyphal extension rate. Nevertheless, such a linear relationship will only be valid for standard culture conditions (temperature, pH, osmotic pressure) which correspond to constant membrane characteristics. In order to relate experimental methods to the previous considerations about the underestimation of turgor pressure in growing tips, we propose a method intended to evaluate only that part of turgor pressure in growing filamentous fungi continuously used for volume expansion. This method is based on the application of different osmotic stepwise increases to the external culture medium in order to evaluate the intensity of the specific osmotic shift which would stop the hyphal extension rate without any plasmolysis, i.e. without decrease of the hyphal volume. The osmotic shift must be as fast as possible in order to prevent active osmoregulation mechanism from the fungi. The perturbation corresponds to a sudden increase of the IIe value from IIer dx
(growing fungus at a constant hyphal growth rate,' dt)
to IIe?_ (stopped hyphae i.e.
dx = 0). It follows from
dt
L1P measurements Previous considerations have allowed to propose a method for L1P evaluation ( !1P = K ~~ with K A~p ). As an example of LJp determination, Fig. 2 shows the hyphal extension rate for an initial water potential value of -2.8 MPa and for three different stepwise decreases in water potential: - 3.1 MPa, - 3.5 MPa, and - 3.9 MPa, respectively. From the plots, the minimum level of water potential required in order to stop growth without modification of the hyphal volume would be between the decrease values -3.5 MPa and -3.9 MPa. So LJp will be (+3.5 -2.8) < Pt < (+3.9 -2.8) i.e. 0.7 < Pt < 1.1 (in MPa). The procedure has been repeated for three other initial water potentials and the results are presented in Table 1. Analysis of the fungal !1P values proposed in Table 1 shows that Aspergillus oryzae exhibits relatively high 3.0
c
'E
2.5
(jj a. E 2; 2.0
2
~
equation (7) that:
c 1.5 0
'iii
(8)
Hence the difference between the two external levels in osmotic pressure gives the part of the turgor pressure which is continuously used for volume expansion i.e. the !1P value of equation (3). The total turgor pressure value could then be experimentally determined by the sum of the experimental !1P value and of the Pr value given by the capillary pro~e techniques or by a direct measurement of the osmotIc step increase leading to the incipient plasmolysis (Money 1990).
c
Q)
xQ) '(ii
1.0
,.,a.
J::
I
0.5
0.0 0
5
10
15
Time after an osmotic shock (min)
Fig. 2. Evolution of the hyphal extension rate for different stepwise decreases in water potential: . : - 3.1 MPa, 0 : -3.5 MPa, + : -3.9 MPa, (initial water potential value was -2.8 MPa). Microbiol. Res. 154 (1999) 1
85
Table 1. Evaluation of LJP values for Aspergillus orizae cultivated at different levels of water potential. Water potential of the culture medium (MPa)
-0.3 1.60 (0.34)*
Hyphal extension rate (f.unlmin) Water potential of the solutions used to stop growth (MPa)
LJp values (MPa) K =LJP/Vh
*
1.94 (0.30)
-4.2 3.08 (0.60)
-0.5
-3.1
-4.9
-1.0
-3.5 -3.9
-5.1 -5.6
0.2
0.7 < Pt < 1.1 0.36 - 0.57
-7.0 1.43 (0.41) -7.3
0.9
values into brackets represent confidence intervals at 95%.
3.5.,----------------------,
0.0 + - - - - + - - - t - - - - + - - - + - - - i - - - - - t - - - - - j - - ' 0.8 1.0 1.2 1.4 0.6 0.2 0.4 0.0
AP (Pa)
Fig. 3. Representation of the hyphal extension rate as a function of the pressure LJP.
AP values, which depend on the water potential value of the media. Such high values for AP are in contradiction with previous theoretical considerations which have concluded that a minuscule difference in water potential suffices to account for water inflow during hyphal growth and that this does not significantly influence turgor pressures as measured by a pressure probe (Money 1994). Similar values have been previously reported (Griffin 1981; Luard and Griffin 1981). Maximal AP values (about 1.3 MPa) were found for water potential values of the external medium of -4 MPa (the one having the highest Vh ). For higher as well as for lower values of external water potential (-0.3 or -7 MPa) the AP was found to decrease drastically to 0.5 MPa and 0.1 MPa, respectively (like behaviour of Vh ). The measured AP part of the turgor pressure is plotted in Fig. 3 against hyphal extension rate. It can be seen that the higher the AP, the higher the apical rate. The variability of the jjp measurements has not allowed to clearly show a linear relation between the two parame86
-2.8
Microbiol. Res. 154 (1999)
1
ters as expected from eq. (6). Nevertheless the relationship between the two parameters is clearly an increasing function. Further experiments will allow us to explore this relation. The theory developed here allows a better understanding of turgor pressure in fungi and mobile microorganisms. For such organisms a main part of the turgor pressure (AP) was shown to be continuously used for cell volume expansion. On the basis of this theoretical approach, a method allowing the evaluation of the jjp of mobile microorganisms has been proposed. This method does not give a precise value but defines a range for the AP values. Nevertheless, the use of osmotic stepwise changes of weaker intensity would certainly improve the resolution of the method. In order to measure the whole turgor pressure of mobile microorganisms, it would be necessary to combine the capillary probe technique in order to evaluate Pr' and the osmotic step technique in order to evaluate .1P. The turgor pressure is the sum of both values. Another method to evaluate the whole turgor pressure is to use the osmotic step technique until the cell shows incipient plasmolysis as proposed previously (Money 1990).
Acknowledgments This work was supported by funds from the Burgundy district. We are indebted to Pr. Hector Jorquera from the Catholic University of Santiago (Chile) for helpful advices and discussions.
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water mold Achyla: interpl ay of turgor and wall strength. Proc. Natl. Acad. Sci . USA 89, 4245-4249. Money, N. P., Harold, F M. (1993): Two water molds can grow without measurable turgor pressure. Planta 190,426-430. Money, N. P. (1990): Measurement of hyphal turgor. Exp. Mycol. 14,416-425. Money, N. P. (1994): Osmotic adjustment and the role of turgor in mycelial fungi, In: The Mycota I: Growth, Differentiation and Sexuality, Wessels and Meinhard (Eds.), Springer-Verlag Berlin Heidelberg. Nielsen, J. (1992): A simple morphologically structured model describing the growth of filamentous microorganisms. Biotechnol. Bioeng. 41, 715-727. Oh, K., Matsuoka, H., Nemoto, Y, Sumita, 0 ., Takatori, K. , Kurata, H. (1993) : Determination of anti-aspergillus activity of antifungal agents based on the dynamic growth rate of a single hypha. App\. Microbiol. Biotechnol. 39, 363-367. Robertson, N. F (1958): Observation of the effect of water on the hyphal apices of Fusarium oxysporum. Ann. Bot. London. 22, 159-173. Robertson, N. F , Rizvi, S. R. H. (1968): Some observations on the water relations of the hyphae of Neurospora crassa. Ann. Bot.-London. 32, 279-291. Scott, W. J. (1957): Water relation of food spoilage microorganisms. Adv. Food Res. 7, 83-127. Suutari, M. , Liukkonen, K., Laakso, S. (1990): Temperature adaptation in yeasts: the role of fatty acids. J. Gen. MicrobioI. 136, 1469-1474. Trinci, A. P. J. (1971): Influence of the width of the peripheral growth zone on the radial growth rate of fungal colonies on solid media. J. Gen. Microbiol. 67,320-344. Trinci, A. P. J. (1974): A study of the kinetics of hyphaI extension and branch initiation of fungal mycelia. J. Gen. MicrobioI. 81,225-236. Trinci, A. P. J. (1978): Wall and hyphal growth. Sci. Prog.-Oxf. 65,75-99. Wessels, J. G. H. (1993): Wall growth, protein excretion and morphogenesis in fungi. New Phytol. 123,397-413. Wilson, 1. M., Griffin, D. M. (1979): The effect of water potential on the growth of some soil basidiomycetes. Soil BioI. Biochem. 11,211-212. Woods, Duniway, 1. M. (1986): Some effects of water potential on turgor and respiration of Phytophthora cryptogea and Fusarium moniliformis. Phytopathology 76, 1248-1254. Zhu, G. L. , Boyer, J. S. (1991 ) : Turgor and elongation growth in Chara cells. Plant Physiol. 96, 38.
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