Sterols in Fungi: Their Functions in Growth and Reproduction

Sterols in Fungi: Their Functions in Growth and Reproduction CHARLES G. E L L I O T Botany Department, University of Glasgow, Scotland. I. Introduction

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11. Functions of Sterols: Possible Approaches to the Problems . . . . . . . 111. Sterols in Model Systems

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IV. Subcellular Distribution of Sterols in Fungi, and States of Binding . . V. Effects of Sterols on Metabolism and Vegetative Growth A. Pythium and Phytophthora . . . . . . . .

VI. VII. VIII. IX. X. XI. XII.

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B. Saccharomyces and Other Fungi . . . . . . . . 144 . . . . . . 148 Effects of Sterols on Asexual Reproduction Sexual Hormones ofkhlya . . . . . . . . .I49 Effects of Sterols on Sexual Reproduction in Homothallic SpeciesofPythium and Phytophthora . . . . . . . . . . . .I52 Reproduction in Heterothallic Species of Pythium and Phytophthora . 156 162 Sterols and Sexual Reproduction in Ascomycetes and Basidiomycetes Conclusion . . . . . . . . . . . . . 165 . . . . . . . . . . .lG6 Acknowledgements References . . . . . . . . . . . . . 166

I. Introduction

My interest in the subject of this review began with the observation that Phytophthoru cuctorum would grow on a simple medium containing sucrose, asparagine, mineral salts and thiamin, but that on this medium it remained purely vegetative. On the other hand, when grown on oatmeal agar, oospores were produced in abundance. The factors in oats responsible for the difference were found to be sterols (Elliott et ul., 1964). Addition of sterols to the simple basal medium changed the mode of growth from vegetative to reproductive. Similar findings were reported about the same time for various species of Phytophthoru and Pythium (Hendrix, 1964, 1965; Haskins et al., 1964; Harnish et ul., 1964; Leal et ul., 1964). It is worth noting that, in 121

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1937, Leonian and Lilly had found that some substance extracted by alcohol from peas was required for reproduction of Phytophthoru. However they considered the active compound in the extract not to be a sterol but some unknown compound which they could not separate from sterols. Since that time, advances in our knowledge of the chemistry and biosynthesis of sterols in other organisms, the development of analytical techniques (particularly gas-liquid chromatography), and the availability of a wide range of pure sterols, have clarified the problem, and it is now clear that it is indeed the sterols which are the required growth factor in Phytophthoru spp. Species of Pythium and Phytophthoru are unable to synthesize sterols. Thus sterols are not detectable in mycelium grown in the absence of sterols (Elliott et ul., 1964; Schlosser and Gottlieb, 1966; McCorkindale et ul., 1969) and incorporation of labelled sterol precursors (acetate and mevalonate) into sterol-like material does not occur (Hendrix, 1966, 1975b; Schlosser et ul., 1969). Richards and Hemming (1972) reported that mevalonate was incorporated into farnesol, geranylgeraniol, dolichols and ubiquinones in Phytophthoru cuctorum. Squalene was not labelled; but D. Gottlieb (personal communication) finds that squalene is synthesized, but is not cyclized to sterol. Langcake (1975) reports that Phytophthoru infeestuns can convert lanosterol to a compound which he thought was possibly cholesterol, but cholesterol, cholestanol, sitosterol and stigmasterol are not converted to other sterols. Knights and Elliott ( 1976) find that A7 and A5*’-sterolsare converted to the corresponding A5-sterolsby Phytophthoru cuctorum. Species of Pythium and Phytophthoru are all plant pathogens, and it is evident that they obtain the sterols necessary for their reproduction from their hosts. It is perhaps surprising that more fungi cannot synthesize sterols, as they mostly inhabit environments where sterols would be expected to occur. The sterol content of spores of Plusmodiophoru brassicue varies significantly according to the sterol composition of the host, suggesting at least partial heterotrophy (Knights, 1970). The rusts however synthesize sterols. The predominant sterols in their uredoand A’-ergostenol spores are A’-stigmastenol, A7*24(28’-stigmastadienol (Jackson and Frear, 1968; Nowak et ul., 1972). A7-Sterolswere found in rust-infected leaves and not (or only exceptionally) in healthy leaves (Nowak et al., 1972; Lin and Knoche, 1974). This indication that the fungus synthesizes sterols was confirmed by the finding that these par-

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ticular sterols became labelled when germinating uredospores were supplied with [14Cl-mevalonateor acetate (Lin et al., 1972). I t is notable that, unlike other basidiomycetes, the rusts do not contain ergosterol. Those oomycetes which synthesize sterols (e.g. species of Achlya, Saprolegnia) also do not contain ergosterol, but rather fucosterol and its relatives (McCorkindale et al., 1969). Ergosterol was not detected either in two chytridiomycetes and two hypochytridiomycetes (Bean et al., 1972). The occurrence and biosynthesis of sterols in fungi have been reviewed by Weete (1973, 1974) and also by Goodwin (1973). A list of fungi and the sterols found in them is given by Bean (1973). The lipids of yeast were reviewed by Rattray et al. (1975). Biological aspects of sterols in fungi were reviewed by Hendrix ( 1970). 11. Functions of Sterols: Possible Approaches to the Problems

The object of this review is to discuss the function of sterols in fungi, with special reference to sexual reproduction ; the effect on sexual reproduction is the most striking aspect of the sterol requirement in Phytophthora. A general approach to this problem in fungi might be to consider what happens when sterol synthesis is inhibited. This inhibition might be the result of gene mutation, so that some step or steps in the biosynthesis of sterol cannot be carried out. As already indicated it appears that pythiaceous fungi cannot convert squalene to sterols. Species of Pythium and Phytophthora are very favourable organisms for studying the function of sterols. One can simply compare the mycelium grown with and without sterol. Evidently, in these fungi, sterols are not essential for vegetative growth. If sterols are essential for

FIG. 1. Structure of stigmastane, indicating the numbering of carbon atoms in sterols.

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CHARLES G. E L L I O l l

I

IV

&

H b:H

11

111

V

VI

HO@H,C kH,

&OH VIII

VI I

IX

FIG. 2. Ring systems of sterols. See Table 1 for sterols in which these occur. TABLE 1. List of principal sterols mentioned in the text, with systematic names and index to their structures shown in Figs. 2 and 3. Common name

Systematic name C,

sterol

C,

sterols

B- Norcholesterol

B-nor-5-cholesten-3~-ol

Coprostanol Cholestanol Cho lest ero I A’-Cholestenol 7 - Dehvdrocholesterol Epicholcsterol Zymosterol

5/hholestan-S/J-ol 5a-cholestan-3/?-ol 5-cholesten-3~-ol 5a-cholest- 7-en-3p-01 5,7-cholestadien-3~-ol 5-cholesten-3a-ol 5a-cholesta-8,24-dien-3/3-ol

Campesterol A5-Ergostenol A7-Ergostenol

(24R)-24-methyl-5-cholesten-3~-ol (24S)-24-methyl-5-cholesten-3/3-ol (24S)-24-methyl-5a-cholest-7-en-3g-ol

C,

Ring system

Side chain

I

A

I1 I11 IV

A A A

V VI VI I VIII

A A B

A

Jterols

IV IV V

C

D D

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STEROLS IN FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

+A+* A

C

G

B

D

E

F

H J FIG. 3. Side chains of sterols.

K

TABLE 1 . 4 co n t i n u ed) Common name

Ergosterol Episterol Ergostadienol AS.l.24 (28)-

Ergostatrieiiol

Systematic name

Ring system

(24R)-24-methyl-5,2ZE-cholestadien- IV

E

VI

E

3/3-01 (24R)- 24- methyl-5,7,22E-cholestatrien3/3-01 24-methylene-5a-cholest-7-en-3P-01

V

(24R)-24-inethyl-5a-cholesta-8(9),22E-VIII dien-3/3-ol. 24-inethylene-5,7-cholestadien-3/3-ol

F

E

VI

F

C,, sterols IV (24R)-24-ethyl-5-cholesten-3/3-ol V (24R)-24-ethyl-5a-cholest7-en-3/3-01 (24R)-24-ethyl-5,7-cholestadien-3~-olVI IV (24S)-24-ethyl-5,22E-cholestadien-

G G G H

(24S)-24-ethyl-5,7,22E-cholestatrien-

VI

H

(24R)-24-ethyl-5,22E-cholestadien-

IV IV

J

IX

B

3/3-01

8/3-01 24E-ethylidene-5-rholesten-3/3-ol 3/3-01

Lanosicrol

Side chain

C,, sterol 4a,4/3, 14a-trimethyl-5a-cholesta-8,24: dirn-3~-ol

K

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CHARLES G . ELLIOlT

vegetative growth in other organisms, then pythiums and phytophthoras must make some substance which can do the same job. But it is apparent that this hypothetical substance cannot fulfil the role in reproduction which sterols have. In species of Saccharomyces, mutants in which the later stages of ergosterol synthesis are blocked, are known. These mutations were selected as conferring resistance to polyene antibiotics (see p. 145). Other ways in which sterol synthesis can be inhibited are to subject the organism to anaerobic conditions (see p. 1461, or to use drugs which inhibit particular steps in biosynthesis (pp. 147 and 162). An alternative and complementary approach is to consider the effect of molecular shape on activity. The approach is complementary because, when the terminal stages of synthesis are blocked, a precursor of different structure may accumulate and it may have some but different activity. Both the configuration of the ring system (Fig. 2) and of the side chain (Fig. 3) are important. The configurations of the ring systems are illustrated in photographs of models in Fig. 4(a-f). There is a fundamental difference in shape between molecules with the 5a and 58 configuration. In the former, there is a regular alternation of the carbon atoms above and below the plane through all the rings. In the latter, the molecule is bent at the A ring end (Figs. 4(aHb)).The position of the hydroxyl group at C-3 also is important. Ifit is in thepposition, the oxygen atom lies in the plane of the rings of the 5a or A5 sterols (i.e. an equatorial configuration; Figs. 4(b) and (c)).A double bond between C-5 and C-6 flattens the B ring somewhat, and alters the position of the C-4 hydrogens relative to those at other positions in ring A (see Figs. 4(b) and (c)),but a more extreme flattening of the B ring occurs with a double bond between C-7 and C-8 (with or without As).I t will be noticed that A7 brings the “axial” methyl groups (C-18 and C- 19) closer together (compare Figs. 4(d) and (el with (a), (b) and (c)). A double bond between C-8 and C-9 results in greater joint flattening of the B and C rings (Fig. 4(f)). An indication of the overall degree of flatness of the molecule is given by the number of the axial ahydrogen atoms which are coplanar. For Sa-cholestanol, those attached to carbons 1, 3, 5 , 7, 9, 12 and 14 are coplanar; for A5cholestenol, 1, 3, 9, 12 and 14; for A8-cholestenol,1,3, 12 and 14, and for A7-cholestenol,1, 3 and 14. In the last instance, it is notable how far the a-hydrogen attached to C- 12 is from this plane. The numbering of the carbon atoms is indicated in Fig. 1.

STEROLS IN FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

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The shape of the side chain is modified by substituents at C-24and double bonds at various positions. An ethyl or methyl group which replaces one of the hydrogen atoms at C-24can be either in the a or p position (Figs. 3, C and G;compare with D and K).The ethyl group of sitosterol (Figs. 2 and 3, IVG) and stigmasterol (IVH) is in the a position; that of poriferasterol (IV K),and the methyl group of ergosterol (VI D)are orientated. In the R and S terminology, sitosterol is (24R)-24-ethyl-5-cholesten-3p-o1. But, under the rules of terminology, the double bond at C-22 causes the ethyl group of stigmasterol to be designated S, and stigmasterol is (24S)-24-ethyl-5,22E-cholestadien3p-01. These variations in the shape of the sterol molecule can result in great variation in biological activity. A very high degree of specificity in their activity is one of the principal characteristics of steroids (Grant, 1969). A third method of approach is to study changes in sterol content or state of binding during the growth cycle, and to corrblate these with metabolic changes in the mycelium. In higher organisms, sterols have three principal functions; as precursors of other steroids, as hormones, and as membrane components (Heftmann, 1971). Heftmann (1970, 1971) points out that plants, as C,, and C,, well as animals, produce sterols and from them CZ1, steroids, and he argues that the fundamental biochemical similarity between all living organisms implies that these compounds have similar roles in plants and animals. In recent years, there have been great advances in our understanding of the action of steroid hormones at molecular level (Jensen and DeSombre, 1972;O’Malley and Means, 1974).A great deal is known about how these hormones bind to cytoplasmic receptors which are characteristic of the target tissues, and how these hormone-receptor complexes then bind to chromosomes leading to synthesis of RNA and specific proteins. At the physiological level, one is struck by the remarkable diversity of effects that hormones induce. We are still far from understanding how events at the molecular level account for this diversity, except that it is evident that a hormone can switch on a sequence of genetic events. Thus, administration of ecdysone to Drosophilu larvae brings about puffing (the sign of gene activity) first in one band of the giant chromosomes, followed by puffing in other bands in a definite order (Ashburner, 1970),so that a complex pattern of metabolic activity can be built up. One of the physiological effects of hormones is on transport across

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FIGS. 4(a-1). Photographs of models of sterols to illustrate the configuration of the ring systmi. (a) Coprostanol; (b) Cholestanol; (c) Cholesterol; (d) A’-Cholestenol; (e) 7-Dehydrocholesterol; (D A8-Cholestenol.

STEROLS IN FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

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membranes. Indeed, this is the principal effect of the mineralocorticoids and the vitamin D complex, but oestrogens too, for example, modify transport into the tissues that show the more dramatic responses to the hormone. Formerly, the possibilities that hormone action resulted from direct effects on permeability or on enzyme activity were considered (see Tepperman and Tepperman, 1960; Riggs, 1970). In work on fungal sterols, we are faced with a particular difficulty. As will be discussed later in this review, sterols as components of membranes have a pronounced effect on permeability, in model systems at least. Therefore we have to try to distinguish whether the effects of sterols on, say, species of Phytophthora, result from direct consequences of the presence of sterols in the plasma or mitochondria1 membranes, or whether there are effects which can only be attributed to a hormone-type action. On the whole, it seems that sterols in model membranes affect simple diffusion, but hormones in cells control active energy-requiring transport of specific ions or molecules. 111. Sterols in Model Systems

Nes ( 19741, in an admirable article, has reviewed the occurrence of sterols and sterol-like substances, and drawn attention to the small range of structures found in the dominant sterols of eukaryotes. Evidently only a few structures can fulfil the particular role which fiee sterols perform in the membranes where they occur. Few organisms are known which do not use sterols for this role. One is Tetruhymena pynyormis, which produces instead tetrahymenol, a compound which can adopt a shape similar to that of a sterol. When supplied with exogenous cholesterol, Tetrahymenu pyntormis incorporates it into its membranes (converting it into A5~7~22-cholestatrienol) and the amount of tetrahymenol synthesized is greatly decreased (Connor et al., 1969). Evidently the sterol is a more satisfactory molecule for performing the role in membranes. However, Nozawa et al. (1975) found that, when T. pyriiormis is grown with ergosterol, the replacement of tetrahymenol by ergosterol is accompanied by extensive changes in phospholipid and fatty-acid composition, and also by ultrastructural changes visible in the electron microscope. Some mycoplasmas require an exogenous source of sterol for growth (Edward and Fitzgerald, 1951; Smith and Lynn, 1958); these are now placed in the genus Mycoplasma. The genus Acholeplasma comprises organisms which do not require sterols. Achole-

STEROLS IN FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

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plasma laidlawii and A. granularum synthesize cartotenoids which Nes (1974) supposes play a similar role to sterols, but A. axanthum does not make pigmented carotenoids (Tully and Razin, 1969, 1970). Sterols are adsorbed into the membranes of both the sterol requirers and nonrequirers (Smith and Rothblat, 1960; Tully and Razin, 1969). The effect of exogenous sterol supplied in serum to A. laidlawii is to decrease the amount of carotenoid in the membrane (Smith, 1963) bht, when the sterol is added without serum, it is incorporated without significantly affecting the carotenoid content (Razin and Cleverdon, 1965).

FIG. 5. Diagram showing possible mode of interaction between cholesterol and lecithin with stearoyl and linolenyl chains (Vandenheuvel, 1963). CCC indicates the carbon atoms of glycerol; P, phosphorus; N, nitrogen ofcholine; and 0, the hydroxyl oxygen of' cholesterol.

The way in which cholesterol can fit into a phospholipid layer has been discussed by Finean ( 19531, Vandenheuvel (1963) and Brockerhoff ( 1974). The dimensions of the cholesterol molecule are such that, if the 3-hydroxyl of the sterol is associated with the nitrogen of the choline of the lecithin, the end of the cholesterol side chain fits neatly under the fatty-acyl chain where it bends at the cis double bonds (as in linoleic acid; Fig. 5; Vandenheuvel, 1963). Brockerhoff (1974) proposed a model based on hydrogen bonding between the 3hydroxyl group of the sterol and the carboxyl oxygen of the fatty-acyl chain. Edwards and Green (1972) showed that extra carbon atoms in the side chain decrease the ability of sterols to pack into phospholipid layers, campesterol being less readily incorporated than cholesterol, and sitosterol than campesterol. This could be related to the well known fact that ergosterol and sitosterol are less readily absorbed in the human intestine than cholesterol (Glover and Morton, 1958). Our understanding of the role of sterols in membranes has been es-

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pecially advanced by the use of physicochemical techniques applied to model systems-the lipid monolayer spread on water, and the bilayer “liposome” produced by mixing lipid with salt solution-as well as on membranes themselves. The membranes of mycoplasmas in particular are admirable objects for such experiments (Razin, 1973). The physicochemical studies were reviewed by Oldfield and Chapman (1972). Demel el al. (1967) and Chapman et al. (1969) showed that incorporation of cholesterol into a lecithin monolayer decreased the area occupied by each molecule. The magnitude of the affect increased with increasing proportion of cholesterol (De Kruyff et al., 1972). Darke et al. (19721, in a study using nuclear magnetic resonance, showed that cholesterol and lecithin formed a complex in which their molar ratio was 1 : 1 ; when the proportion of cholesterol in a bilayer was less than half, its distribution was clustered. However, Verkleij et al. (1974) found, using freeze-fractured liposomes, that lecithin bilayers containing only 1&15 mole per cent cholesterol were homogeneous. Rand et al. (1975) described a 1 : 1 molar complex between cholesterol and lysolethicin, and emphasized that their interaction was quite different from the interaction of cholesterol with the diacyllecithins. Addition of cholesterol to a lecithin decreases the amount of movement which the fatty-acyl chains of the lecithin can undergo. Thus the temperature at which the transition between the more rigid gel phase and the less rigid crystalline phase occurs is lowered, and the amount of heat adsorbed at the transition point is decreased (Ladbrooke et al., 1968). More recently, the use of electron spin resonance (ESR also called electron paramagnetic resonance, EPR), in which the freedom of movement of the electron of an introduced free radicle can be studied, has provided much information. For example, Hsia and Boggs (1972) and Mailer et al. ( 1974) showed that cholesterol restricts the freedom of movement of the hydrocarbon chains of fatty acyl groups, and Butler et al. (1970) compared the effects of a number of steroids. Kroes et al. ( 1972) similarly showed increased viscosity in erythrocyte membranes from guinea pigs fed a cholesterol-supplemented diet; their erythrocytes contained about twice the control amount of cholesterol. Feinstein et al. (1975) used a fluorescent probe to show that cholesterol decreased membrane fluidity. Rothman and Engelman (1972) showed with models how the presence of cholesterol in the membrane restricts the movement of the carboxyl half of the fatty-acyl chain, while some movement is still possible at the free end.

STEROLS I N

FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

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Work with artificial membranes has shown that sterols affect their permeability. When they contain cholesterol, their permeability to water is decreased (Finkelstein and Cass, 19671, the rate at which liposomes swell in hypotonic solutions is diminished (De Gier et al., 1968) and the rate of loss of sequestered glucose is also decreased (Demel et al., 1968). Both cells of Acholeplasma laidlawii, and liposomes prepared from their lipids, showed lower permeability to glycerol and erythritol when the cells has been grown in cholesterol-containing medium (De Kruyff et al., 1972; McElhaney et al., 1973). The latter authors, and also De Kruyff et al. (1973), emphasized the effect of fatty-acid composition on membrane permeability, but incorporation of cholesterol had no effect on the fatty-acid composition, and thus the diminished permeability with cholesterol could be attributed to cholesterol (De Kruyff et al., 1972). Rottem et al. (1973) adapted the sterol-requiring species Mycoplasma mycodes var. Capri to grow without sterol, and showed that the adapted cells were more fragile osmotically, and were more permeable to erythritol. The effects of other sterols besides cholesterol on molecular spacing in monolayers and on permeability of liposomes have been studied by Demel et al. (1972a, b). Their papers give no indication of the variability of the results, but it appears that the effects of sterol structure on both phenomena are very similar and are comparable to the effects on cell permeability (De Kruyff et al., 1972, 1973). The ESR studies by Butler et al. ( 1970) also give essentially similar ordering of the compounds. In general, cholesterol (Figs. 2 and 3, IVA) has the greatest effect in all of these systems, with cholestanol (IIIA), A’cholestenol (VA), 7 -dehydrocholesterol (VIA) and B-norcholesterol (IA) similar. Molecules without a side chain (5a-androstan-3p-01 and 5-androst-en-Sp-01) have little effect, as is also the case with 3ahydroxysterols (e.g. epicholesterol; VIIA) and coprostanol (IIA) with its 58 configuration. Of the sterols with extra carbon atoms in the side chain, sitosterol (with a saturated side chain; IVG) has a similar effect to cholesterol, and stigmasterol (IVH) and ergosterol WID) (with a double bond at C-22) are less effective. These conclusions are in agreement with the effects of steroids on growth of mycoplasmas (Smith, 1964; Smith and Lynn, 1958; Rottem et al., 1971). I t is interesting to note that, in agreement with Nes’s (1974) hypothesis, carotenoids have similar effects to sterols on permeability. Huang and Haug (1974) grew A . laidlawii on acetate-containing

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CHARLES G. ELLIOlT

medium (and thus increased the mount of carotenoids) and on propionate (thereby lowering the content of carotenoids). The carotenoid-enriched cells had less fluid membranes (as indicated by the ESR spectrum) and the cells were tougher to lyse and less permeable to glycerol. The effect of sterol composition on permeability and fragility of membranes has also been studied with erythrocytes and their ghosts by Bruckdorfer et al. (1968a, b). Erythrocytes readily lost cholesterol to sterol-depleted plasma or pure phospholipid dispersions. When these treated cells were re-incubated with dispersions of lecithin and sterol, the sterol was absorbed into the membranes, different sterols being taken up to different extents. Ergosterol was taken up to a remarkably poor extent. I t was found (Bruckdorfer et al., 1969) that the erythrocytes incub'ated in lecithin alone (which lost 3348% of their cholesterol) were very fragile and permeable to glycerol. As compared with cells re-incubated with cholesterol, those in which there was partial replacement of cholesterol (IVA) by A'-cholestenol (VA), 7 -dehydrocholesterol (VIA) or B-norcholesterol (IA) showed considerably decreased fragility, while incorporation of stigmasterol (IVH)or ergosterol (VIE) effected slight increases and decreases, respectively, although they replaced cholesterol to a very limited extent. The S-oxosteroids increased fragility and also permeability to glycerol. Coprostanol (IIA) and 7 -dehydrocholesterol (VIA) caused some decrease in permeability, but A'-cholestenol (VA) and Bnorcholesterol (IA) had little effect. Edwards and Green (1972) found that, in a replacement system, sitosterol (IVG) and campesterol (IVC) were incorporated into erythrocyte membranes to a lesser extent than cholesterol, the enlarged side chain being inimical to adsorption. In higher plant tissues, the effects of sterols on permeability have been studied by Grunwald (1968, 1971). He found (1968) that cholesterol was highly effective in reversing the methanol-induced leakage of betacyanin from beetroot cells, but sitosterol (IVG) and stigmasterol (IVH) were less effective; ergosterol (VIE) increased the leakage. With barley roots (Grunwald, 197 11, cholesterol was most effective at 10 pM at decreasing ethanol-induced permeability (but it increased leakage at 100 pM);campesterol (IVC)was less effective and stigmasterol (IVH) and sitosterol (IVG) had little effect. The evideuce already discussed shows that the occurrence in

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membranes of 3/3-hydroxysterolswith 5a- or A5-configurationsand a side chain decreases their permeability, and cholesterol is one of the most effective in this respect. But it must be remembered that not all membranes are alike in their lipid composition. Besides species differences, the membranes of different organelles are different, in sterol as well as phospholipid composition (Dod and Gray, 1968; Siekeviu, 1970). The differences are no doubt related to the structural requirements associated with the different enzymic proteins which are bound to the membranes; the lipid composition would require to be adapted to the physiological activity of the membrane. Also, Smith ( 1969) has argued that sterols in mycoplasmas are actively associated with transport across the membranes; especially, he believed that the sterol glucosides found in those species which use glucose as an energy source are sterols caught in the act of moving glucose into the cell. More recently other transport mechanisms have been found (see Razin, 1973, p. 66). Wiley and Cooper (1975) studied the effect of cholesterol enrichment on transport of Na' and K+across erythrocyte membranes, and suggested that the excess cholesterol could influence the position of the protein carrier within the membrane, or decrease its ability to move within a more viscous micro-environment. IV. Subcellular Distribution of Sterols in Fungi, and States of Binding

A number of fungi absorb sterols from their medium, even those which synthesize them (Hartman and Holmlund, 1962). Study of the uptake of sterols from solutions of various concentrations provides evidence as to the mechanism of uptake. With simple physical adsorption, we expect, from the Freundlich adsorption isotherm, a linear relation between the logarithms of the concentration supplied and the conceytration in the adsorbing surface. The evidence in Phytophthora cactorum (Elliott and Knights, 1974) is in agreement with this expectation, as are the much more extensive data on mycoplasmas (Smith and Rothblat, 1960) and mammalian tissue-culture cells (Rothblat et al., 1967). The uptake of cholesterol by Acholeplcwmu laidlawii was also shown to follow first-order kinetics by Gershfeld et al. (1974). Uptake of various sterols by Phytophthera infestans was investigated by Langcake (1975) who found that cholesterol (IVA) and cholestanol (IIIA) were taken up much more than sitosterol (IVG) and stigmasterol (IVH),

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while ergosterol (VIE) was very poorly taken up. Hendrix (1975a) found that sitosterol and cholesterol were taken up less readily by Achlya spp. than by Pythium spp. The sterol taken up by Phytophthora and Pythium spp. is recoverable partly as free sterol and partly as ester (Hendrix et al., 1970; Gain, 1972; Elliott and Knights, 1974; Hendrix, 1975b). Hendrix et al. (1970) considered that all of the sterol could be readily extracted from the mycelium, but Elliott and Knights (1974) found that about 10%of the cholesterol taken up from the medium was not recovered with a 7 h Soxhlet extraction with acetone, but it was recovered if the acetoneextracted mycelium was hydrolysed with 6-N HCl or by pyrogallol in methanolic potassium hydroxide. We have also shown (B. A. Knights and C. G. Elliott, unpublished observations) that the residue remaining after prolonged extraction with acetone contains sterol by combustion of the residue and analysis of the carbon dioxide produced for radioactivity. Cholesteryl oleate added to cultures growing without sterol is taken up by P. cactorum much more slowly than free cholesterol (Elliott and Knights, 1974). We believe that the ester must be converted to free sterol before it can be absorbed. The free sterol content of the mycelium reaches a constant value per unit weight about 12 h after addition of free sterol to the culture, and despite the slower rate of uptake of oleate the free sterol content per unit weight reaches the same constant value within 24 h of adding the ester (Fig. 6). The amount of sterol ester in the cells follows a different pattern according to whether the culture is supplied with free sterol or with ester. With free sterol supplied, much sterol is taken up, and much is found as ester, in harvests made soon after addition of the sterol to the cultures; later the amount of ester declines. Presumably the ester formed in the early stages is de-esterified, thus keeping the free sterol content constant as the weight of mycelium increases. With oleate supplied, less sterol is taken up at first, and little of it is found as ester (Fig. 6; Elliott and Knights, 1974). These results are consistent with the view that free sterols are found in membranes, while the sterol present in the cell in excess of that required free in the membranes is esterified and located elsewhere (Nes, 1974).We have not investigated the distribution of free sterol and ester in P. cactorum, but Sietsma and Haskins (1968) fractionated cells of Pythium acanthium grown with cholesterol, and reported that 79%of the

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;---fp---.---:--

-0-0

Time ( h ) from addition of cholesterol to medium

FIG. 6. Changes with time in sterol and sterol ester content of mycelium of Phytop h h m cactorum, following addition of cholesterol or cholesteryl oleate to six-day-old indicates changes in the content of free sterol in ecetone extracts of cultures. U mycelium (free cholesterol supplied in medium); o----O of sterol ester in extracts of mycelium (free cholesterol in medium);. --- Aof free sterol in acetone extracts of mycelium (cholesteryloleate supplied in medium);A---Aof sterol ester in extracts of mycelium (cholesteryl oleate in medium). Data of Elliott and Knights (1974).

sterol was in the protoplast membrane, 18%in mitochondria and 3%in endoplasmic reticulum. Their assay was by the Liebermann-Burchard assay, and esters were not investigated. Langcake (1975)reported that 71% of the cholesterol taken up by Phytophthora infeestuns was in the supernatant after centrifugation of blended mycelium for 15 min at 2 O O O g and 86% of the sterol of this was sedimented after 30min at 160,000g. The subcellular distribution of ergosterol in the ascomycete Aspergdlus niger was studied by Barr and Hemming (1972).They found that about half of the free sterol was in the mitochondria1 fraction (identified by the presence of ubiquinone), but the largest proportion of ester was in the heaviest fraction (sedimentingat 500 g after 10 mid. More critical information comes from the work on yeast (Saccharomyces cereviseae) by Hossack et al. (19731, who found that the membranous fraction (identified by phosphatidylinositol kinase) contained primarily free sterol, while the fraction consisting of low-density vesicles contained sterol esters and very little free sterol. Such vesicles are present in the growing hyphal tip (Bartnicki-Garcia, 1973kin yeast in the young bud (Moor, 1967)-and they contribute directly to the extending plasmalemma. I t seems possible that the sterol esters of the

138

CHARLES

G. ELLIOlT

vesicles serve as a ready source of the sterol required for the new membrane. Hallermeyer and Neupert ( 1974) found that ergosterol was present in the outer mitochondria1 membranes of Neurospora crassa, but there was no sterol in the inner membrane. Assay of the amounts of free sterol and ester in mycelium is however dependent on the methods of chemical extraction used. Adams and Parks ( 1967) showed that different extraction procedures removed different amounts of sterol from yeast. Saccharomyces cerevisiae contains a mannan in the cell wall which binds sterol (Thompson et al., 1973), making it water soluble and resistant to extraction with the usual lipid solvents; this bound sterol can be extracted after hydrolysis with alkaline pyrogallol (Adams and Parks, 1968). Sterol-binding polysaccharides were also described from Rhizopus arrhizus, Penicillium roquefortii and Saccharomyces carlsbergensis by Pillai and Weete ( 1975). Hence, studies on variation in the sterol content of mycelium during the growth cycle, using a single method of extraction only (as was done by Van Etten and Gottlieb (1965) with Penicillium atrouentum) are of limited value. In our analysis of sterols in Neurospora crassa (Elliott et al., 19741, we extracted the dried mycelium first with acetone, and then subjected it to alkaline pyrogallol hydrolysis. The acetone extract contained “readily extractable” free sterol and sterol esters; the alkaline pyrogallol extract was of “tightly bound” sterol. The occurrence of a large tightly bound fraction was characteristic of conidia and of senescent mycelium. It appeared that there was transfer of sterol from a readily extractable to a tightly bound state with the onset of senescence. Sterols were synthesized during active growth of the mycelium, with the amount of ester more or less parallel to free sterol. When accumulation of dry matter ceased, the free-sterol fraction continued to increase in size for a couple of days, apparently at the expense of the esters. Increase of free sterol at the expense of esters was noted in yeast by Madyastha and Parks (1969). In Phycomyces blakesleeanus, the composition of the free-sterol fraction is relatively constant during growth of the culture, but there are marked changes in the ester fraction. Thus, the proportion of episterol varies from 31 to 37% in the free-sterol fraction but from 45 to 76% in the ester fraction at different stages of growth; ergosterol forms from 49 to 66% of the free sterols but from 18 to 48% of the esters. The proportion of ergosterol esterified increases during the first 30 h of incubation, then declines somewhat and increases again after 45 h (Bartlett and Mercer, 1974). The composition of ester and free-sterol

STEROLS IN FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

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fractions differ also in Saccharomyces cereuisiae (Barks et af., 19741, and in Leptosphaeria typhae (Alais et ul., 1974).Atallah and Nicholas (1974)have suggested that esters may be important during biosynthesis; hence, perhaps, the greater diversity in the esterified sterols. DupCron et a f . (1972) found a small proportion of the sterol of potato tuber tissue in the supernatant after centrifugation of the material at 100,OOOg. This had a very large amount of sterol ester. They considered the occurrence of ester in the cytosol reflected a function in transport of sterol round the cell. The amount of sterol ester did not decline during starvation in Phycomyces blahleeanus (Bartlett and Mercer, 1974); thus sterol ester can act as a reserve for sterol but not for energy production. In potato tuber during storage, the sterol esters increase while the glucosides decrease; the free-sterol content remains more or less constant (DupPron et ul., 1971). Synthesis of sterols and sterol esters was followed during growth of Saccharomyces cereuisiae by Bailey and Parks (1975).They found that synthesis of C,, sterols continued throughout the logarithmic phase of growth, but the amount of sterol ester remained at a constant low value until the onset of the resting phase when it rapidly increased. While ergosterol formed the major sterol at an early stage of growth (92%of all sterol), it decreased to 40%at a later stage, concomitant with the appearance in substantial quantities of ergosterol precursors, especially A5.7-ergostadienoland zymosterol, and these were found mainly in the ester fraction. While zymosterol (Figs. 2 and 3, VIII B) is readily methylated at C-24, its esters are not, and, in general, esters of the precursors are not metabolised to ergosterol. Thus Bailey and Parks (1975)suggest that sterol esters may have an important role to play in the regulation of sterol synthesis. Alais et al. (1974)compared the sterols of Leptosphaeria typhae grown in light and in the dark. In the light-grown cultures, ergosterol was the only sterol found in the free state but, in the dark-grown cultures, there were small amounts of episterol, A7-ergostenol and ( ?) 24-ethyl-5cholestenol. The ester fractions also differed, the dark-grown cultures containing a considerable proportion of C,, and C,, sterols. It is to be noted that light induces formation of sexual structures in this fungus. Safe (1973)investigated the sterols of Mucor rouxii in the filamentous and yeast-like phases induced by growth under aerobic and anaerobic conditions, respectively, and found differences in sterol composition and binding. Under aerobic conditions, 90% of the sterol readily extractable with chloroform-methanol was ergosterol, but the tightly bound fraction (obtained by alkaline pyrogallol hydrolysis of the

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CHARLES G. ELLIOTT

chloroform-methanol extracted mycelium) contained only 33% ergo(25%). Under anaerobic consterol together with A7J4(28)-ergostadienol ditions, several sterols appeared in the readily extractable fraction as well as in the tightly bound fraction, but their proportions in the two fractions were different. Differences in the relative proportions of ergosterol and di-unsaturated sterols in the two fractions were also found in Neurospora crassa (Elliott et al., 1974). Safe and Caldwell(1975) reported further studies on the distribution of sterols between the cell wall and cytoplasm of Mucor rouxii in relation to the mode of binding, and they studied a yeast-like phase induced by phenethyl alcohol as well as that induced by anaerobiosis. Changes in the binding state of sterols in yeast were described by Adams and Parks (1967). Their technique was to grow cells anaerobically and then aerate them in a medium containing KH,PO,, 1% glucose and ['4C-methyllmethionine, so that the ergosterol synthesized was labelled at c-28. Cyclic changes in the amount of sterol extractable after saponification with 40% potassium hydroxide and after hydrolysis with hot 0.1-N HCI, were observed. The amounts of sterol recovered by these two methods, when added together, gave a constant value from the fourth hour after the beginning of aeration; up to this time, sterol was being synthesized. It was suggested that the sterol extracted with the base and acid treatments represented different pools, and that there was a shift of some sterol from one pool to the other according to the metabolic state of the cells. Starr and Parks ( 1962) observed that the time of occurrence of the drop in the amount of base-extractable sterol of cells aerated in glucose was determined by the glucose concentration, and was associated with exhaustion of sugar from the medium and a switch to respiratory utilization of the ethanol which had accumulated during fermentative metabolism. When the cells were aerated with acetate as energy source, the amount of baseextractable sterol increased to a maximum value which then remained constant (Adams and Parks, 1967). Also, when cells were aerated with glucose in the presence of acriflavine, which prevents adaptation to respiratory competence, the drop in base-extractable sterol did not occur (Adams and Parks, 1969). Synthesis of sterols during ascus formation in Saccharomyces cerevisiae has been investigated by Esposito et al. ( 19691, Henry and Halvorson (1973) and Illingworth et al. (1973). Incorporation of labelled acetate into lipid occurs in two stages (Esposito et al., 19691, the second of

STEROLS IN FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

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which co-incides with the appearance of the ascospores (Henry and Halvorson, 1973). Illingworth et al. (1973) noted an increase in free sterol proportional to the amount of new membrane formed, but there was a much greater increase in the amount of sterol ester, which was associated with the development of vesicles. Crystals observed in hyphae of Neurospora crassu (Tsuda and Tatum, 196 1) and Phymatotrichum omnivorum (Baniecki and Bloss, 1969) and considered to be ergosterol, have recently been shown to be proteinaceous (Hoch and Maxwell, 1974).

V. Effects of Sterols on Metabolism and Vegetative Growth A. P Y T H I U M

A N D PHYTOPHTHORA

An immediate effect of the uptake of sterol by Pythium sp. is to make the cells less permeable. Cholesterol-grown cells “leak” nucleosides and proteins less than ones grown without sterol (Sietsma and Haskins, 1968). Child et al. ( 1969) compared leakage of material from mycelium grown with and without cholesterol; after an initial loss attendant on transferring cholesterol-grown cells to fresh medium or water, there was no further leakage of material absorbing at 260 nm, of nitrogen or of protein, and no change in conductivity of the suspending fluid, whereas loss continued with cells grown without added cholesterol. Loss of carbohydrate however was greater with cholesterol-grown cells. Species of Phytophthora and Pythium grow faster in the presence of sterols than on medium without added sterols. This is true both for dry weight and hyphal extension. The amount of increase in growth with sterols is not as great as with lecithin, but the growth rate with lecithin and sterol is greater than on medium supplemented with lecithin only (Hohl, 1975). With Pythium acanthium, addition of cholesterol to the medium shortens the lag phase of growth or accelerates growth in the early exponential phase; the total mycelial mass grown with or without cholesterol is the same (Child et al., 1969), or eventually the mycelial mass without cholesterol may exceed that grown with cholesterol (Brushaber et al., 1972). In several other Pythium species and in Phytophthora cactorum, cholesterol-grown cultures attained a greater maximum weight (Schlosser and Gottlieb, 1968; Elliott, 1972a). Sterols require to be added to the medium for asexual reproduction

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CHARLES G. ELLIOlT

by sporangia (Hendrix, 1965, 1967; Chee and Turner, 1965; Elliott, 1972a; Langcake, 1974). Their incorporation also increases the ability to survive at high and low temperatures (Haskins, 1965; Sietsma and Haskins, 1967). Sterols -increased the number of propagules produced by Pythium sylvaticum, but those grown without sterols survived in soil as well as those grown with sterols (Kaosiri and Hendrix, 1972). Various sterols have different effects on vegetative growth and they affect aspects of growth differently (Langcake, 1974). Langcake’s results with P. inzstans are summarized in Table 2. The difference between cholesterol and stigmasterol is particularly intriguing. The effect of sitosterol is intermediate but generally much closer to that of TABLE 2. Ellects of several sterols on growth of Phytophthoru infeestuns (Langcake, 1974, 1975)

Stcrol

;Idclrd None

Cfiolrstrrol Cholrst;inol Si Iostrrol Stigniastrrol Ergosrcrol L;iii~~tc-r~l

Stcrol content ofmycelium (% ofdrv wt)

Dry weight of eight-day cultures (mg)

Colony diam. at 14 days (cm)

Sporangium production’

-

15.8 28.3 27.9 27.8 24.6

12.0 39.9 54.0 46.1 58.9 35.1 15.5

0.6 28.6 20.5 33.3 25.1 22.1 15.2

0.19 0.19 0.09 0.11

Trace -

-

6.0

~

A plug I cm in diameter was cut from the culture and shaken in 2 ml water. The number of sporangia liberated was counted in a 10 pl sample.

cholesterol. Cholesterol gives heavier cultures than stigmasterol (although the difference in weight is not actually significant) but, with stigmasterol, hyphal elongation is much faster (the difference being highly significant). Could a difference in their effect on the cell membrane account for these phenomena? The more permeable membrane might facilitate deposition of material at the hyphal tip, and so promote faster elongation; the less permeable might retain material within the cell and thereby increase its weight. Sterols appear to promote more active cellular metabolism. Schlosser and Gottlieb ( 1968) investigated glucose metabolism in Pythium ultimum grown with and without cholesterol. They found that glucose uptake was faster in cultures with cholesterol than without, but the difference was less than one would expect from the rate of dry

STEROLS IN FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

143

matter production. However, the cultures containing cholesterol produced 65%more carbon dioxide per unit of glucose consumed, indicating a much greater energy production in the sterol-containing mycelium. Pythium acanthium grown with cholesterol produced a greater quantity of lipid than when grown without it (Brushaber et al., 1972). Sietsma (1971) showed that, in Pythium spp. grown in the presence of cholesterol, most of the enzymes of the tricarboxylic-acid cycle, some aminotransferases and glutamate dehydrogenase showed higher activity than when the
144

CHARLES G. ELLIOlT

weight of tissue). The authors suggested that sterols occurring in high localized concentration in older roots changed the fungus from its aggressive pectolytic enzyme-producing phase to a less aggressive reproductive phase. Defago et al. (1975) showed that the relationship between the amount of cholesterol supplied to Pythium paroecandrum and its pathogenicity depended on other constituents of the host plant. Thus, with sugar beet (Beta vulgaris) and tomato (Lycopersiconesculentum), the severity of disease symptoms decreased with increasing sterol concentration, as the plants contained saponins to which sterol-containing mycelium is sensitive, whereas mycelium without sterol is not sensitive to saponins. Pea (Pisum sutivum) and soy bean (Glycine max) contain no saponins, and here disease severity increased with increasing sterol concentration. With lucerne (Medicago sativa), the severity of symptoms increased with sterol concentration despite the presence of saponins, as the plant contains tannic acid, and cholesterol in the mycelium imparts some resistance to tannic acid. Langcake (1974) tested the suggestion of Elliott and Knights (1969) that, in plant tissues containing a high ratio of sterol precursors (e.g. cycloartenol) to sterols, the precursors would interfere with growth and sporulation of Phytophthora and Pythium spp. and confer resistance on the host tissues. Langcake (1974) found, contrary to expectation, that leaves of the potato varieties Majestic and Ulster Chieftain, which were susceptible to blight (caused by Phytophthora infestand, contained more cycloartenol relative to sitosterol than the resistant Pentland Dell. He also found that extracts of Majestic and Pentland Dell leaves, added to medium at concentrations adjusted to give the same amounts of sitosterol, had a similar ability to promote growth of P . infeestans, despite the difference in their cycloartenol content. B. S A C C H A R O M Y C E S A N D O T H E R F U N G I

The study of sterols in Saccharomyces cerevisiae has recently been considerably advanced by the use of mutants with altered sterol composition, and by a physiological technique which similarly allows replacement of ergosterol by other sterols. Sterol-requiring yeast mutants were described by Karst and Lacroute ( 1973) and by Sprinson and his coworkers (Gollub et al., 1974; Trocha et al., 1974). These mutants are “petite”, and the sterol deficiency is apparently due to a defect in biosynthesis of haem (Bard et al., 1974). Haem is required in sterol syn-

STEROLS IN FUNGI: THEIR FUNCTIONS IN

GROWTH AND REPRODUCTION

145

thesis for oxygenation reactions concerned with removal of the C- 14 methyl group. The other kind of sterol mutants investigated are the polyeneresistant strains. Polyene antibiotics act by complexing with sterols in membranes (Lampen, 1966; Kinsky, 1967). They inhibit growth of fungal cells, but not in species of Phytophthora or Pythium grown without sterols-the antibiotics may even increase growth of Pythium (Defago et al., 1969). However, when these fungi are grown with sterol, they become sensitive to polyenes (Schlosser and Gottlieb, 1966; Van Etten and Gottlieb, 1967; Schlosser, 1972; Sietsma and Haskins, 1968; Child et al., 1969) and soluble material leaks out of the cells. Mutants of Neurospora crassa ( Grindle, 197 31 and Saccharomyces cereuisiue (Woods, 1971; Molzahn and Woods, 1972; Bard, 1972; Parks et al., 1972) resistant to polyene antibiotics contain different sterols from the wild-type sensitive strain, and it appears that each of four genetically different mutants of Sacch. cereuisiae has a different lesion in the biosynthetic pathway from zymosterol to ergosterol (Barton et al., 1974). The sterols of double mutants were as expected from the lesions observed in single mutants (Barton et al., 1975). Another mutant selected for nystatin resistance in the presence of cholesterol by Karst and Lacroute (1974) was found to be unable to convert squalene to lanosterol. Bard (1972) remarked that the sterols in his mutant strains must be effective in meeting the structural and functional requirements of the cell, but that the antibiotic has a greater chemical affinity for the sterols terminal in the biosynthetic pathways, i.e. ergosterol and cholesterol. Fryberg et al. (1974) adapted yeast to grow in concentrations of 30, 100, 200 and 300 units of nystatirdml, and they found that the strains which were resistant to the highest concentrations were blocked earliest in the synthetic pathway (and contained A8*24(28)ergostadienol (Figs. 2 and 3 VIIIF)) while those resistant to the lowest concentrations were blocked at the latest stage (and produced A’***ergostadienol WE), instead of ergosterol). Karst and Lacroute (1973) noted that a sterol-requiring mutant of yeast growing in ergosterol-containing medium was sensitive to nystatin at a concentration of 8 pg/ml but, when grown with cholesterol, sitosterol or stigmasterol, its sensitivity was less, 15 pg/ml being required for inhibition. Several people have investigated the ability of various sterols, when added to the medium together with the antibiotic, to reverse the anti-

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CHARLES G. ELLIOTT

fungal activity of the polyenes (Gottlieb et al., 1960; Perritt et al., 1960; Zygmunt and Tavormina, 1966; Lampen et al., 1960; Gale, 1974) and in some cases the complexing of sterol and polyene in the absence of fungus was studied.-But these investigations have not included a sufficient range of sterols to provide any clue as to why absence of ergosterol confers resistance to nystatin on the yeast mutants. The physiological technique of replacement of ergosterol by other sterols depends on the fact that, during anaerobic growth, yeast cells require an exogenous source of sterol and unsaturated fatty acid (Andreasen and Stier, 1953, 1954).This sterol requirement arises from the need for molecular oxygen in the formation of 3-oxidosqualene, the first step in the cyclization of squalene to sterol. Proudlock et al. ( 1968) found a relatively “all or none” effect in the ability of sterols of various structures to support anaerobic growth. A number Of 5 a or A5 sterols (including, remarkably, 5a-cholestan-3a-ol) supported growth equally well. Hossack and Rose (1976) supplied anaerobic cells with a sterol other than ergosterol, and achieved considerable enrichment of the cells with the foreign sterol. The effects of this modification on the ability of protoplasts to stretch was investigated. The main finding was that protoplasts containing sterols with a double bond in the side chain (ergosterol, stigmasterol) are considerably more stable and burst less readily than those with a saturated side chain (cholesterol, 7-dehydrocholesterol, A’-ergostenol, campesterol or sitosterol). The presence of Azz sterols in the membranes thus endows them with a limited capacity to stretch (Hossack and Rose, 1976). As already noted, ergosterol and stigmasterol decrease the packing of molecules in a film less than sterols with a saturated side chain (Demel et al., 1972a). I t is obvious that species of Phytophthora and Pythium could provide much precise information on the effect of sterols on membrane structure and permeability, since the sterol content can be so directly controlled. I am not aware of such experiments having been reported, although protoplasts of Pythium sp. were prepared by Sietsma et al. (1968, 1969). The amount of sterol supplied to yeast cultures during anaerobic growth affects the performance of the cells when they are subsequently aerated. Cultures grown anaerobically with high concentrations of ergosterol in the medium adapt to aerobic growth more rapidly than,those with only low concentrations-the activities of succinate dehydrogenase and cytochrome oxidase increase linearly with

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time (and not by an S-shaped curve) in the presence of high concentrations of ergosterol (Hebb and Slebodnik, 1958). The effect of ergosterol on the ultrastructure of Saccharoqces cerevisiue was described by Wallace et al. (1968). Adding ergosterol to aerobically-grown cultures produced no visible effect. Cultures incubated anaerobically without sterol or unsaturated fatty acid showed only vague outlines of mitochondria. With oleic or palmitoleic acids or Tween 80, but no sterol, extensive membranous structures appeared in the cells, but not normal mitochondria. However, with Tween 80 and ergosterol, mitochondria profiles were much more clearly defined, and extra membranes were not found. It seems that such mitochondria would be ready to assemble and use the respiratory enzymes immediately they were formed, With the obligate aerobe, Candida parapsilosis, addition of ergosterol and unsaturated fatty acid to the medium during growth under low aeration greatly enhanced the clarity of the mitochondrial profiles without increasing growth rate (Kellermanet al., 1969). The conclusion here seems to be that ergosterol is fulfilling a structural role in mitochondrial membranes. This was also the conclusion of Thompson and Parks (1972) from their investigation of cytochrome oxidase. Associated with an 80-fold purified preparation of the enzyme, and necessary for its activity, was a lipid component which included ergosterol. However, ergosterol itself, when added to the enzyme, did not promote its activity, but unsaturated fatty acids did. Notable use of nystatin-resistant mutants has been made by Thompson and Parks ( 1974) to study physiological activity in relation to sterol structure. Wild-type cells contained ergosterol ; one mutant two others contained As*nncontained A5*7~22J4(28)-erg~~tatetren~l; ergostadienol. When grown fermentatively in glucose-containing media, all strains had similar temperature optima but, when required to respire ethanol, the strains with A8J2-ergostadienol had lower temperature optima, and lower upper limits for growth, than those containing the A5*’-sterols.Differences were also found in the activity of sterol methyl transferase in mitochondria, attendant on the alteration in sterol structure from A5n7to As. The As-sterol appears to be less effective as a repressor of the enzyme than ergosterol. The effects of inhibitors of sterol synthesis are discussed in Section X,p. 162 in connection with sexual reproduction; but here we will note that certain fungicides are inhibitors of sterol synthesis (triarimol,

148

CHARLES G. E L L I O l l

Ragsdale and Sisler, 1972; S-1358, Kato et al., 1974, 1975). Ragsdale (1975) showed that, in Ustilago maydis, triarimol inhibits removal of the 14-methyl group, inserkion of the double bond at C-22 and the reduction of the double bond at C-24(28). The morphological effects were that sporidia enlarged and became multicellular and multinucleate; no more sporidia were budded off from these cells. The controls without triarimol continued sporidial production, the cells remaining unbranched, unicellular and uninucleate (Ragsdale and Sisler, 1972, 1973). The related compound triforine also inhibits ergosterol synthesis in Neurospora c r a m (Sherald et al., 1973). These authors also found that certain mutants of Cladosporium cucumerinum and Ustilago maydis, selected for resistance to triforine, were resistant to triarimol, and its other analogues, ancymidol and EL-241. VI. Effects of Sterols on Asexual Reproduction A well nourished mycelium is a sine qua non for asexual reproduction, and it is perhaps because of this that sterols are required for sporangium formation in species of Phytophthora (see page 14 1).

However, induction of asexual reproduction in fungi presumably involves the switching on of genes which are repressed during earlier stages of growth. We know nothing of the means whereby this is brought about at the chromosomal level. Could a steroid hormone be involved ? In the imperfect ascomycete Stemphylium solani, ergosterol can replace the requirement for light for induction of conidiation (Sproston and Setlow, 1968). The ergosterol was dissolved in 5% dimethyl sulphoxide in 0.1-M KH,PO,; the solvent itself induced some conidium formation. Ergosterol at 0.5 pg/ml further enhanced conidium production, although concentrations above 0.8 ,ug/ml caused inhibition of conidium formation. Ethanol, propylene glycol and ethylene glycol also increased conidiation. Sproston and Setlow ( 1968) suggested that the similar inducing effect of ergosterol and of ultraviolet radiation and solvents could be due to the freeing of internally bound sterol by the latter agents, and this release of free sterol is the triggering mechanism for conidium formation. Baniecki and Bloss ( 1969) reported that ergosterol stimulated conidium production by Phymatotrichum omnivorum but, in this case, it did not substitute for the light requirement; both light and sterol were re-

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149

quired. I t seems that ergosterol itself is not required but that its vitamin D product is, since vitamins D, and D, and dihydrotachysterol were stimulatory to conidiation. VII. Sexual Hormones of Achlya I begin the discussion of the effect of sterols on sexual reproduction in fungi with the sex hormones of Achlya spp., for here the involvement of sterols, through steroid hormones, is clearly understood, thanks to the elegant work of Dr. Alma Barksdale and her associates. The control of reproduction in Achlya spp. by a series of hormones was demonstrated in the classic experiments of Raper ( 1939, 1940). He showed that a hormone, A, produced by female plants of A . bisexualis and A . ambisexualis induced formation of antheridial branches in the male plant. The male plant was then induced to produce a second hormone, B, which had the effect of stimulating production of oogonia by the female plant. Raper supposed there was a third hormone, C, produced by the oogonial initials, which attracted the antheridial branches; when the antheridial branches made contact with the oogonial initials, the antheridia were delimited, and a fourth hormone, D, then led to delimitation of the oogonia from the oogonial initials. Following the work of Raper and Haagen-Smit (1942), hormone A was isolated from culture filtrates and partly characterized by McMorris and Barksdale (1967) who named it antheridiol. Its structure was elucidated by Arsenault et al. (1968), and confirmed by synthesis (Edwards et al., 1969, 1972; Fig. 7, I and 11). 23-Deoxyantheridiol (Fig. 7, I and VI) was also found in culture filtrates of female plants (Green et al., 1971). Antheridiol is active towards species of Achlya and Thraustotheca, but it is not active towards Allomyces and Dictyuchus (A. W. Barksdale, personal communication). Antheridiol is taken up by both male and female plants of Achlya spp., but only male plants react to it (Barksdale and Lasure, 1973). The primary morphological effect of antheridiol on the male plant is to induce branching within the space of two hours or so. Other compounds can also induce branching, but it is only antheridiol-induced branches which can develop into antheridia. In media containing a high concentration of nitrogenous nutrients, however, the antheridiol-induced branches may become vegetative (Barksdale, 1970). The branches are attracted to polystyrene balls soaked in antheridiol, and antheridia are delimited by the hyphae in

150

* +$

CHARLES

G. ELLIOlT

I

* yl-$

0

0

I1

111

V 0

0

IV

VI

CH,OH

(CHJ,CHCOO VI I FIG. 7 . Formulae of antheridiol and its isomers. I, ring system; 11, the 22s 2SR side chain of antheridiol; 111, the 22s 23s side chain; IV, the 22R 23s side chain; V, the 22R 23R side chain; VI, the side chain of 23-deoxyantheridiol. VII, structure of oogoniol- 1 .

STEROLS IN FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

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contact with the balls; thus it appears that the functions of hormone C, postulated by Raper, are fulfilled by antheridiol (Barksdale, 1963). While branching is the first visible result of antheridiol treatment of a male plant, this is of course the consequence of a number of processes at a molecular level. Kane et al. (1973) and Horowitz and Russell ( 1974) showed that the antheridiol-induced branching is inhibited by actinomycin D, and thus depends on RNA synthesis. Silver and Horgen ( 1974) reported that antheridiol treatment induced accumulation over an 8-h period of a species of RNA rich in adenylic acid, presumably messenger-RNA; addition of actinomycin D or cordycepin inhibited its accumulation. Cordycepin (3’-deoxyadenosine) inhibits the addition of polyadenylic acid to RNA and conversion of heterogenous nuclear-RNA to messenger-RNA (Darnel et al., 197 1). Thomas and Mullins (1967, 1969) found that the level of cellulase in cells rose following antheridiol treatment, reaching a maximum in two hours. Cellulase production, as well as branching, was inhibited by puromycin and p-fluorophenylalanine. Mullins and Ellis ( 1974) reported that antheridiol treatment brought about accumulation of vesicles at the points where branches were formed; such vesicles contain cellulase (Nolan and Bal, 1974). Thus antheridiol has similar properties to the more extensively studied steroid hormones of mammals and insects, in that it stimulates production of RNA and protein and induces a series of specific morphogenetic events. The principal sterols of Achlya bisexualis are fucosterol, 24methylenecholesterol, 7 -dehydrofucosterol and cholesterol ( Popplestone and Unrau, 1973). 7-Dehydrofucosterol and fucosterol can be converted to antheridiol by the fungus (Popplestone and Unrau, 1975). Antheridiol and its C-22 and C-23 isomers have been synthesized and it was shown that the configuration of the side chain profoundly affects the activity of the compound (Barksdale et al., 1974). The 22s 23R compound (antheridiol; Fig. 7, I and 11) is active at a concentration of 6 pg/ml, but the 22R 23s ( I and IV) and 22s 23s ( I and 111) compounds have less than one thousandth of the activity of antheridiol (active at 20 ng/ml) and the 22R 23R (I and V) compound has even less activity (active at 400 ng/ml). 7 -Deoxy- 7 -dihydro-antheridiol is active at 0.11 ng/mg. Replacement of the 22-hydroxyl group of 7-deoxy-7dihydro-antheridiol acetate by an 0x0 group decreases its activity to about one-fiftieth (Barksdale et al., 1974). 23-Dehydro-antheriodiol

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(Fig. 7, I and VI) (found in culture solutions) has no activity at 28 ng/ml (Green et al., 197 1). Various male and hermaphroditic strains of Achlya spp. (and one weak female strain) have been found to convert antheridiol to a more polar compound, metabolite A, and in some strains to a second metabolite B. Production of metabolite A is inhibited by actinomycin D and cycloheximide. Evidently an enzyme is produced which destroys the hormone. Neither metabolite is identical with hormone B (Musgrave and Nieuwenhuis, 1975). Hormone B is produced by male strains of A . ambisexualis when stimulated by antheridiol, and it induces formation of oogonia in the female plant. Male plants respond to antheridiol, female plants to hormone B. Hermaphroditic strains of A . heterosexualis produce hormone B without the necessity for stimulation by antheridiol. Such strains also react to both antheridiol and hormone B, which is the basis of their hermaphroditic behaviour (Barksdale and Lasure, 1973). However, it was found that treatment of strain 8-6 of A . heterosexualis with antheridiol greatly stimulates its production of hormone B. 7-Deoxy7 -dihydro-antheridiol also stimulates production of hormone B, but the 22R 23s isomer of antheridiol, and fucosterol, are inactive (Barksdale and Lasure, 1974). The structure of three compounds having hormone B activity, named oogoniol-1, -2 and -3, has recently been described by McMorris et al. (1975). They are C,, steroids, but differ from antheridiol in not having a lactone ring in the side chain, and in being esterified at C-3 (Fig. 7, VII). While hormones A and B control the formation of the male and female organs-antheridia and oogonia-respectively, their conjugation is controlled by a third hormone, the delimiting hormone. A. W. Barksdale (personal communication) believes this hormone D is produced by antheridial branches and acts on both antheridia and oogonia. VIII. Effects of Sterols on Sexual Reproduction in Homothallic Species of Pythiwn and Phytophthoru

The most striking effects of adding sterols to cultures of Pythium and Phytophthora are the ensuing morphogenetic changes. In Phytophthoru cactorum, the outer parts of the colony first become much more extensively branched. Gametangia (oogonia and antheridia) are then produced on these branches and subsequently the oospores develop inside the oogonia.

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The structure of sterols in relation to their activity in effecting reproduction has been extensively studied (Haskins et al., 1964; Elliott et al., 1966; Elliott, 1972b).The results with P. cactorum (Elliott, 1972b, and unpublished work) are as follows. The most active are the C,, plant sterols, which have an ethyl group with the a-configuration at C-24 of the side chain (sitosterol; Figs. 2 and 3, IV G, p. 124) and stigmasterol (IV HI) or a 24-ethylidene group (fucosterol (IV J) and avenasterol). The C,, sterols have much lower activity, i.e. fewer oospores are produced with the same sterol concentration. The C,, sterols (24methyl) are intermediate in their effect. The configuration of the 24methyl group is important, the a configuration conferring greater activity than the p ; thus campesterol (IV C) is more active than A’-ergosterol (IV D), and stigmasterol (IV H) than poriferasterol (IV K)at all concentrations. A double bond in the B ring is essential; cholestanol (111A) is much less active than cholesterol (IV A), and ergostanol (I11D) than A5-ergostenol (IV D). However, A’-cholestenol (V A) and 7dehydrocholesterol (VI A) gave more oospores at high concentrations and fewer at low concentrations than cholesterol (IV A), and the same holds for A’-stigmastenol (V G) and 7-dehydrositosterol (VI G) as compared with sitosterol (IV G). The double bond at C-22 does not appear to have any significant effect as indicated by equal activity of A5-ergostenol (IV D) and brassicasterol (IV E), and of sitosterol (IV G ) and stigmasterol (IV H). Defago et al. (1969) reported that polyene antibiotics could induce oospore formation in Pythium acanthium in the absence of sterols. Hendrix and Gutman (1968, 1969) found oestradiol inhibited production of oospores by cholesterol or sitosterol in six strains of Pythium; there was no significant effect with two other pythia or with Phytophthora cactorum. In most, but not all, cases, oestradiol decreased vegetative growth (colony diameter), but reproduction was inhibited at lower concentrations of oestradiol than was growth rate. Inhibition of oospore formation by oestradiol could be reversed by increasing the concentration of cholesterol or sitosterol, but to a different extent in various fungi. The 17p-hydroxyl group is important in this interference; 17a-oestradiol and 17p-oestradiol-17-acetate are not inhibitory, although 17p-oestradiol-3-acetate is (Hendrix and Guttman, 1972). The sterol side chain occupies the 17s position. Experiments on the effect of C,,, C,, and C,, steroids on the permeability of liposomes were reported by Bangham et al. ( 1965), Sessa and Weissmann (1968) and Heap et al. (1970). The synthetic oestrogen,

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diethylstilbestrol, also inhibits oospore production in P. cactorum ( G .A. Bean and C. G. Elliott, unpublished results). The most notable point about the relative activities of various sterols in promoting oospore production in P. cactorum is that they bear little relationship to their effects on structural stabilization and permeability of lipid films and cell membranes. Thus, in the latter systems, AZ2sterols generally have different effects from those with saturated side chains (e.g. Demel et a/., 1972a, b; Rottem et a/., 1971; Hossack and Rose, 19761, and cholestanol (and sometimes sitosterol also) is generally equivalent to cholesterol. Thus it would seem that the effect on reproduction cannot be simply one on permeability of membranes. It may be, of course, that the composition of higher-plant membranes is such that the rules for the effects of sterols on simple lecithin films and animal membranes do not apply, but Grunwald’s (1968, 197 1) results suggest this is not so.Perhaps the degree of leakiness obtained with the C,, sterols is required for the deposition of the cellulose walls of higher plants and of the oomycetes. Phytophthora species too, being higher-plant pathogens, have access to the predominantly C,, sterols of higher plants. But the evident importance of the C-24 ethyl or methyl group and of its a configuration suggest that this structure has in itself a particular metabolic significance. Thus, it could be that C-29 is involved in the formation of a structure similar to the lactone ring in the side chain of antheridiol. Antheridiol biosynthesis involves oxidation of C-29 of fucosterol to form a carboxyl group. I t is therefore perhaps highly relevant to sexual reproduction that species of Pythium and Phytophthora metabolize cholesterol and sitosterol to more polar compounds (Hendrix et al., 1970; Elliott and Knights, 1974; Hendrix, 1975a, b) although Hendrix (1975b) could find no relationship between sterol metabolism and reproduction; the polar metabolite was formed both by species which produced oospores under the experimental conditions and by those which did not (i.e. heterothallic species in single culture). Polar metabolites are produced from cholesterol and sitosterol supplied to species of Pythium and Phytophthora but not to Achlya bisexualis or A. ambisexualis (Hendrix, 1975a). The metabolite described by Hendrix is slightly more polar than kryptogenin (Fig. 8; Hendrix, 1975a). Heftmann (1971) goes so far as to speak of all A5-sterols being “metabolized to the same hormone”.

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The low activity of cholestanol is particularly interesting as, although few oospores are formed, the number of oogonia produced is as high as with cholesterol, but most of them abort (Elliott, 1968, 1972b). Abortive oogonia are also produced with cholestanol by Pythium periplocum and P. prolatum (Hendrix and Guttrnann, 1972)and by Phytophthora dreschleri (G. A. Bean and C. G. Elliott, unpublished observations). It was found (Elliott, 1968)that cholestanol added with cholesterol lowered the number of oospores compared with that produced with cholesterol alone, with “kinetics” resembling competitive inhibition. But this was so only at high concentrations of sterol; at low concentrations, the substances acted synergistically. I considered that these results show that sterols have effects on sexual reproduction during at least two separate stages of development (gametangialformation and conjugation), and that the steric requirements for activity at these stages are different. Such an interpretation is compatible with a hormone-type action, but not with a simple permeability model.

HO FIG. 8. Structure of kryptogenin.

It will not be easy to prove that an endoge.noushormone is involved in regulation of reproduction in these homothallic species. In heterothallic species, there is a much better chance of identifying a hormone produced by one of the interacting strains. Indeed Kouyeas (1953) observed oospore formation in the heterothallic Phytophthora paracitica in one or both members of a mated pair when they were separated by a porous filter. Also, he reported oospore formation by one strain when it was grown in the culture filtrate of the other. However, similar experiments reported by Stamps (19551, Apple (1959)and Brasier (1972) gave negative results. In the Zygomycetes, the hormonal role of trisporic acid was apparent from work with heterothallic species, in which the two strains make characteristic contributions to synthesis of the compound ( Gooday, 197 4). Werkman and Van den Ende ( 197 4) investigated the effects of homothallic species on the precursors of tri-

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sporic acid, and concluded that the control of sexual development in homothallic species was not essentially different from that in heterothallic forms. IX. Reproduction in Heterothallic Species of Pythium and Phytophtlroru

Before describing reproduction in the Pythiaceae and the little that is known of its control, it would seem desirable to give some account of the system of “relative sexuality” as it exists in Achlya spp. An understanding of the morphological events at mating is essential for an investigation of their chemical control. The various isolates of Achlya ambisexualis and A . bisexualis can be placed in a single series as regards their interactions one with another, ranging from strong male on the one hand to strong female on the other. In an intermediate position are strains which can act as male or female depending upon whether they are paired with an isolate which lies further to the female or male side (Barksdale, 1960). The strains differ in their production and response to antheridiol and hormone B (Barksdale and Lasure, 1973). In pairings between a homothallic and a female heterothallic strain, the homothallic strain produces antheridial branches which interact with oogonia produced on the heterothallic strain. When male strains were paired with homothallic strains, it was not generally possible to say whether any of the oogonia were produced as a result of interstrain interaction, or whether they were all due to self stimulation by the homothallic strain; but in one particular combination, both mature and abortive oogonial initials were induced by the male (Barksdale, 1960). Mating in the heterothallic species Pythium sylvaticum was described by Papa et al. (1967). Isolate 1063-7 was always the male (antheridial) parent, and isolate 1063-8 the female (oogonial)parent. At the boundary between the two strains, the appearance of the male side was “smooth”, and the female “rough” due to penetration of male hyphae some distance into the female culture. Pratt and Green ( 197 1) showed that there were differences in the strength of the sexual response among a large number of isolates; they could be arranged in an approximate gradient. In any one mating, one strain generally behaved as male, one as female. Thus, the situation in Pythium sylvaticum resembles that in Achlya bisexualis; but sexuality in Phytophlhora is more complicated. Heterothallic species of Phytophthora can normally be assigned to one

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or other of two mating types or compatibility groups, namely A1 and A2 (Savage et al., 1968). Mating occurs between an A1 strain and an A2 strain, and not between two A l s or two A2s. Strains reacting with neither tester were isolated by Shepherd and Pratt ( 1973). Within each

of the mating types, differences in sexual potency occur. Galindo and Gallegly ( 1960) grew pairs of strains of P. infestans into opposite sides of a plain agar block and traced the origin of the antheridial and oogonial branches. They found that any one isolate behaved chiefly or entirely as male in some matings, as female in others, and in others the same isolate produced both antheridia and oogonia. In each mating type (A1 and A2), the strains could be arranged in a series, strong male (acting as male to most other strains) to strong female (female to other strains). I t is to be noted that Galindo and Gallegly’s results indicate that all oospores are the result of crossing between the two mating types. Savage et al. (1968) reported that either mating type could produce the antheridia and oogonia in the interspecific crosses P. infestans A1 x P. capsici A2 and P . capsici A1 x P. infestans A2 ; they implied that the phenomenon was a general one. Kouyeas (1953) arranged strains of P . parasitica in a series depending on the strength of their sexual reaction. Oogonium size was characteristic of some strains. Kouyeas (1953) used the distribution of oogonial sizes in crosses to determine the frequency with which each of the interacting strains functioned as female parent. Huguenin (1973) labelled one of the interacting strains of P. palmivora with the fluorescent dye, calcofluor white. According as the oogonium, the antheridium, both or neither were fluorescent, the origin of the antheridia and oogonia could be determined. Three A1 strains (26, K and 570) and two A2 strains (L and 36) were studied. When paired with strain L, 570 formed the oogonial parent in 7 1.5%of pairings, K formed the oogonia in 36.8% of pairings and 26 in 3.1%. When paired with strain 36, K formed the oogonia in 100%of pairings, 570 in 73.0% and 26 in 62.0% of pairings. These relationships are shown diagramatically in Fig. 9. It will be noted that the relative sexual expression of strains K and 570 differs according to the strain they are paired with. In addition, a substantial proportion of self fertilization was observed, particularly of the A2 strain when it carried the fluorochrome. Stamps ( 1953) reported that, in some matings between strains of P. cryptogea and P . cinnamoni, all oospores were hybrids, but in other matings selfing of the qptogea or the cinnamoni partner could occur as well.

CHARLES G. ELLIOlT

158

Brasier (1972)studied the interaction between a number of strains of P . palmivora. He scored the numbers of oospores formed on each side of the line where the two isolates met, and found significant variation between strains in their-mean score, both among the A1 mating-type strains and among the A2s. This indicated a gradient in sexual potency in both mating types, with, one might suppose, decreasing ability of strains to act as female parent, and increasing maleness, along the gradient. Brasier, however, thought that the result of the interstrain

9

A1

A2

1

L

6

FIG. 9. Relative sexuality of three A1 strains of Phytophthorapalmiuora when paired with two A2 strains. From Huguenin (1975).

interaction was to make each strain produce selfed oospores. He noted that some strains produced oospores in single culture much more readily than others I$ Stamps, 1953; Apple, 1959)and that the strains which produced the most oospores in paired matings were those which produced the most oospores in single culture. He also found that some strains of each mating type (again those which tended to produce oospores in single culture) produced oospores when grown in combination with the homothallic P. heveae; no interspecific hyphal pairing was detected. I t seems hardly possible to draw a general conclusion from these morphological observations on interstrain pairings. Is the effect of the strains on one another to stimulate one to act as male parent, the other as female in a strict interstrain relationship? Can one strain act as both male and female parent in a cross? To what extent can one strain induce the other to undergo self reproduction? It seems that different species (and different strains of one species) do different things. But genetical evidence should provide more definite evidence on the occurrence and extent of selfing. Sansome (1970)discussed the possibility of selfing to account for the peculiar segregation ratios observed for mating type and other

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characters in P. drechsleri by Galindo and Zentmyer (1967).However, one must ensure that the characters used to evaluate the extent of selfing are suitable. Mating type in P. drechsleri is unsuitable because its inheritance has unusual features, whereas drug resistance showed simple Mendelian inheritance in the same crosses (Khaki and Shaw, 1974). Khaki and Shaw (1974)crossed strains resistant to p-fluorophenylalanine or chloramphenicol with wild type; the resistant F, progeny were crossed with both parents, and F, progeny of different mating type were intercrossed. Segregation in crosses of the F, to the sensitive r+being the wild-type drug-sensitive allele parent [Rr+(A2)x r+~+(Al), and R the resistant mutant] would be sensitive to selfing, which would give rise to deviation from the expected 1 : 1 ratio or to heterogeneity TABLE 3. Segregation for drug resistance in Phytophlhora drechsleri, showing the results of backcrosses of F, (and for mutant C1 of two heterozygous F, cultures) to a sensitive parent. Data of Khaki and Shaw (1974).

Mutant FA 1

c1

C 1(F2) c 2

Total

Resistant

Semitive

68 60 25 18 43 214

51 48 19 25 32 181

between crosses. Khaki and Shaw's (1974)data (Table 3) are homogenous (& = 3.14,P 0.7-0.5)and the totals give ax;,, value of 2.75 (P 0.1-0.05)against the expected 1 : 1. However, there is a fairly consistent excess of resistant offspring. If a proportion p of the resistant parents undergo selfing, the expected proportions, 4 and 4, become 4 + @ and 4- &, and the data give a value for p of 16.7%. Such a hypothesis however is unnecessary. There is no indication of selfing of the heterozygous resistant F, (of the other mating type) in the backcrosses to the resistant parents, as these crosses gave no sensitive offspring. The conclusion then is that all oospores are of hybrid origin, but it would be much more certain if doubly marked strains were available. Khaki and Shaw (1974)collected oospores from the region where the two cultures met (D.S. Shaw, personal communication). It should be noted however that, when the wild-type strains of P. drechsleri (6500A1

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and 6503 A l l are mated by inoculating them on opposite sides of a petri dish, oospores are found not only where the two cultures meet but throughout much of the A1 culture. Oospores are not formed in either strain grown by -itself under otherwise similar conditions. The effect of sterols on reproduction in heterothallic species have received far too little attention. Child and Haskins (1971) found that addition of cholesterol and a number of other sterols to paired cultures of Pythium sylvaticum and P. catenulatum greatly stimulated oospore production; in the absence of sterols, only a weak mating reaction occurred. They noted that, in these matings, oospores were produced not only where the strains met but also within the area occupied by one of the strains. In a mating between P. sylvaticum 1063-7 and 1063-8, oospores were formed within 1063-7 which Papa et al. ( 1967 had shown functioned as male (antheridium producing) in this mating. Child and Haskins (1971)also found that, when P. sylvaticum 1063-7 was grown by itself with cholesterol in the medium, oospores were produced; this did not occur in the absence of cholesterol, nor did it occur when strain 1063-8 was grown with cholesterol. Similar results were found with single cultures of the P. catenulatum strains, the responsive strain again being that which had the morphology of the male strain. They supposed that sterol regulated promotion of oogonial structures. Thus, a tendency to femaleness was induced in male strains, but maleness was not induced in female strains. One might speculate that the increased sterol supply stimulated the female strain to produce more of a hormone (analogous to antheridiol) which induced the male strain to produce another hormone (like hormone B) to which both it and the female strain responded by producing oogonia. Cholesterol could also directly stimulate production of hormone by the male strain, leading to selfing. Pratt and Mitchell (1973)investigated the effect of cholesterol on matings of Pythium sylvaticum and Phytophthora capsici. The strains were inoculated singly into medium with or without cholesterol ( 10 mg/l), and allowed to grow into opposite sides of plain agar blocks between the two nutrient media. With the Pythium isolates, addition of cholesterol to the male culture only had little effect, but a great increase in the number of oospores maturing in the plan agar block was observed when cholesterol was added to the female cultures, and a still greater stimulation occurred when it was added to both. This result is compatible with Child and Haskins’s (1971)results if the effect of

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cholesterol is to stimulate the female strains to produce an antheridiollike hormone. With Phytophthora capsici, Pratt and Mitchell ( 1973)found that the number of oospores formed was greatly increased by adding cholesterol to the A1 strain, more still by adding it to the A2 strain, and most of all when added to both. They suggested that addition of cholesterol to only one strain led to increased oospore production because either strain might function as both male and female. Their interpretation was that the effect of cholesterol on oospore development relates to the amount present in the gametangial initials, supposing that a large amount occurred in the larger oogonium, a small amount only in the smaller antheridium. In conclusion, the results with Pythium species seem to be fairly easily interpreted in terms of a hormonal system similar to that in Achlya spp. In the heterothallic phytophthoras, there is a separation of strains into two mating types, within each of which there is a gradation from strong males to strong females. The mating-type system, which seems to be superimposed on the relative sexuality system, is in some ways similar to the mating-type system in the heterothallic ascomycetes such as Neurospora crmsa, but it is not such an effective barrier to self fertilization as obtains in N . crassa. Although much more work needs to be done, one can make the general hypothesis that the interaction of strains of different mating type results in crossing along the line where the two cultures meet, and the interstrain reaction may stimulate selfing in one or both of the partners further back from the junction. One can hardly doubt that such a system is hormonally controlled. An interesting point is that A2, but not Al, cultures of Phytophthoru spp. can be induced to form oospores by volatile metabolites of Trichoderma spp. (Brasier, 197 1, 1975; Pratt et al., 1972). More recently, it has been reported that oospores can be induced to form in A2 cultures (but not A l ) by treatment with the fungicide chloroneb (Noon and Hickman, 1974) and even simply by cutting the hyphae with a scalpel (Reeves and Jackson, 1974). It thus seems that a variety of agents, perhaps relatively non-specific, can overcome the barrier to self compatibility in A2 strains, whereas with A1 strains this is not so. Damage to A2 hyphae of a kind to induce branching is, maybe, the stimulus to oogonium formation. But stimulation of A1 strains is only possible by A2 strains. The natural interaction of strains must be specific. Further genetical evidence is needed to clarify the extent of crossing and selfing.

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X. Sterols and Sexual Reproduction in Ascomycetes and Basidiomycetes The clearest evidence for involvement of sterols in reproduction in ascomycetes is in the work of Nelson et ul. (1967) on Cochliobolus curbonum. Perithecia of this fungus are produced in culture by inoculating two compatible mating types on opposite sides of a strip of sterilized senescent maize leaf lying on the surface of Sach’s agar (Nelson, 197 1). When SKF 3301-A, a compound known to inhibit sterol synthesis in other organisms, was added four days after mating, perithecial development was inhibited by appropriate concentrations of the drug. Its addition seven days after mating impaired ascus development, and at 11 days ascospore formation was affected. At the same time, vegetative growth and conidium formation (normally sparse in this kind of experiment) were greatly increased. Chromatographic differences in the non-saponifiable extract of matings with and without SKF 3301-A were noted. The inhibition of development was prevented by adding squalene, sitosterol, ergosterol, cholesterol or cholestanol. According to Holmes and Di Tullio (19621, SKF 3301-A inhibits sterol synthesis at a stage between mevalonic acid and squalene. Mating is not successful if the two strains of Cochliobolus are paired on filter paper lying on Sach’s agar, but Fries and Nelson (1972) found that a chloroform-methanol extract of maize leaves would stimulate perithecial development if applied to the filter paper at any time prior to perithecial initiation; addition of a water extract of the leaves also further stimulated perithecial production. Sterols from the chloroform-methanol extract were effective, but not as effective as the crude extract; fatty acids from the saponified extract had no effect. When sterols were added to matings on filter paper, perithecia were produced, provided that zinc was also added (Nelson, 1971). The effects of inhibitors of sterol synthesis were investigated in Sorduriujmicolu (Elliott, 1969). In medium containing SKF 3301-A, there was an abrupt change in hyphal growth rate when the colony had covered about half the petri dish, and where this change occurred a ring of perithecia developed ; subsequently perithecia were produced in concentric rings. Except at the highest concentrations tested, perithecial production exceeded that in the drug-fiee controls. O n the other hand, a different compound, AY9944, inhibited perithecium formation at concentrations which had little effect on hyphal growth rate.

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This compound inhibits saturation of the double bond at C-7 in animal material (Chappel et al., 1964; Horlick, 1966) and in high concentration it also inhibits reduction of A'* (Lutsky et al., 1975). In the alga Chlorellu ellipsoidea it inhibits conversion of A* to A7 in the sterol molecule as well as reduction of A14 and insertion of Azz (Dickson and Patterson, 1972). However, it is not possible to say whether the effect on reproduction in Sordariufimicola is related to any effect on sterol synthesis; there was no evidence of annulment of the inhibition by cholesterol (Elliott, 19691, but it seems unlikely that the cholesterol added was sufficiently taken up by the fungus. Zearalenone (Fig. 101, a metabolite of Gibberella zeae (Fusariumroseum) which has oestrogenic properties in animals (see Mirocha and Christensen, 1974), was found to affect sexual reproduction in some 39 ascomycetes and also several species of Pythium and Phytophthoru (Nelson, 1971). Nelson (1971) studied particularly the effect on Cochliobolus curbonum, and found it to stimulate perithecial production at concentrations of 0.01 to 1.Opg per culture. It was most effective when applied just prior to the time the sexual process was initiated, and its stimulatory abilities were quantitatively affected by the time of application. Higher concentrations (10 pg or more per mating) decreased the number of perithecia formed. Concentrations of the

Go I

HO

\

HO

0& O H

0 I1

I11

FIG. 10. Structural formulae of oestrone (I) and alternative ways of presenting the structural formula of zearalenone (11 and 111).

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CHARLES G . ELLIOlT

compound which stimulated sexual development had no effect on vegetative growth. These points suggest that zearalenone is serving as a hormone-type regulato-r. Wolf and Mirocha ( 1973) showed that zearalenone affects reproduction in Gibberella zeae, the fungus which produces it, the exogenous compound augmenting the effect of that produced endogenously. Synthesis of zearalenone by G. zeae was inhibited by “Dichlorvos”, which also inhibits perithecial production; this effect was annulled by zearalenone (Wolf et al., 1972; Wolf and Mirocha, 1973). It seems highly significant that an oestrogen should have a hormone-like effect on reproduction. The sterols which affect reproduction in Cochliobolus carbonum could be metabolized to smaller molecules with hormonal effects, just as in animals. Various ascomycetes and Fungi Imperfecti have remarkable abilities to transform steroids (Charney and Hertzog, 1967). Such faculties would be meaningless if they were not required for the fungus’s own metabolism. My colleagues and I are approaching the question of involvement of sterols in reproduction by examining sterile mutants of Neurospora crassa (Elliott et al., 1974). We suppose that, if it were possible that sterility resulted from altered sterol metabolism, qualitative or quantitative differences in sterol content as compared with the fertile wild type might be detectable. Yanagishima ( 1969) reported that Saccharomyces cerevisiae secretes hormones which cause swelling of cells of opposite mating type. It was also reported that testosterone causes swelling of cells of the a but not the a mating type and that oestradiol causes swelling of a but not a (Yanagishima et ul., 1970). The yeast hormones were considered to be steroids (Takao et al., 1970), but the a hormone was subsequently identified as n-octanoic acid (Sakurari et al., 1974). On the other hand, a factor produced by a cells which causes elongation of a cells (Duntze et al., 1970) and which inhibits DNA synthesis in them (Throm and Duntze, 1970; Bucking-Throm et al., 1973) is a peptide of molecular weight of between one and two thousand daltons (Duntze et al., 1973). The sterols of Coprinus lagopus were studied by Defago et al. (1971). They found that monokaryotic and vegetative dikaryotic mycelium, whether grown in shaken or static culture, contained mostly A59’ergostadienol (50 to 70 times as much as of ergosterol), but that the fruiting bodies contained about equal quantities of these two

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sterols. In shake cultures, sterol synthesis continued for a longer period, and the sterol content reached a higher value per culture, than in static cultures. However, in shake culture, the amount of sterol per unit weight decreased between the third and ninth days and then rose; the primordia of the fruiting bodies appeared at about 15 days. In static culture, the sterol content per unit weight increased between two and six days, and fruiting bodies were then initiated; the sterol content of the mycelium then declined. In this study, the sterol was recovered only by one method, that is by extraction of the non-saponifiable material. Holtz and Schisler (197 1, 1972) did not detect any sterol in the vegetative mycelium of Agaricus bisporus, but free sterols (ergosterol, A5n7ergostadienol and A7-ergostenol) were found in fruit bodies. Sterols were, however, found in both vegetative mycelium and fruit bodies by Byrne and Brennan (1975). XI. Conclusions

Work on model membranes, and on the homone system of Achlya spp., have together provided a framework with which we can interpret the effects of sterols on growth and development in species of Pythium and Phytophthora. We have reached the stage where we can formulate hypotheses and test them. More work needs to be done with model systems on the C,, arid C,, sterols but, as far as the information goes, it is clear that stigmasterol lowers permeability less than cholesterol in such membranes. These sterols appear to have different effects on vegetative growth of Phytophthora in25tans (Langcake, 1974, 1975).With more information of this kind, we could see how their effects are correlated with the results with the models. In particular, we need information on the permeability of fungal membranes; work with yeast protoplasts shows the way in which this might be carried out. As regards sexual reproduction, the comparison of cholestanol and cholesterol in P. cactorum indicates that sterols have separate effects on reproduction at oogonium formation and at conjugation, which is readily comprehensible on the basis of a hormonal system like that in Achlya spp., but not if reproduction depended primarily on permeability of membranes. There is obviously a great deal to be clarified about reproduction in the heterothallic Pythium and Phytophthora; what we do know enables us to formulate a hypothesis on lines similar to those for Achlya.

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The role which sterols play in controlling reproduction in ascomycetes is not yet clear. The work with zearalenone suggests that any steroid hormone which might be involved is more likely to be a small molecule (CI9or C,,),not a true sterol. The importance of sterols as essential structural components of membranes, and their consequent importance in efficient metabolism, are well established in yeast, but their precise functions are still hardly understood. In the zygomycetes, control of reproduction by trisporic acid is well understood (Gooday, 1974). The precise function of sterols here is unknown. Bu'Lock and Osagie ( 1973) observed an increased ergosterol content in mated, as compared with single, cultures of Blukesleu tn'sporu, but this could be attributed merely to the autocatalytic stimulation of isopentenoid synthesis by trisporic acid. In the basidiomycetes, consideration of the problem of sterol function has hardly begun. XII. Acknowledgements It is a pleasure to acknowledge my indebtedness to my colleague Dr. Brian A. Knights for advice on many points of chemistry mentioned in this review, and also because it is his collaboration which has made possible the work on sterols with Phytophthoru and Neurosporu done in this Department. Much of this work has been supported by grants from the Science Research Council. I am also grateful to Professor A. H. Rose for his comments on the draft of this review. REFERENCES

Adams, B. G . and Parks, L. Adanis, B. G. and Parks, L. Adams, B. G . and Parks, L. Alais. J., Lablache-Conibier, 13, 2833.

W. W. W. A.,

(1967). Journal ofCell Physiology 70, 161. (1968). Journal $Lipid Research 9, 8 . (1969). Journal ofBacteriology 100, 370. Lacoste, L. and Vandewalle, B. (1974). Phytochernistry

Andreasen, A. A. and Stier, T. J. B. (1953).Journal ofCellular and Comparative Physiology 41, 23.

Andrrascn, A. A. and Stier, T. J. B. (1954).Journal ofCellular and Comparative Physiology 43, 271.

Applr. J . L. (1959). fh.vtopathology 49, 37. Arsrnault, G . P., Bieniann, K., Barksdale, A. W. and McMorris, T. C. (1968). Journal / h i Amrriran Chrinicnl Society 90, 5635. Ashburncr. M. (1970). Advances in Insect Physiology 7 , 1. Arallah. A. M . and Nicholas, H. J. (1974). Lipids 9, 613. Bailey R. B. and Parks, L. W. (1975). Journal of BacterioloRy 124, 606.

of

STEROLS IN FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

167

Bangham, A. D., Standish, M. M. and Weissmann, G.(1965).JournalofMolecular Biology 13, 253. Baniecki, J. F. and Bloss, H. E. (1969).Phytopathology 59, 680. Bard, M. (1972).Journal of Bacteriology 111, 649. Bard, M.,Woods, R. A. and Haslam, J. M. (1974).Biochemical and Biophysical Research Communications 56, 324. Barksdale, A. W. (1960).American Journal of Botany 47, 14. Barksdale, A. W. (1963).Mycologia 55, 627. Barksdale, A. W. (1970).Mycologia 62, 411. Barksdale, A. W. and Lasure, L. L. (1973).Bulletin ofthe Torrey Botanical Club 100, 199. Barksdale, A. W. and Lasure, L. L. (1974).Applied Microbiology 28, 544. Barksdale, A. W., McMorris, T. C., Seshadri, R., Arunachalam, T., Edwards, J. A., Sundeen, J. and Green, D. M. (1974).Journal of General Microbiology 82, 295. Barr, R. M. and Hemming, F. W. (1972).Biochemical Journal 126, 1193. Bartlett, K. and Mercer, E. I. (1974).Phytochemistry 13, 1115. Bartnicki-Garcia, S. (1973).Symposium of the Society for General Microbiology 23, 245. Barton, D. H. R., Corrie, J. E. T., Widdowson, D. A., Bard, M. and Woods, R. A. (1974).Journal of the Chemical Society, Perhin Transactions I, 1326. Barton, D. H. R., Gunatilaka, A. A. L., Jarman, T. R., Widdowson, D. A., Bard, M. and Woods, R. A. (1975).Journal ofthe Chemical Society, Perhin Transactions I, 8 8 . Bean, G. A. (1973).Advances in Lipid Research 11, 193. Bean, G. A., Patterson, G. W. and Motta, J. J. (1972).Comparative Biochemistry and Physiology 43B, 935. Brasier, C. M. (1971).Nature New Biology 231, 283. Brasier, C. M. (1972).Transactions of the British Mycological Society 58, 237. Brasier, C. M. (1975).New Phytologist 74, 183. Brockerhoff, H. (1974).Lipids 9, 645. Bruckdorfer, K. R., Demel, R. A,, d e Gier, J. and Van Deenen, L. L. M. (1969). Biochimica el Biophysica Acta 183, 334. Bruckdorfer, K. R., Edwards, P. A., and Green, C. (1968a).European Journal of Biochemistry 4, 506. Bruckdorfer, K. R., Graham, J. M. and Green, C. (196813).European J o u m l ofBiochemistry 4, 512. Brushaber, J. A., Child, J. J. and Haskins, R. H. (1972).CanadianJournal $Microbiology 18, 1059. Bucking-Throm, E., Duntze, W., Hartwell, L. H . and Manney, T. R. (1973).Experimenla1 Cell Research 7 6 , 99. Bu’Lock, J. D. and Osagie, A. U. (1973).Journal of General Microbiology 76, 77. Butler, K. W., Smith, I . C. P. and Schneider, H. (1970).Biochimica et Biophysica Acta 219, 5 14. Byrne, P. F. S. and Brennan, P. J. (1975).Journal $General Microbiology 89, 245. Chapman, D., Owens, N. F., Phillips, M. C. and Walker, D. A. (1969).Biochimica et Biophysica Acta 183, 458. Chappel, C., Dubuc, J., Dvornik, D., Givner, M., Humber, L., Kraml, M., Voith, K. and Gaudry, R. (1964).Nature, London 201, 497. Charney, W. and Herzog, H. L. (1967).“Microbial Transformations of Steroids: a Handbook”. Academic Press, New York. Chee, K. H. and Turner, N. A. (1965).New ZealandJoumlof Ap’culturalResearch 8, 104. Child, J. J., Dcfago, G. and Haskins, R. H. (1969).Canadian Journal ofMicrobiology 15, 599.

168

CHARLES G. ELLIOTT

Child, J . J . and Haskins, R. H. (1971).Canadian Journal $Botany 49, 329. Conner, R. L., Mallory, F. B., Landrey, J. R. and Iyengar, C. W. L. (1969). Journal o f Biologral Chemistry 244, 2325. Darke, A., Finer, E. G., Flook, A. G. and Phillips, M. C. (1972).Journal ofMolecular Biology 63, 265. Darnell, J . E., Philipson, L., Wall, R. and Adesnik, M. (1971).Science, New York 174, 507. Dklago, G., Child, J. J. and Haskins, R. H. (1969).Canadian Journal ofMicrobiology 15, 509. Dklago, G., Fazeli, A. and Schweizer, H. (1971).Zentralblatt f u r Bacteriologie, Pnra.si(rnkzrnde. Infeektionskrankheiten und Hygiene, Abteilung 2, 126, 1. Dkfago, G. and Kern, H. (1975).Phytopathologische Zeitschrii 84, 34. Dklago, G., Memmen, K. F. and Kern, H. (1975).Phytopathologische Zeitschrii 83, 167. De Gier, J., Mandersloot, J . G. and Van Deenen, L. L. M. (1968).Biochimica et Biophysica Actn 150, 666. De Kruyll; B., De Greef, W. J., Van Eyk, R. W. V., Demel, R. A. and Van Deenen, L. L. M. (1973).Biochimica el Biophysica Acta 298, 279. Dr Kruvll; B., Demel, R. A. and Van Deenen, L. L. M. (1972).Biochimica et Biophysica Actn 255, 33 1. Demel, R. A., Bruckforfer, K. R. and Van Deenen, L. L. M. (1972a).Biochimica et Bioph?.cirn Actn 255, 3 11. Denid. R. A., Bruckforfer, K. R. and Van Deenen, L. L. M. (1972b).Biochimica et Biophvrira Actn 255, 32 1. Demel, R. A., Kinsky, S. C., Kinsky, C. B. and Van Deenen, L. L. M. (1968).Biochimica el Biophy.sicn A d a 150, 655. Dernel, R. A.. Van Deenen, L. L. M. and Pethica, B. A. (1967).Biochimica et Biophysica Arla 135, 11. Dirkson, L. G. and Patterson, G. W. (1972).Lipids 7, 635. Dod, B. J . and Gray, B. M. (1968).Biochimica et Biophysica Acta 150, 397. Duntm, W., MacKav, V.and Manney, T. R. (1970).Science, New Yorek 168, 1472. Duntze, W., Stotzler, D., Bucking-Throm, E. and Kalbitzer, S. (1973).European Journal 01 Biochemi.ttry 35, 357. Dupkron, R., Brillard, M. and Dupkron, P. (1972). Comptes Rendus Hebdomadaire des Sbnnre.r de 1’Acnd;mie des Sciences, Paris 274D. 2321. Dupkron, R., Dupkron, P. and Thiersault, M. ( 197 I ) . Comptes Rendus Hebdomadaire des Sbnnrrs de lilcndbmie des Sciences, Paris 273D,580. Edward, D. G . ff. and Fitzgerald, W. A. (1951).Journal of General Microbiology 5, 576. Edwards, J . A., Mills, J. S. Sundeen, J. and Fried, J. H. (1969).Journal ofthe American Chrmiral Societ? 91, 1248. Edwards, J . A., Sundeen, J.. Salmond, W., Iwadare, T. and Fried, J. H. (1972). 7’e/mhrdrorr Letters, 79 1. Edwards, P. A. and Green, C. (1972).Federation o f European Biochemical Societies Letters 20, 97. Elliott, C. G. (1968).Journal of General Microbiology 51, 137. Elliott, C. G. (1969).Journal of General Microbiology 56, 331. Elliott, C. G. (1972a).Transactions of the British Mycological Society 58, 169. Elliott. C. G. (197%). Journal of General Microbiology 72, 321. Elliott, C. G.. Hendrie, M. R. and Knights, B. A. (1966). Journal $General Microbiology 42. 425.

STEROLS IN FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

169

Elliott, C. G., Hendrie, M. R., Knights, B. A. and Parker, W. (1964). Nature, London 203, 427.

Elliott, C. G . and Knights, B. A. (1969).Journal ofthe Science ofFood and Ap’culture 20,

406. Elliott, C . G. and Knights, B. A. (1974). Biochimica et Biophysica Acta 360, 78. Elliott, C. G., Knights, B. A. and Freeland, J. A. (1974). Biochimica et Biophysica Acta360, 339. Esposito, M. S . , Esposito, R. E., Arnaud, M. and Hahorson, H. 0. (1969). Journal of Burltriology 100, 180. Feinstein, M. B., Fernandez, S . M. and Sha’afi, R. I. (1975). Biochimica el Biophysica Acla 413, 354. Finean, J . B. (1953). Experientia 9, 17. Finkelstein, A. and Cass, A. (1967). Nature, London 216, 717. Fries, E. R. and Nelson, R. R. (1972). Canadian Journal of Microbiology 18, 199. Fryberg, M., Oehlschlager, A. C. and Unrau, A. M. (1974). Archives ofBiochem’stry and Biophysics 160, 83. Gain, R. E. (1972). Mycologia 64, 198. Gale, E. F. (1974). Journal of General Microbiology 80, 451. Galindo, A. J. and Gallegly, M. E. (1960). Phytopathology 50, 123. Galindo, A. J. and Zentmyer, G. A. (1967). Phytopathology 57, 1300. Gershfdd, N. L., Wormser, M. and Razin, S. (1974). Biochimica et Biophysica Acta 352, 371. Glover, J . and Morton, R. A. (1958). British Medical Bulletin 14, 226. Gollub, E. G., Trocha, P., Liu, P. K. and Sprinson, D. B. (1974). Biochemical and Biophysiral RPsearch Communications 56, 47 1. Gooday, G . W. (1974). Annual Review of Biochemistry 43, 35. Goodwin, T. W. (1973). In “Lipids and Biomembranes of Eukaryotic Microorganisms”, (J. A. Erwin, ed.), pp. 1-40. Academic Press, New York. Gottlieb, D., Carter, H. E., Wu, L. and Sloneker, J. H. (1960). Phytopathology 50, 594. Grant, J. K. (1969). Essays in Biochemistry 5, 1. Green, D. M., Edwards, J. A., Barksdale, A. W. and McMorris, T. C. (1971). Tetrahedron 27, 1199. Grindle, M. (1973). Molecular and General Genetics 120, 283. Grunwald, C. (1968). Plant Physiology 43, 484. Grunwald, C. (197 1). Plant Physiology 48, 653. Hallermeyer, G. and Neupert, W. (1974). Hoppe-Seyler’s Zeilschnift fur PhysiologiJche Chemie 355, 279. Harnish, W. N., Berg, L. A. and Lilly, V. G. (1964). Phytopathology 54, 895. Hartman, R. E. and Holmlund, C. E. (1962). Journal ofBacteriology 84, 1254. Haskins, R. H. (1965). Science, New York 150, 1615. Haskins, R. H., Tulloch, A. P. and Micetich, R. G. (1964). Canadian Journal of Mirrobiolom 10, 187. Heap, R. B.-,-Symonds, A. M. and Watkins, J. C. (1970). Biochimica et Biophysica Acta 218, 482. Hebb, C. R. and Slebodnik, J. (1958). Experimental Cell Research 14, 286. Heftmann, E. ( 1970). “Steroid Biochemistry”, Academic Press, New York. Heftmann, E. (1971). Lipids 6, 128. Hendrix, J. W. (1964). Science, New York 144, 1028. Hendrix. J. W. (1965). Phytopathology 55, 790. Hendrix, J. W. (1966). Mycologia 58, 307.

170

CHARLES G. ELLIOTT

Hendrix, J. W. (1967). Mycologia 59, 1107. Hendrix, J. W. (1970). Annual Review of Phytopathology 8, 111. Hendrix. J . W. (1975a). Canadian Journal of Microbiology 21, 735. Hendrix, J . W. (1975b). Mycologia 67. 663. Hendrix, J. W.. Bennett, R. D. and Heftmann, E. (1970). Microbios 5, 11. Hendrix, J . W. and Guttinan, S.-M. (1968). Science, New Yor& 161, 1252. Hcndrix, J . W. and Guttman, S.-M. (1969). Microbios 2, 137. Heridrix, J . W. and Guttman, S.-M. (1972). Phytopathology 62, 763. Henry, S. A. and Halvorsen, H . 0. (1973).Journal ofBacten’ology 114, 1158. Hoch, H. C. and Maxwell, D. P. (1974). Canadian Journal ofhficrobiology 20, 1029. Holmes, W. L. and Di Tullio, N. W. (1962). AmericanJournalofClinical Nutrition 10,310. Hohl, H. R. (1975). Phytopathologische Zeitschntt 84, 18. Holtz, R. B. a d Schisler, L. C. (1971). Lipids 6, 176. Holtz, R. B. a d Schisler, L. C. (1972). Lipids 7, 251. Horlick, L. ( 1966). Journal of Lipid Research 7, 116. Horowitz, D. K. and Russell, P. J. (1974). Canadian J o u d of Microbiology 20, 977. Hossack, J. A. and Rose, A. H. (1976).J o u m l of Bateriology 127,67. Hossack, J . A., Wheeler, G. E. and Rose, A. H. (1973). In “Yeast, Mould and Plant Protoplasts”, (J. R. Villaneuva, I. Garcia-Acha, S . Gascon and F. Uruburu, eds.), pp. 2 1 1-227. Academic Press, New York. Hsia, J . C. and Boggs, J. M. (1972). Biochimica et Biophysica Acta 266, 18. Huang, L. and Haug, A. (1974). Biochimica et Biophysica Acta 352, 361. Hugueniii, B. (1973). Cuhiers de l’O@ce de la Recherche ScientiFque et Technique Outre-mer, ririr Biologit- 20, 59. llling\h.orth, R. F., Rose, A. H. and Beckett, A. (1973).Journal oflaten’ology 113, 373. Jackson, L. L. and Frear, D. S. (1968). Phytochemistry 7, 651. Jensen, E. V. and De Sombre, E. R. (1972). Annual Review OfBichemistry 41, 203. Kane, B. E., Reiskind, J. B. and Mullins, J. T. (1973). Science, New York 180, 1192. Kaosiri, T. and Hendrix, J. W. (1972). C a n u d i a n J o u d ofBotmy 50, 891. Karst, F. and Lacroute, F. (1973). Biochemical and Biophysical Research Communications 52. 741.

Karst, F. and Lacroute, F. (1974). Biochemical and Biophysical Research Communicutions 59, 370.

Kato, T., Tanaka, S., Ueda, M. and Kawase, Y. (1974). Agricultural and Biological Chemislry 38, 23 7 7. Kato, T., Tanaka, S . , Ueda, M. and Kawase, Y. (1975). Agricultural and Biological Chemishy 39, 169. Kellerman, G. M., Biggs, D. R. and Linnane, A. W. (1969). J o u m l of Cell Biology 42, 378.

Khaki, 1. A. and Shaw, D. S. (1974). Genetical Research 23, 75. Kinsky, S . C. (1967). In “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.), vol. 1, pp. 122-14 1. Springer-Verlag, Berlin. Knights, B. A. (1970). Phytochemistry 9, 701. Knights, B. A. and Elliott, C. G. (1976). Biochim’ca et Biophysica Acta 441, 341. Kouyeas, V. (1953). Annales, Institut Phytoputhologique Benuhi 7 , 40. Kroes, J., Ostwald, R. and Keith, A. (1972). Biochimica et Biophysica Acta 274, 71. Ladbrooke, B. D., Williams, R. M. and Chapman, D. (1968). Biochim’caet Biophysica Acta 150, 333.

Lampen, J. 0. (1966). Symposium ofthe Society for General Micsobiology 16, 111. Lampen, J . 0.. Arnow, P. M. and Safferman, R. S. (1960).J o u m l ofBacteriology 80,200.

STEROLS IN FUNGI: THEIR FUNCTIONS IN G R O W H AND REPRODUCTION

171

Langcake, P. (1974). Transactions of the British Mycological Society 63, 573. Langcake, P. (1975). Transactions of the British Mycological Soceity 64, 55. Leal, J. A., Friend, J. and Holliday, P. (1964). Nature, London 203, 545. Leonian, L. H. and Lilly, V. G. (1937). American Joumul of Botany 24, 700. Lin, H.-L., Langenbach, R. J. and Knoche, H. W. (1972). Phytochemistry 11, 2319. Lin, H.-K. and Knoche, H. W. (1974). Phytochemistry 13, 1795. Lutsky, B. N., Hsiung, H. M. and Schroepfer, G. J. (1975). Lipids 10, 9 McCorkindale, N. J., Hutchinson, S. A., Pursey, B. A., Scott, W. T. and Wheeler, R. ( 1969). Phytochemistry 8, 86 1. McElhaney, R. N., d e Gier, J. and Van der Neut-Kok, E. C. M. (1973). Biochimica et Biofhysica Acta 298, 500. McMorris, T. C. and Barksdale, A. W. (1967). Nature, London 215, 320. McMorris, T. C., Seshadri, R., Weihe, G. R., Arsenault, G. P. and Barksdale, A. W. (1975). Journal ofthe American Chemical Society 97, 2544. Madyastha, P. B. and Parks, L. W. (1969). Biochimica et Biophysica Acta 187, 858. Mailer, C., Taylor, C. P. S., Schreier-Muccillo. S. and Smith, I. C. P. (1974). Archives of Biochemistry and Biophysics 163, 67 1. Mellano, H. M., Munnecke, D. E. and Sims, J. J. (1970). PhytoPathology 60, 943. Mirocha, C. J. and Christensen, C. M. (1974). Annual Review of Phytopathology 12, 303. Molzahn, S. W. and Woods, R. A. (1972). Journal of General Microbiology 72, 339. Moor, H. (1967). Archiv f u r Mihrobiologie 57, 135. Mullins, J. T. and Ellis, E. A. (1974). Proceedings ofthe National Academy ofSciences ofthe United States of America 71, 1347. Musgrave, A. and Nieuwenhuis, D. (1975). Archives of Microbiology 105, 3 13. Nelson, R. R. (197 1). I n “Morphological and Biochemical Events in Plant-Parasite Interaction”, (S.Akai and S. Ouchi, eds.), pp. 18 1-205. Phytopathological Society of Japan, Tokyo. Nelson, R. R., Huisingh, D. and Webster, R. K. (1967). Phytopathology 57, 1081. Nes, W. R. (1974). Lipids 9, 596. Nolan, R. A. and Bal, A. K. (1974). Joumul ofBacteriology 117, 840. Noon, J. P. and Hickman, C. J. (1974). Canadian Journal ofBotany 52, 1591. Norman, C. and Calderone, R. A. (1971). Phytopathology 61, 904. Nowak, R., Kim, W. K. and Rohringer, R. (1972). Canadian Journal ofBotany 50, 185. Nozawa, Y.,Fukushima, H. and Iida, H. (1975). Biochimica et Biophysica Acta 406, 248. Oldfield, E. and Chapman, D. ( 1972). Federation of European Biochemical Societies Letters 23, 285.

O’Malley, B. W. and Means, A. R. (1974). Science, New York 183, 610. Papa, K. E., Campbell, W. A. and Hendrix, F. F. (1967). Mycologia 59, 589. Parks, L. W., Anding, C. and Ourisson, G. (1974). European Joumul ofBiochemistry 43, 451.

Parks, L. W., Bond, F. T., Thompson, E. D. and Starr, P. R. (1972). Journal ofLipid Research 13, 3 11. Perritt, A. M., Phillips, A. W. and Robinson, T. (1960). Biochemical and Biophysical Research Communications 2, 432. Pillai, C. G . P. and Weete, J. D. (1975). Phytochemistry 14, 2347. Popplestone, C. R. and Unrau, A. M. (1973). Phytochemistry 12, 1131. Popplestone, C. R. and Unrau, A. M. (1975). Canadian Journal of Chemistry 52, 462. Pratt, B. H., Sedgley, J. H., Heather, W. A. and Shepherd, C. J. (1972). Australian Journu1 of Biological Sciences 25, 861. Pratt, R. G. and Green, R. J. (1971). Canadian Journal ofBotany 49, 273.

172

CHARLES G. ELLIOTT

Pratt, R. G. and Mitchell, J. E. (1973). Canadian Journal ofBotany 51, 595. Proudlock, J. W., Wheeldon, L. W., Jollow, D. J. and Linnane, A. W. (1968). Biochim’ca el Biophysica Acta 152, 434. Rand, R. P., Pangborn, W. A., Purdon, A. D. andTinker, D. 0. (1975). CanadianJournal of Biochemistry 53, 189.Raper, J. R. (1939). American Journal of Botany 26, 639. Raper, J. R. (1940). American Journal of Botany 27, 162. Raper, J. R. and Haagen-Smit, A. J. (1942). Journal ofBiologica1 C h i s t r y 143, 311. Ragsdale, N. N. (1975). Biochimica et Biophysica Acta 380, 81. Ragsdale, N. N. and Sisler, H. D. (1972). Biochemical and Biophysical Research Communications 46, 2048. Ragsdale, N. N. and Sisler, H. D. (1973). Pesticide Biochemistry and Physiology 3, 20. Rattray, J. M. B., Schibeci, A. and Kidby, D. K. (1975). Bacteriological Reviews 39, 197. Razin, S. (1973). Advances in Microbial Physiology 10, 1. Razin, S. and Cleverdon, R. C. (1965). J o u m l of General Microbiology 41, 409. Reeves, R. C. and Jackson, R. M. (1974). Journal of General Microbiology 84, 303. Richards, J. B. and Hemming, F. W. (1972). Biochemical Journal 128, 1345. Riggs, T. R. (1970). In “Biochemical Actions of Hormones”, (G. Litwack, ed.), vol. 1, pp. 157-208. Academic Press, New York. Rothblat, G. H., Hartzell, R., Mialhe, H. and Kritchevsky, D. (1967). In “Lipid Metabolism in Tissue Culture Cells”, (G. H. Rothblat and D. Kritchevsky, eds.), pp. 129-149. Wistar Institute, Philadelphia. Rothman, J. E. and Engelman, D. M. (1972). Nature New Biology 237, 42. Rottem, S., Pfendt, E. A. and Hayflick, L. (1971). Journal of Bacteriology 105, 323. Rottem, S., Yashouv, J., Ne’eman, Z. and k i n , S. (1973). Biochim’ca et Biophysica Acta 323, 495.

Safe, S. (1973). Biochimica et Biophysica Acta 326, 471. Safe, S. and Caldwell, J. (1975). Canadian J o u m l of Microbiology 21, 79. Sakurari, A., Tamura, S., Yanagishima, N., Shimoda, C., Hagiya, M. and Takao, N. (1974). Ap‘cultural and Biological Chemistry 38, 231. Sansome, E. (1970). Transactions of the British Mycological Society 54, 101. Savage, E. J., Clayton, C. W., Hunter, J. H., Brennerman, J. A., Laviola, C. and Gallegly, M. E. (1968). Phytopathology 58, 1004. Schlosser, E. (1972). Phytopathologische ZeitschriJ 74, 9 1. Schlosser, E. and Gottlieb, D. (1966). Journal ofBacteriology 91, 1080. Schlosser, E. and Gottlieb, D. (1968). Archiufir Mikrobiologie 61, 246. Schlosser, E., Shaw, P. D. and Gottlieb, D. (1969). Archivfir Mikrobiologie 66, 147. Sessa, G. and Weissmann, G. ( 1968). Biochimica et Biophysica Acta 150, 173. Shepherd, C. J. and Pratt, B. H. (1973). Australian J o u m l of Biological Sciences 26, 1095. Sherald, J. L., Ragsdale, N. N. and Sisler, H. D. (1973). Pesticide Science 4, 719. Siekevitz, P. (1970). New England Journal of Medicine 283, 1034. Sietsma, J. H. (1971). Biochimica et Biophysica Acta 244, 178. Sietsma, J. H . and Haskins, R. H. (1967). Canadian Journal ofMicrobiology 13, 361. Sietsma, J . H. and Haskins, R. H. (1961). Canadian Journal ofBiochemistry 46, 813. Sietsma, J. H., Eveleigh, D. E. and Haskins, R. H. (1968). Antonie van Leeuwenhoek 34, 331.

Sietsma, J. H., Eveleigh, D. E. and Haskins, R. H. (1969). Biochimica et Biophysica Acta 184, 306.

Silver, J. C. and Horgen, P. A. (1964). Nature, London 249, 252. Smith, P. F. (1963). Journal of General Microbiology 32, 307.

STEROLS I N FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

173

Smith, P. F. (1964). Journal ofLipid Research 5, 121. Smith, P. F. (1969). Lipids 4, 331. Smith, P. F. and Lynn, R. J. (1958). Journal ofBmteriology 76, 264. Smith, P. F. and Rothblat, G. H. (1960). Journal of Bmteriology 80, 842. Sproston, T. and Setlow, R. B. (1968). Mycologia 60, 104. Stamps, D. J. (1953). Transactions of the British Mycological Society 36, 255. Starr, P. R. and Parks, L. W. (1962). Journal of Bacteriology 83, 1042. Takao, N., Shimoda, C. and Yanagishima, N. (1970). Development, Growth and Dttferentiation 12, 199. Tepperman, J. and Tepperman, H. M. (1960): Pharmucological Reviews 12, 301. Thomas, D. des S. and Mullins, J. T. (1967). Science, New York 156, 84. Thomas, D. des S. and Mullins, J. T. (1969). Physiologia Plantarum 22, 347. Thompson, E. D., Knights, B. A. and Parks, L. W. (1973). Biochimica et Biophysica Acta 504, 132.

Thompson, E. D. and Parks, L. W. (1972). Biochimica et Biophysica Acta 260, 601. Thompson, E. D. and Parks, L. W. (1974). Journul ofBacteriology 120, 779. Throm, E. and Duntze, W. (1970). Journal ofBacteriology 104, 1388. Trocha, P. J., Jasne, S. J. and Sprinson, D. B. (1974). Biochemicaland Biophysical Research Communications 59, 666. Tsuda, S. and Tatum, E. L. (1961).Journal ofBiophysical and Biochemical Cytology 11, 17 1. Tully, J. G. and Razin, S. (1969). Journal ofBmteriology 98, 970. Tully, J. G . and Razin, S. (1970). J o u m l $Bacteriology 103, 751. Vandenheuvel, F. A. (1963). Journal ofthe American Oil Chemists Society 40, 455. Van Etten, J. L. and Gottlieb, D. (1965). Journal ofBacteriology 89, 409. Van Etten, J. L. and Gottlieb, D. (1967). Journal of General Microbiology 46, 377. Verkleij, A. J., Ververgaert, P. H. J. Th., de Kruyff, B. and Van Deenen, L. L. M. (1974). Biochimica et Biophysica Acta 373, 495. Wallace, P. G., Huang, M. and Linnane, A. W. (1968).Journal of Cell Biology 37, 207. Weete, J. D. (1973). Phytochemistry 12, 1843. Weete, J. D. (1974). “Fungal Lipid Biochemistry”. Plenum Press, London and New York. Werkman, B. A. and Van den Ende, H. (1974).Journal $General Microbiology 82, 273. Wiley, J. S. and Cooper, R. A. (1975). Biochimica et Biophysica Acta 413, 425. Wolf, J. C., Lieberman, J. R. and Mirocha, C. J. (1972). Phytopathology 62, 937. Wolf, J. C. and Mirocha, C. J. (1973). Canadian Journal of Microbiology 19, 725. N o d s , R. A. (1971). Journal ofBacteriology 108, 69. Yanagishima, N. (1969). Planta 87, 110. Yanagishima, N., Shimoda, C., Takahashi, T. and Takao, N. (1970). Development, Growth and Differentiation 11, 217. Zygmunt, W. A. and Tavormina, P. A. (1966). Applied Microbiology 14, 865.