THE MORPHOGENESIS AND POSSIBLE EVOLUTIONARY ORIGINS OF FUNGAL SCLEROTIA

BY H. J. WILLETTS School of Botany, University of New South Wales,

Australia, 2033

(Received 6 July 1972)

CONTENTS

I. Introduction . . . . 515 11. Types of development of sclerotia . . 5x7

(I) Loose type . . . . 517 (2) Terminal type . . 517 (3) Lateral type. . . 518

(a) Simple . . . 518 (b) Strand . . . 518

111. Factors and processes involved in sclerotial development (I) Initiation . . .

(a) Staling substances . (b) Morphogenetic factors . (c ) The possible role of sulphur bonds (d) Polyphenols and polyphenol oxidases (e) Possible biochemical control .

(2) Developmental stage . (a) Translocation of nutrients . (b) Exudation . (c ) Interweaving of hyphae .

.

.

(3) Maturation . .

* 519 519

. 520

. 520

. 521

. 522

. 523

. 524 * 524 * 525 . 526 * 527

IV. Evolutionary origins of sclerotia . . 528 ( I ) Typhulu spp. . . . 528 (2) Sclerotinia spp. . . . 529 (3) Other relevant examples . . 530

V. Conclusions. . . * * 531 VI. Summary . . . . . . . . 531

VII. References . * 533

I. INTRODUCTION

Several recent general reviews have been written on the morphogenesis of fungi (Turian, 1966; Taber, 1966; Butler, 1966; Smith & Galbraith, 1971) and there are others that have considered limited aspects of the subject such as the hyphal cell wall (Bartnicki-Garcia, I 968), the mechanism of cellular extension and branching (Robertson, 19654 b; Park & Robinson, 1966) and the extent to which development can be causally linked with the physiology and biochemistry of the cell (Morton, 1967). These reviews indicate that our knowledge of the genetical, physiological, biochemical and, to a lesser extent, morphological aspects of the morphogenesis of

H. J. WILLETTS fungi is limited. Thus, at the present time it is possible only to speculate on many of the processes involved in morphogenesis although the information becoming available in this field indicates that a better understanding should be obtained in the near future.

In some fungi, hyphae become interwoven to form small hyphal aggregates which have the ability to resist adverse conditions for longer periods than the ordinary hyphae of the mycelium. There may be a further increase in the number of hyphae in the aggregate, coalescence may take place between adjacent hyphal knots and so a young sclerotium develops. In section, different regions can often be seen and it appears that growth has been co-ordinated to some degree to produce the sclerotium. The term ‘sclerotium’ in this context has been used broadly and includes many diverse fungal bodies. It is sometimes used in a more restricted manner. De Bary (1887) and Lohwag (1941) used the term for resting vegetative bodies that produce external reproductive structures and forms that are obviously related. This definition excludes structures such as microsclerotia and bulbils which these workers referred to as sclerotioid structures. For convenience, I will use the term ‘sclerotium’ for fungal structures which, although they show considerable differences in development and anatomy, are alike functionally in that they are able to survive periods of adverse conditions that are too severe for ordinary vegetative mycelium. Thus, they serve as persistent resting stages which, when conditions are suitable, reproduce the fungus by means of mycelial growth and/or by the production of sexual or asexual spores. Many fungal plant pathogens form sclerotia and their success as pathogens is partly due to the ability of the sclerotium to survive severe conditions and then to reproduce and multiply. Reviews on the survival of sclerotia under adverse environmental conditions have been written by Coley-Smith & Cooke (1971) and Willetts (1971).

Garrett (1970) pointed out that the sclerotia of root-infecting fungi are usually small, subspherical and regular in shape and act as infective propagules directly. Infection is by hyphae or hyphal strands. Examples which were given by Garrett of this group are Sclerotium rolfsii, Verticillium dahliae, Phymatotrichum omnivorum and S. cepivorum. The sclerotia of airborne pathogens are variable in size and shape and they produce large numbers of infective propagules in or on fruit bodies. Examples of this group are Sclerotinia spp., Claviceps and Typhula spp,

An understanding of the factors responsible for the initiation of sclerotia may be of considerable economic importance as it could lead to a method of repressing sclerotial formation on infected crop plants and hence would reduce the chances of survival of the fungus.

The ways in which hyphae become interwoven to form sclerotia and complex fungal structures such as the fruiting bodies of Ascomycetes and Basidiomycetes are, presumably, similar. The sclerotium is a more convenient structure for morphogenetic studies than are ascocarps and basidiocarps. It is of significance in this respect that some workers consider that sclerotia are abortive fruiting bodies and this has been discussed in more detail in the text. Thus, data obtained on the morphogenesis of sclerotia are of direct relevance to developmental studies of other fungal structures. Obviously our understanding of sclerotial development depends greatly on our

The mmphogenesis and possible evolutionary m&im of fungal sclerotia 5 I 7 knowledge of the growth and differentiation of the hypha, which is the structural unit of the sclerotium and all other fungal structures.

In this paper I have attempted to review our limited understanding of the processes involved in sclerotial morphogenesis, to discuss the possible evolutionary origins of sclerotia and to point out areas where further work is desirable.

11. TYPES OF DEVELOPMENT OF SCLEROTIA

Interest was shown in the development of sclerotia by some of the early workers, particularly Brefeld (1877) and de Bary (1887), but the difficulties of studying any but the early stages discouraged further work in this field until comparatively recent years. Soon after initiation the hyphae of the sclerotial primordium become intricately interwoven and the periphery usually becomes darkly pigmented, obscuring further development. Townsend & Willetts (1954) attempted to overcome these difficulties by studying the formation of initials in slide-cultures. A selection of sclerotium-form- ing fungi was studied by means of the Vernon (193 I) slide technique and three types of sclerotial development were distinguished. Most of the fungal species that have been studied fit into the three groups.

In recent years, transmission and scanning electron microscopy has made significant contributions to our understanding of the development and structure of sclerotia. The surfaces of sclerotia provide very good material for scanning electron microscope studies (Willetts, 1968u, 19694.

( I ) Loose type This type of development is illustrated by Rhizoctoniu soluni (Townsend & Willetts,

1954). There is no definite pattern of organization of the hyphae to form an initial. Irregular branching of the mycelium takes place, followed by intercalary septation and hyphal swelling to give what looks like a chain of chlamydospores. The cells of the mature sclerotia have dense contents with numerous vacuoles and the walls darken because of the accumulation in them of melanin pigment.

Blakeman & Hornby (1966) described the sclerotium of Mycosphaerellu ligulicolu as another example of the loose type of development.

( 2 ) Terminal type The sclerotia that are included in this group are formed by a well-defined pattern

of branching at the tip of a hypha or tips of closely associated hyphae. There is con- densed terminal growth and abundant branching of the tips to give a knot of hyphae, which are closely adpressed and often held together by anastomoses. Several initials coalesce to form a compact sclerotium. Examples of fungi whose sclerotia develop in this way are Botrytis cinereu, B. ullii, Sclerotium cepivorum (Townsend & Willetts, 1954), Pyronemu domesticum (Moore, 1962), Sclerotiniu fructicolu (Willetts, 1968 b), S. sclerotiorum and S. trifoliorum (Willetts & Wong, 1971).

Arimura & Kihara (1968) described the ultrastructure of sclerotia of Sclerotiniu sclerotiorum ; Jones (1970) examined the vegetative and sclerotial cell walls using electron microscope, infrared and biochemical techniques.

5'8 H. J. WILLETTS

( 3 ) Lateral type The important feature of this type of development is that the initials are formed by

the interweaving of numerous side (intercalary) branches or bud-like outgrowths of one or several main hyphae and are therefore in positions lateral to the main hyphae. There is a fairly clear distinction between the lateral and terminal types of development. Some lateral sclerotia arise from a single leading, primary or secondary hypha (sin@ type) and others by the branching of several or many hyphae united to form a strand (strand type). Fungi that produce lateral sclerotia are: Sclerotium rolfsii, Sclerotinia gladioli (Townsend & Willetts, I 954) ; Phymatotrichum omnivorum (King & Loomis, 1929 ; Reichert & Hellinger, 1949); Leptosphaeria salvinii (Kato, 1959) ; Aspergillus alliacus (Rudolph, 1962) ; Verticillium dahliae (Isaac, 1949) ; Colleto- trichum coccodes (Blakeman & Hornby, 1966) ; Macrophominaphaseoli(Wyl1ie & Brown, 1970); Sclerotinia minor (Willetts & Wong, 1971).

( a ) Simple The sclerotial initials of Sclerotinia gladioli and S. minor are formed from numerous

side branches of only one main hypha. The branches become closely interwoven, filled with dense contents and regular septa are formed. Several hyphal knots that develop in this way may become joined to form one sclerotium, but sometimes only one knot or initial gives rise to a mature sclerotium. This is partly due to the absence of hyphae bridging the space between initials and also to the short length and compact nature of the hyphal components. The angle of hyphal branching of S. minor is wide and these initials are not usually formed in close proximity. This reduces the chances of coalescence (Willetts & Wong, 1971).

(b ) Strand The sclerotia of Sclerotium rolfi i develop from strands which consist of as many

as twelve hyphae. The sclerotial initials are produced by multiple, intercalary branch- ing of the hyphae of the strand; sometimes an initial forms at the intersection of two strands. The branches become interwoven to form a knot of hyphae and num- erous septa are produced. When a white, small but compact initial has developed, differentiation of the tissue takes place and several regions can be distinguished in sections of a mature sclerotium (Townsend & Willetts, 1954; Chet, Henis & Kislev,

The sclerotium of Phymatotrichum omnivorum develops from a strand which is differentiated into three zones (King & Loomis, 1929) and is probably the most advanced member of this group (Townsend & Willetts, 1954). The sclerotium is formed by the rapid division and growth of the central tissue.

Remsberg (1940) found that the sclerotium of Typhula spp. forms from the mycelium as a tuft of branching hyphae which becomes a ball of enlarged hyphal cells. The sclerotium is compacted by further branching and a peripheral layer, which later

1969).

The morphogenesis and possible evolutionary origins of fungal sclerotia 5 I 9 becomes distorted, develops. Scurti & Converso (1965) made a detailed study of the fine-structure of sclerotia of Typhula sp.

Leptosphaeria salvinii also develops from a strand which is in the form of a thick cord of parallel hyphae (Kato, 1959).

111. FACTORS AND PROCESSES INVOLVED IN SCLEROTIAL DEVELOPMENT

Townsend (1952) distinguished three stages during the growth of a sclerotium; initiation (i.e. the formation of small, discrete initials), development (i.e. growth to full size), and maturation (i.e. surface delimitation, internal changes and pigmentation of the peripheral hyphae). These stages will be considered separately below.

Environmental factors have been shown to affect sclerotial development, but there have been few attempts to interpret the considerable data that have accumulated on the effects of light, nutrients, temperature, and H-ion concentration, on the initiation, development and maturation of sclerotia. Light may lead to the initiation and further development of sclerotia (Tarurenko, 1954; Carlile, 1956; Rudolph, 1962)~ but its role is poorly understood in both physical and biochemical terms. Townsend (1957) found that there are different nutrient requirements for initiation, growth and matura- tion of sclerotia. Sclerotial initials may form on a comparatively poor medium, but for further development of substantial numbers of sclerotia there must be more nutrients available than is essential for mycelial growth only. The source and concentration of nutrients, particularly those supplying carbon and nitrogen, have been shown to be important (Townsend, 1957; Rudolph, 1962). Temperatures best suited for mycelial growth need not necessarily be the optimum for sclerotial production, although growth of the mycelium and sclerotia takes place over a similar temperature range (Hawker, 1957). The H-ion concentrations that are most favourable for vegetative growth are usually those that are optimal for the production of sclerotia (Townsend, 1957; Rudolph, 1962). However, it is not the purpose of this review to consider general factors associated with the formation of sclerotia but, instead, the possible effects of specific morphogenetic factors on initiation and, also, several processes that appear to have particular significance in respect to development and maturation.

( I ) Initiation The comparatively large size of some sclerotia and what appears to be their differen-

tiation into distinct tissues suggest some degree of co-ordination during development. However, Corner (1950) regarded the sclerotia of Typhula spp. as arising as the result of a stimulus for concrescence followed by aimless growth, without the usual form factors. A mass of interwoven hyphae would be produced in this way. The distinction between the medulla, cortex and rind arises owing to different densities of mycelium and varying degrees of agglutination and pigmentation in the respective regions of the sclerotium. Corner’s conclusions may also be applied to sclerotia of other genera. However, regardless of subsequent development, concrescence or initiation is a necessary phase in the ontogeny of a sclerotium. Conditions that trigger off the growth of a young sclerotial initial may also be the same as, or similar to those that

33 B R E 47

520 H. J. WILLETTS are responsible for the formation of other multihyphal structures such as fruit bodies.

Apart from the data available on the effects of environmental factors on the initia- tion of sclerotia, there have been reports of many other factors that induce the formation of sclerotial primordia. Thus, growth of the mycelium against a mechanical barrier, physical damage due to the tearing of the mycelium, chemical barriers that arise from staling or specific morphogenetic substances have been found to be associated with initiation. Some of the latter are reviewed below.

( a ) Staling substances Bedi (1958) carried out a study of the effect of staling substances on the formation

of sclerotia of Scbotinia sclerotiorum. He found that there was a great increase in the number of sclerotia of this fungus when its own staling products (obtained from other cultures) were added to the growth medium. Also, sclerotia formed sooner and were larger in size than those on control plates. No sclerotia developed when staling products were removed from plates or prevented from accumulating. Mutants not normally forming sclerotia produced them when the growth medium was supple- mented with staling substances. However, the substance(s) that accumulates in the medium and induces initial formation is completely undefined from such experiments.

(b) Mmphogenetic factors Brandt & Reese (1964) found that Verticillium albo-atrum produces a diffusible

morphogenetic factor (DMF) which stimulates the production of microsclerotia and also of melanin. When added to cultures in low concentrations, it inhibits hyphal elongation and sporulation. The hyphae swell, become constricted, septation is increased and the cell walls become thickened. These are all processes that are part of microsclerotium development. Above a certain concentration, the non-volatile DMF inhibits development. Near ultraviolet light suppresses the synthesis of the morphogen and subsequently the formation of microsclerotia and melanin.

Goujon (1968, 1969, 1970) and Geiger & Goujon (1970) presented evidence for a morphogenetic factor (MF), produced by the mycelium of Sclerotium rolfsii, that induces sclerotium formation. This substance arrested the growth of leader hyphae and initiated branching in lateral positions where sclerotia developed later. The factor was detected after about four days and moved from old to young cells. From the results obtained with inhibitors of protein synthesis, it would seem that the morpho- genetic factor is a protein or a complex of proteinaceous substances.

Wheeler & Sharan (1965) studied the production of sclerotia by Sclerotium ro&ii and they suggested that the actively growing hyphae at the margin of the colony control the initiation of sclerotia. This was investigated further by Wheeler & Waller (1965) and they concluded that from a common intermediate metabolite(s), different metabolic pathways arise resulting in either vegetative growth or sclerotial formation.

The morphogenesis and possible evolutionary origins of fungal sclerotia 52 I

(c ) The possible role of sulphur bonds In biological systems, sulphur bonds or groups are of great importance in maintain-

ing the structure and to a great extent, the activities of proteins. The sulphydryl (-SH, thiol) group and its oxidized counterpart the disulphide (-S-S-) group are the most important and have been shown to be of great significance in the morpho- genesis of organisms (Brachet, 1964). The fungal cell wall is made up of microfibrillar components associated with material of a non-fibrillar nature. There is evidence for polysaccharide-protein complexes in the walls of yeasts (Nickerson, Falcone & Kessler, 1961) and possibly a chitin-glucan complex in SchixophylZum (Wessels, 1965). In the polysaccharide-protein complexes there are sulphydryl groups and -S-S- linkages and Nickerson & Falcone (1959) suggested that more plastic, localized areas may arise in the cell walls of Cundidu ulbicuns by the rupture of covalent bonds between fibrillar elements. The rupture is affected by reduction of the disulphide (-S-S-) to the sulphydryl (-SH) group under the enzymic action of protein disulphide reductase. Growth of a non-budding strain of C. ulbicuns in sulphur-deficient medium stimulated budding and it was suggested that low availability of sulphur gave greater plasticity to the cell wall. Only a few -SH groups were found in the walls of fungi that form mycelia (Robson & Stockley, 1962) but more were observed near the hyphal tips (Zalokar, 1959; Robson & Stockley, 1962). Pitt (1969) found -SH groups near the tips of phialides of Penicillium notutum but none was detected elsewhere in the walls of this species.

Chet, Henis & Mitchell (1966) carried out experiments to determine the effect of five sulphur-containing amino acids on the production of sclerotia by Sclerotium rolfsii. They found that the sulphur compounds, including glutathione, inhibited the formation of sclerotia but induced the production of basidia of the perfect stage. The latter are infrequently formed on host plants and had never previously been seen in culture. Iodoacetic acid induced early initiation of sclerotia which were large in size. When both iodoacetic acid and L-cysteine were added to the same culture medium in the ratio of I : 30 respectively, their effects were cancelled out and normal growth obtained. Chet et ul. suggested that the sites at which these compounds acted were possibly the -SH groups of the cell wall or of certain enzymes with morphogenetic functions. In later work, using radioactive techniques, Chet & Henis (1968) showed selective accumulation of iodoacetic acid in regions where sclerotial initials and therefore sclerotia would develop subsequently, while L-cysteine was evenly distri- buted through the mycelium. The different distributions account for the very different concentrations of the two compounds needed to cancel each other and give normal growth. Sclerotia were found to be induced by chelating agents and potassium iodate; the sclerotia were formed in concentric circles. Chet & Henis suggested that sclerotium formation could be repressed by a sulphydryl-containing copper-linked entity, the activity of which was increased by L-cysteine but neutralized by iodoacetic acid, potassium iodate or Na,EDTA when the concentrations of these reached a certain level. They used this model to explain the induction of sclerotia in areas where mycelium is torn or grows in contact with a mechanical barrier; thus there was local

33-2

522 H. J. WILLETTS inactivation of the repressor in the former instance and accumulation of the neutralizer in the latter.

Trevethick & Cooke (1971) carried out some experiments similar to those of Chet & Henis (1968) to determine the effect of selected amino acids, inhibitors of sulphydryl groups and chelating agents on the formation of sclerotia by Sclerotium rolfi i , Sclerotinia delphinii and S. sclerotiorum. The production of sclerotia of the first two species was inhibited by all the compounds used and variable results were obtained for the last one. They concluded that the compounds used by them do not directly affect sclerotial morphogenesis by altering the activity of a repressor but may have a more indirect effect.

From the results of an investigation which has been in progress in our laboratories for the past two years, A. L. Wong and I support the suggestion made by Trevethick & Cooke (1971) that the compounds used by them and by Chet & Henis (1968) block a major biochemical pathway, the indirect result of which is the stimulation of sclerotial production. Substances that do not affect this pathway or, alternatively, cancel out the blocking effect of inhibitors will lead to normal vegetative growth and the repression of sclerotial production. Several isolates of Sclerotinia sclerotiorum and S. minor, in addition to one of Sclerotium rolfsii and of Botrytis cinerea, were used and it is apparent that the acceptance of data obtained from only one isolate of a species could be most misleading. The results of our investigation are being prepared for publication.

( d ) Polyphenols and polyphenol oxidases Smith & Galbraith (1971) reviewed aspects of differentiation in fungi and drew

attention to the connexion between the development of certain specialized structures that serve a survival function and the accumulation of secondary metabolites. Pheno- lics come into this category and there is evidence to show that they and their associated enzymes are connected with the morphogenesis of ascocarps and basidiocarps of certain fungi. If the sclerotia of some fungi are indeed abortive fruiting bodies (Corner, 1950) then information obtained on the initiation of sporocarps would have direct relevance to the morphogenesis of sclerotia. Also, some sclerotia may have originated from conidiophores (Townsend & Willetts, 1954; Willetts, 1969 b) and polyphenol oxidases have been implicated in the differentiation of conidiophores and in sporulation (Jicinska, 1968).

Hirsch (1954) suggested a correlation between tyrosinase metabolism and the initia- tion of protoperithecia by Neurospora massa. Horowitz et al. (1970) presented results in support of a model in which tyrosinase is repressed in growing Neurospora by a rapidly turning over or an unstable protein. This would account for the absence of detectable tyrosinase activity in rapidly growing vegetative cultures. The concentra- tion of this repressor in the cell would be drastically reduced by a slight but prolonged inhibition of general protein synthesis. Thus inhibition of protein synthesis could act as a stimulator of tyrosinase synthesis. Esser (1968) found a correlation between polyphenol-oxidase activity and the development of perithecia by Podospma anserina and he suggested that laccase alone has a morphogenetic effect on fruit-body develop- ment. The melanin-free mutant, P. albospora had the same polyphenol-oxidase

The morphogenesis and possible evolutionary origins of fungal sclerotia 523 spectrum as wild types and gave white perithecia and spores. He concluded that melanin itself is merely incidental in the process of initiation of perithecia as the last stages in the production of the pigment from indol-5-6-orthoquinone proceed without enzymes.

Leonard (1971) demonstrated that the formation of basidiocarps by Schizophyllum commune is closely correlated with polyphenol-oxidase activity of the fungus. Leonard pointed out, when discussing his results, that this association of polyphenol-oxidase activity with fruit-body formation need not necessarily indicate that the enzyme is actually participating in the process of morphogenesis either directly or indirectly, before or during initiation, but it seems likely that polyphenol oxidases do more than simply accompany the process of fruit-body formation by Schizophyllum.

Wilson (1968), working with Hypomyces sp., also obtained a positive correlation between tyrosinase activity and formation of perithecial primordia - the greater the activity the larger the number of primordia formed. When liquid cultures were supplemented with commercial tyrosinase, large numbers of primordia were formed. He discussed several ways in which the tyrosinase may function and suggested that possibly the effect of the enzyme on cellular membranes is of prime importance. Cory (1967) has shown that tyrosinase affects the permeability and organization of cell membranes of red blood cells. This appears to present a fruitful approach for further work on sclerotium initiation.

Brandt (1964) demonstrated a close correlation between melanin synthesis and microsclerotial development by Verticillium, but there is very limited published work to suggest a role for polyphenol oxidase in the morphogenesis of sclerotia. A. L. Wong and I are at present studying various aspects of the morphogenetic effects of phenolics and associated enzymes on the initiation of sclerotia. Tyrosinase activity has been detected in sclerotial initials of Sclerotinia sclerotiorum and the activity increased as the sclerotia developed. No tyrosinase activity was found in vegetative cultures grow- ing in liquid medium.

( e ) Possible biochemical control The incomplete information that is available on the important aspect of biochemical

control of initiation does not prevent speculation on the role of the compounds discussed above. Possibly phenolics and polyphenol oxidases, and also sulphur linkages and certain enzymes associated with sulphur bonding are involved, in sequence, in the formation of initials. At certain concentrations and activities poly- phenols and their enzymes may inhibit growth of the apices of main hyphae. This could come about by changing the permeability of the membrane, by inducing a thickening of the cell wall at the tip or by reducing plasticity of the wall. This does not infer that phenolics are always responsible for the inhibition of growth but merely that they sometimes, or possibly often, are involved. After inhibition at the tips, pseudodicho- tomous branching and/or subapical branching close to the tip takes place to give a terminal initial. Alternatively, branching is initiated in intercalary positions of lateral branches to form lateral-type sclerotia. Vegetative hyphae growing on the surface or submerged in the substrate will be more affected by the accumulation of

524 H. J. WILLETTS any inhibitory substance in the medium than aerial hyphae and it is perhaps signi- ficant that most sclerotia develop in aerial positions. Although some sclerotia and stromata develop in or closely adpressed to the substrate, initiation of these may involve factors other than phenolics or chemical inhibitors of growth. Such sclerotia and stromata are often formed behind the margin of the colony where possibly toxins have not accumulated in effective concentrations or have diffused out of the substrate.

One of the main prerequisites for sclerotial initiation is the formation of a large number of hyphal branches. To understand this we need to be informed on the structure and physiology of hyphal tips, apical growth and lateral branch development. The limited relevant literature has been reviewed by Robertson (1965a, b), Morton (1967) and Burnett (1968), and they speculated on the mechanism of mycelial growth. One very important aspect associated with branch formation is the change in the plasticity and rigidity of walls in regions where the sclerotium initial is to develop. The ability of the wall to be moulded is possibly related to changes in the sulphur linkages of the protein of the protein-carbohydrate complexes of the cell wall and could be influenced by factors such as sulphur availability or increased activity of specific enzymes, of which protein disulphide reductase may be one. We have observed in our laboratories (Willetts & Wong, 1971) that sclerotial initials of Sclerotinia minor often arise in the region where anastomosis of two branches takes place. The lysis of the walls of the tips at the region of contact must be by means of wall-degrading enzymes present, possibly, in the vesicles at the apices of hyphae (Bracker, 1967). It is tempting to suggest that after lysis of the cross-walls, the activities of these enzymes are then turned to the lateral walls in the region of the anastomosis. The resultant softening of the lateral walls would permit more ready branching in these areas. However, anastomoses do not always precede initial formation and, in some fungi, anastomoses are relatively infrequent in the early stages of sclerotium formation.

( 2 ) Developmental stage ( a ) Translocation of nutrients

During this phase the maximum size of the sclerotium is attained and a compact structure develops. Townsend (1957) showed that different environmental conditions are required for initiation and for further development of sclerotia. Hawker (1957) suggested that maturing sclerotia may inhibit the formation of other initials, either by using up the available nutrients or by the production of inhibitory substances. Translocation of nutrients to the primordia is obviously of great importance as energy must be available for the growth of the large number of hyphae that give the body its compact structure. Wilcoxson & Sudia (1968) reviewed the literature on translocation in fungi and discussed the movement of nutrients into sclerotia. There is a strong polarity of transport of s2P into sclerotia of Rhizoctonia solani and Sclerotium rolfsii (Littlefield, Wilcoxson & Sudia, 1965 ; Littlefield, 1967; Wilcoxson & Subba- rayudu, 1968). The movement of nutrients to the developing sclerotia is usually through only a few hyphae which take on a translocatory function. Goujon (1967) described two types of hyphae in S. rolfsii, translocatory and lateral types; Cooke

The morphogenesis and possible evolutionary origins of fungal sclerotia 525 (1970) studied [14C]glucose uptake by sclerotia of Sclerotinia sclerotiorum and empha- sized the importance of translocatory hyphae in the development of the sclerotia. He found that even very young sclerotia depended on a few translocatory hyphae for the uptake of 14C from a liquid medium. Littlefield (1967) showed that there was translocation of from old to young regions of cultures of Rhizoctonia solani but this stopped when the mycelium had filled the culture plate. When sclerotial initials began to develop, translocation was again apparent and the direction was almost entirely towards the sclerotium. Cytoplasmic streaming is a means of transport of nutrients in fungi but active movement may take place without streaming (Littlefield et al., 1965; Thrower & Thrower, 1968). Wilcoxson & Sudia (1968) and Thrower & Thrower (1968) suggested that transport of nutrients in fungi is usually from regions of higher nutrient concentration to physiologically active hyphae and thus follows a concentration gradient. Conditions that favour evaporation also favour translocation. Developing sclerotia are metabolically very active and they exude water.

(b ) Exudation Numerous small drops of a clear liquid are frequently seen on the surfaces of

developing sclerotia. Remsberg (1940) observed a crystalline residue when exudates from sclerotia of some Typhula species were dried on a glass slide. Recently more data have been obtained on exudation, particularly from the sclerotia of Sclerotinia sclero- tiorum. Working with this fungus, Cooke (1970) found that there is a rapid movement of water and nutrients into a young sclerotium and that most of the material is moved into the developing sclerotium within about three days of initiation. Water loss was greatest when the initials began to increase in size, which corresponds with the time of maximum translocation (Cooke, 1969).

Jones (1970) detected polyphenol-oxidase activity, salts, amino acids and proteins in exudates from sclerotia of Sclerotinia sclerotiorum and Colotelo, Sumner & Voegelin (1971 b) obtained similar results. They detected cations, lipids, ammonia, amino acids, proteins and a number of different enzymes including catalase, peroxidase, polyphenol oxidase and P-glucosidase. These same workers (1971 a ) claimed that a membrane of undetermined origin and nature formed around the exudate drops. Kybal(1964) and Cooke (1969) suggested that the active and selective exudation of water and sugars from the surfaces of immature sclerotia helps to keep osmotic balance within the sclerotial cells and also maintains a gradient whereby sugars continue to move into them. Colotelo et al. (1971b) presented the hypothesis that exudation during early stages of development could possibly be a physical phenomenon whereby soluble compounds are expelled at the time of cell wall thickening and cell wall dehydration. At later stages the exudates could contain cell contents liberated by the disruption of peripheral or rind cells. This could account for the complex nature of the exudates. A skin or crust of dried up cells is present over the outer surfaces of many sclerotia and the former contents of these hyphae would accumulate in liquid drops on the outside of the sclerotium.

The suggestion that exudation is associated with the translocation of water and nutrients and the removal of excess amounts of these so that internal physiological

526 H. J. WILLETTS balance is maintained, is an attractive idea to explain the way that sclerotia compete for nutrients with other centres of active growth. Thus, some stimulus is provided either from within or from outside the hyphae that leads to the initiation of the sclerotial primordium. It can be assumed that at the site where the initial is to develop, sufficient nutrients are available for branching to take place. The nutrients may be available at this site through the cessation or slowing down of growth of the hyphae at the margin, thereby permitting the establishment of new regions of growth. Presumably, once this region of increased metabolic activity has arisen, the initial will be able to compete successfully with centres of active growth elsewhere in the colony, such as at the margin where, in the meantime, growth may have been resumed. The ability to compete will, however, depend on the maintenance of a concentration or pressure gradient. If there is, for some reason, even a brief interruption in the flow of nutrients to the developing sclerotium then probably the initial will be unable to re-establish sufficient ‘pull ’ to provide energy for further growth and differentiation of the young sclerotium. In culture, many initials fail to mature and probably they have been unable to obtain the necessary nutrients. A concentration or pressure gradient to the initial is maintained at first by the rapid use of nutrients during the formation of new branches and later, when excess nutrients are being translocated into the young sclerotium, by their conversion into different, less active or insoluble substances and/or by exudation. An increase in the permeability of the membranes of sclerotial hyphae would facilitate more ready secretion of a complex exudate.

( c ) IntHweaving of hyphae Soon after a sclerotial primordium is initiated the hyphae become interwoven and

a compact structure begins to take form. Usually, the hyphae of the same mycelium grow away from each other so that they are well dispersed over the substrate, thereby reducing competition. This does not apply to hyphae that form a sclerotium or other multi-hyphal structure. I have often observed how in sclerotium-forming fungi two hyphae meet and then continue to grow together instead of one growing over or under the other. A strand of several hyphae or a network of hyphae is built up in this way. The hyphae do not necessarily stay together but after a period of growth they may separate and then become associated with other strands. This tends to bind the mycelium into a compact tissue. Observations of the growth of these hyphae over long periods suggest to me that they stick together after coming in contact. A possible adhesive is the gelatinous substance that has been found to be a feature of many sclerotia. Whetzel(1945) used the presence or absence of a mucilaginous matrix as a diagnostic characteristic of the Sclerotiniaceae ; Corner (1950) reported its presence in varying amounts in Typhula spp. According to Whetzel’s classification, the sclerotia of Sclerotinia sclerotiorum and S. fructicola are not mucilaginous. However, using histochemical techniques B. D. Kosasih (unpublished) found a thick layer of mucilage over the surface of medullary hyphae of these two species, although the interhyphal medullary spaces are not filled with the mucilage. A. L. Wong (unpublished) obtained large amounts of mucilage from these sclerotia when extracting proteins for electro-

The morphogenesis and possible evolutionary origins of fungal sclerotia 527 phoretic studies. Also, in mutant forms of S. sclerotiorum, which form loose, fluffy initials that never mature, no mucilage appears to develop.

Work on the slime moulds (Sonneborn, Sussman & Levine, 1964) indicates that adhesion of cells is specific and necessary to produce morphogenetic interaction. Probably the adhesive materials are mucopolysaccharides and enzymes are produced that enable cells to stick together (Gerisch, Malchow, Wilhelms 8z Luderitz, 1969). A system of this type may also operate in the morphogenesis of sclerotial tissues.

Anastomoses (hyphal fusions) are a common feature of sclerotia and serve as permanent unions between hyphae. The work of Buller (1958) is still the main source of reference on hyphal fusions. Numerous small protuberances develop in regions of sclerotial initiation with corresponding ones on adjacent hyphae suggesting mutual stimulation and attraction. Apart from binding the hyphae together anastomoses also facilitate the movement of nutrients and organelles into the developing sclerotium.

( 3 ) Maturation Most sclerotia grow to their maximum size, i.e. pass through the developmental

phase, within a few days from initiation, and metabolic activity, as measured by 14C0, evolution, decreases (Cooke, 1971). The maturation stage is usually completed in only a few days. During maturation, water containing certain dissolved substances such as trehalose, mannitol, inositol and traces of glucose (Cooke, 1969) continues to be exuded from the surface resulting in decreased hydration of the sclerotium. There is only limited movement of nutrients into the young sclerotium and probably those which are not needed to supply energy for formation of hyphae accumulate to form endogenous reserves or are exuded. The reserves enable the cells to become nutrition- ally independent of the substrate and their period of viability, under conditions of starvation, is thereby increased. The reserves are found in large food vacuoles, and in lipid bodies. Sclerotial walls may become thickened and are of different chemical composition from vegetative hyphal walls (Chet, Henis & Mitchell, 1967). These changes increase resistance to desiccation and, possibly, biological degradation. The thickened walls may also serve as stores of insoluble materials which can be utilized under conditions of starvation or of active growth as at the time of germination.

Most sclerotia become pigmented after they have reached maximum size. The colour of sclerotia varies, but most are dark brown on account of the melanin-like pigment which accumulates in and between the walls of those peripheral hyphae that form the rind. When the rind is removed the newly exposed hyphae usually become pigmented and a new rind develops. The tips of the hyphae that form the rind become swollen, tightly arranged and the walls become thickened. The greater density of the hyphal tips at the surface may be partly due to the higher oxygen level at the periphery compared with the centre of the tissue. The rind has been shown to seal off the sclerotium effectively from its surroundings (Coley-Smith, 1959; Chet, 1969) and when there is no further movement of substances along the translocatory hyphae connecting it with the underlying mycelium the sclerotium becomes isolated both physiologically and nutritionally. A persistent resting stage is formed which has a high level of endogenous reserves, an impermeable outer layer in which the individual

H. J. WILLETTS cells of the cortex and medulla are surrounded by a partially permeable barrier. The sclerotium is able to survive in a balanced and efficient manner in the absence of nutrients and then, under suitable conditions, germinates by the formation of mycelium and/or spores. Thus, the sclerotium is a good example of the type of isolated system that was described by Wright (1970) in her discussion on the evolution of substrate control in differentiation.

IV. EVOLUTIONARY ORIGINS OF SCLEROTIA

Superficially, sclerotia show considerable diversity but the anatomy of most is alike. Several terms such as prosoplectenchyma, paraplectenchyma and pseudo- parenchyma have been used to describe the different regions, i.e. the medulla, cortex and rind. Corner (1950) pointed out that these are basically plectenchyma which is agglutinated to different degrees. Also, different mycelial densities partly determine what name is to be used. Thus, no clear distinction can be made between the 'tissues'. Although, in general, the structures of sclerotia are alike, there are ontogenetic differences between them, as has been described earlier. This may be explained most simply if it is assumed that the sclerotia of fungi have developed as a result of conver- gent evolution from different origins.

In mycological literature there are a number of references to the origins of sclerotia of specific fungi, bur1 am not aware of any general account. Most data are available for the genus Typhulu and for members of the family Sclerotiniaceae. I will discuss these separately as their sclerotia possibly illustrate two lines of development whereby structures that serve the same function and are alike anatomically have evolved from different origins. Some other relevant examples are also given.

(I) Typhula spp. Corner (1950), in his monograph on Cluouriu and allied genera, concluded that the

sclerotia of Typhulu spp. are abortive fruit bodies which have assumed the role of resting structures, capable of resisting adverse environmental conditions, particularly low temperatures. Both Remsberg (1940) and Corner (1950) have described Typhulu as a low-temperature genus and the agglutination of the hyphae increases the resis- tance of the structure against severe conditions. Corner distinguished four kinds of sclerotia in Typhulu according to the degree of agglutination of the sclerotial hyphae from the periphery inwards. Possibly, suboptimal temperatures were mainly respon- sible for the sterility of the fruit bodies by inhibiting the differentiation of the hymen- ium. Corner demonstrated the production in culture of sterile fruit bodies of Typhulu at suboptimal temperatures; Noble (1937) found that if an adequate supply of nut- rients was available, sclerotia of T. trifolii formed at 13OC. but not at 23OC. Sclerotia of Typhulu that developed in cultures incubated at low temperatures were crowded and sometimes coalesced to give a stromatic crust, but at higher temperatures they were scattered. However, other environmental factors such as light may interact with low temperatures and have a controlling effect on development of the hymenium.

The upright, rhizomorph-like structure that would be produced if no hymenium

The morphogenesis and possible evolutionary origins of fungal sclerotia 529 developed on the fruit body of Typhula could become a sclerotium-like outgrowth if there were further reduction in its size. Corner observed tissue at the base of the fruit body of Pistillaria setipes which was similar to a small Typhula-like sclerotium except that it was not agglutinated. The absence of sclerotia was used to distinguish the genus Pistillaria from that of Typhula. Species of the latter normally produce one fruit body per sclerotium when conditions are satisfactory, but on occasions, as many as twenty may be produced. However, it is not essential that sclerotia are formed before fruit bodies develop - Remsberg observed basidiocarps of T. idahoensis and T. pertenius that had grown directly from mycelium in nature.

(2) Sclerotinia spp. Townsend & Willetts (1954) suggested that sclerotia of some fungi that have a

terminal type of sclerotial development, e.g. Botrytis spp., may have developed from conidiophores. In later work (Willetts, 1969b), observations of stromata and conidia of ~c le ro t in ia~r~c~ico~a indicated that they are alike in their general growth requirements and basic structure. However, stromata and sclerotia usually develop in moist places while conidia are formed under drier conditions. This may partly explain the forma- tion of stromata of S. fructicola on the surface of and submerged in the substrate and the development of macroconidia in corresponding areas above the medium (Willetts, 1969b). Sclerotia, because of their dense mycelial growth and accumulation of food reserves, require more nutrients to develop and mature than do conidia. Where supplies of nutrients are limited there is probably competition between them, and conidia are formed more readily. Sclerotia develop when there are sufficient nutrients to support the growth of both spores and sclerotia, or when sporulation is partly or completely inhibited by some factor such as temperature or light.

It is not difficult to envisage the development of sclerotia from a large number of interwoven conidiophores which have not differentiated into chains of conidia because of unsuitable conditions. There is a close morphological similarity between young sclerotial initials and the spreading conidiophores of several of the Sclerotiniaceae that produce conidia. Examples are found in Septotinia where the conidial fructifications are composed of massed, branching, hyaline, septate conidiophores (Whetzel, I 945) : in Streptotinia where the conidiophores are characteristically twisted together (streptoform) (Whetzel, 1945) : in Sclerotinia fructicola where a close similarity between branching conidiophores and early stages in stromatal initial formation is apparent (Willetts, 1969b). When cultures of S. fructicola were incubated at high humidities, septum formation was inhibited in conidiophores and conidial chains did not develop (Willetts & Calonge, 1969 a). Under these conditions conidiophores branched repeatedly and stroma-like structures developed (Willetts, 1968 c). Regular constrictions were observed along some stromatal medullary hyphae of 5'. fiuctigena which gave them the same appearance as young undifferentiated chains of conidia (Willetts & Calonge, 1969b). Whetzel(1945) found that conidia of Botryotiniu formed more profusely in a dry atmosphere than under conditions of high humidity. Byrde (1954) and Hulatt (1956) concluded that a period of exposure to low humidity is needed before the spore chains of S. f.uct@na are able to differentiate.

530 H. J. WILLETTS Whetzel (1945) included fifteen genera in the Sclerotiniaceae and nine of these do

not form conidial fructifications or, at least, conidia were not found by him. The nine species that do not produce conidia (or if they do, only do so rarely and under condi- tions that are not normally found in nature or the laboratory) are possible examples of the selection of sclerotium-producing strains and the loss of the ability to form conidia. This could be associated with the colonization of substrates in moist and/or cold habitats. Under adverse conditions, the sclerotium has a survival advantage over the conidium which is lower in food reserves and less resistant to freezing, desiccation, irradiation, etc. When conditions become suitable for infection of plants in the spring or autumn, large numbers of propagules are produced on the sclerotia by the develop- ment of apothecia bearing asci and ascospores. Thus, reproduction by asexual spores is not essential to these fungi and probably the ability to produce such spores has been lost.

Two different types of sclerotia were described by Whetzel (1945) for Stromatinia. The first mantles the affected portion of the host and from it apothecia develop. The second type is small, black, borne free on the mycelium and does not produce apothecia. The latter was named a ‘sclerotule’ and has been seen only when the fungus is growing on artificial media. The structures of the sclerotule and the mantling sclerotium are similar. In recent work, we have observed the development of small aggregates of spore-like cells by Sclerotinia sclerotiorum, S. trifoliorum and S. minor, in addition to the production of ‘typical’ sclerotia, terminally in the first two species and laterally in the third (Willetts & Wong, 1971). We suggested that the aggregates may be spore masses and could indicate the origin of the sclerotia of these fungi from branched conidiophores and conidial chains, associated with a loss of the ability to produce asexual spores. In S. minor there appears to be the formation of sclerotia from lateral (vegetative) hyphae and by the modification of former sporogenous tissue. Thus, there could be different methods of sclerotial ontogeny even within one species.

( 3 ) Other relevant examples Langeron & Vanbreuseghem (1955) regarded the sclerotia of Aspergillus and

Penicillium as immature perithecia which never completed development. They drew attention to the external resemblance of the sclerotia of these two genera to the perithecia of Eurotium (Aspergillus) and Carpenteles (Penicillium). Rudolph ( I 962) studied the effect of physiological and environmental factors on the development of sclerotia of Aspergillus. He found sexual structures and spores inside some sclerotia and he also induced changes in the form of the sclerotia by ultraviolet irradiation. He concluded that the sclerotia of this fungus are sterile stromata or other tissues associa- ted with sexual reproduction which, in the past, may have contained cleistothecia and asci.

Leakey (1964) described four species of a new genus Dactuliophora from Africa which produce sclerotia in a cup-like sclerotiophore. The sclerotia develop from a mass of plectenchyma within the leaf tissue. Leakey concluded that the sclerotium of this genus is analogous to the conidium, and the sclerotiophore to the conidiophore of a hyphomycete. Mukiibi (1969) found that the structure of the sclerotium of

The morphogenesis and possible evolutionary origins of fungal sclerotia 53 I Dactuliophora is similar to that of Botrytis and the sclerotia of both fungi have a terminal type of development. Mukiibi suggested that the sclerotiophore may be regarded as a synnema and the sclerotium as a ‘propagule of unified conidia’. Thus the genus Dactuliophora could give support to a theory that some sclerotia have developed from conidia or conidial elements which remain together to form one large structure. Without further changes the compact nature of the multiple conidium will be more resistant to adverse conditions than the single-celled conidium and over a period of time the selection of other modifications will make it more adapted to a harsh environment.

V. CONCLUSIONS

It is tempting to speculate on the relationship of different ontogenetic types with origins from different fungal structures. It seems that the loose type of sclerotium is primitive and has arisen from ordinary vegetative mycelium by the accumulation of food reserves in hyphae together with thickening and pigmentation of the hyphal walls. The terminal type of sclerotium could have originated from the interweaving of potentially sporogenous (asexual) tissue which failed to differentiate into spores. The lateral type appears to have developed by the localized, active branching of vegetative hyphae to give hyphal aggregates. Evidence has been given in the text to support the theory that some of these sclerotia are degenerate sexual reproductive structures such as pycnidia, perithecia or basidiocarps which, by selection, have become resting structures. In many instances sporocarps grow from sclerotia when environmental conditions become suitable and the presence of a sclerotium in the life-cycle of a fungus may merely delay sexual reproduction. This has obvious advan- age to the organism. The sclerotium appears to provide an interesting example of convergent evolution whereby analogous structures, which have become adapted to resist adverse conditions, have evolved.

VI. SUMMARY

I . Fungal sclerotia are able to survive adverse conditions for long periods and they are formed by many important plant pathogens. An understanding of the factors involved in their initiation and development may lead to a method of repressing their formation in nature, thereby reducing the chances of survival of fungi that depend on them as persistent resting stages in their life-cycles. Also, data on sclerotial morpho- genesis may be applicable to other multihyphal fungal structures.

2. There are three types of sclerotial development. The most primitive and least common is the loose type, which is illustrated by Rhizoctonia solani. The sclerotium forms by irregular branching of the mycelium followed by intercalary septation and hyphal swelling. When mature, it consists of loosely interwoven hyphae that are rich in food reserves and darkly pigmented. The main types of development are terminal and lateral. The former develops from the coalescence of initials that are produced by a well-defined pattern of branching at the tip of a hypha or tips of closely associated hyphae, e.g. Botrytis cinerea. Lateral sclerotia are formed by the interweaving of side

532 H. J. WILLETTS branches of one or several main hyphae. When only one main hypha is involved the sclerotium is of the lateral, simple type, e.g. Sclerotiniagladioli. If several main hyphae give rise to a sclerotium, the term strand type has been used. Sclerotium rolfsii is the classical example.

3. There is a considerable literature on the effects of environmental conditions on the initiation, development and maturation of sclerotia but few attempts have been made to interpret the data.

Phenolics and/or polyphenol oxidases have been found to be connected with mor- phogenesis of the protoperithecium of Neurospora crassa, the perithecium of Podospora anserina and of Hypomyces sp. and the basidiocarp of Schixophyllum commune. A close correlation has been shown between melanin synthesis and microsclerotial develop- ment by Verticillium but there appears to be no literature on the role of phenolics and polyphenol oxidases in the morphogenesis of sclerotia. Possibly these substances may inhibit growth of the apices of main hyphae by changing the permeability of the membrane, by inducing a thickening of the cell wall at the tip or by reducing the plasticity of the wall. Such a check in growth could trigger-off the formation of initials close to the margin of the colony or elsewhere in the culture.

Sulphydryl groups and disulphide bonds are of great significance in morphogenesis of organisms and are probably involved in sclerotial initiation. The formation of a large number of hyphal branches is a prerequisite for sclerotial initiation and mycelial branching is possible only if there is plasticity of hyphal walls. The ability of the wall to be moulded is possibly related to changes in the sulphur linkages of the protein of the protein-carbohydrate complexes of the cell wall and could be influenced by sulphur availability or the activity of specific enzymes.

4. After a sclerotial primordium has been initiated, further increase in size will depend on the continued, active translocation of nutrients to the site of development. Movement of nutrients to sclerotia is through a few translocatory hyphae. Presumably, nutrients will continue to move into the young sclerotium as long as a concentration or pressure gradient is maintained. Energy and substances for the formation of new branches are supplied in this way and as the requirements for hyphal branches are reduced, excess nutrients become available for conversion to inactive or insoluble reserves and for exudation. The exudates are often complex, consisting of proteins, including enzymes, lipids and carbohydrates.

Many sclerotia have a mucilaginous matrix in which the medullary hyphae are embedded. Sclerotium-forming, fungal species that are not regarded as having such a matrix appear to secrete a layer of mucilage over the surface of sclerotial hyphae. This mucilage could have a morphogenetic function and serve as an adhesive which loosely binds hyphae together. More permanent unions are by hyphal fusions or anastomoses.

5. The sclerotium matures within a few days of attaining its maximum size. The rind effectively seals off the medullary hyphae from the surroundings and the trans- locatory hyphae cease to function. Thus the sclerotium is isolated both physiologically and nutritionally. The endogenous reserves enable the structure to exist in the absence of exogenous nutrients and then, when conditions become suitable, to germinate.

The morphogenesis and possible evolutionary origins of fungal sclerotia 533 6 . The sclerotium appears to provide an example of convergent evolution whereby

analogous structures, which have become adapted to resist adverse conditions, have evolved. Data are available mainly for Typhula spp. and ScZerotinia spp. Sclerotia may be degenerate sexual reproductive structures, hyphal aggregates that have developed from closely interwoven conidiophores and undifferentiated conidia or they may be modified vegetative structures.

I am grateful to Professor L. E. Hawker and Dr M. F. Madelin of the University of Bristol; Professor D. J. Anderson, Dr R. S. Vickery and Miss J. Tarran of the University of New South Wales, for their critical reading of the manuscript. My sincere thanks are due to Mr A. L. Wong for many stimulating discussions and for drawing my attention to several important references.

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