Biol. Rev. (197x), 46, pp. 387-407 387

THE SURVIVAL OF FUNGAL SCLEROTIA UNDER ADVERSE ENVIRONMENTAL CONDITIONS

BY H. J. WILLETTS School of Botany, University of New South Wales, Australia, 2033

(Received 3 May 1971)

CONTENTS I. Introduction . . .

11. Concept of sclerotium . . . . 111. Longevity of sclerotia . IV. Structure of mature sclerotium . .

(I) Rind . . . . (2) Cortex and medulla .

( I ) Desiccation. . (2) Extremes of temperature . (3) Physiological features that influence survival against several different factors (4) Radiations . ( 5 ) Starvation . (6) Resistance to toxic chemicals and enzymic activities of other organisms

VI. Conclusions .

V. Structure and physiology in relation to survival .

.

VJI. Summary . VIII. References .

. 387

. 388

. 388

. 390 * 390 . 392 . 393 . 394 . 395 . 398 . 400 . 401 . 401 * 403 . 404

404

I. INTRODUCTION

Much information has accumulated on factors affecting the survival of fungi under adverse conditions and several excellent reviews have been written on this subject (Hawker, 1 9 5 7 ~ ; Park, 1965 ; Mazur, 1968; Sussman, 1968). Specialized resistant bodies are important as a means of survival (Hawker, 1957a), and these may be spores, parts of hyphae that possess special characteristics, or complex vegetative structures, such as rhizomorphs and sclerotia, formed by the aggregation of hyphae. Some resting bodies are formed only when conditions become adverse. The survival of an organism involves not only the ability to retain viability when the habitat is unsuitable, but inherited adaptations such as the ability for rapid mycelial growth, abundant spore production, colonization of a wide variety of substrates, etc., are also important. These characteristics have been described by a number of workers by such terms as ‘ competitive saprophytic ability’ (Garrett, 1956), ‘genetic variability, disseminability and responsiveness’ (Sussman, 1968).

It appears that, in general, sclerotia can survive under more severe conditions and for longer periods than any of the other resistant bodies. The structure and physiology of sclerotia have many features that enable these bodies to endure, sometimes for

388 H. J. WILLETTS several years, such adverse environmental conditions as Sussman (1968) describes as ‘ deleterious agents’. These include : extremes of temperature ; desiccation ; starvation ; a variety of toxic chemicals that may either be naturally present in the soil or atmosphere, or that may accumulate as a result of the activities of other organisms; and solar radiations. These factors all interact with each other and are seldom constant for more than a brief interval.

The purpose of this paper is to review the available data on the structure and physiology of sclerotia and to relate them to the survival of these bodies under extreme environmental conditions of the type referred to as deleterious agents.

11. CONCEPT OF SCLEROTIUM

The term ‘ sclerotium’ includes many diverse fungal bodies which are particularly characteristic of certain taxa such as Sclerotinia, Claviceps and Typhula. They are formed in various ways by the aggregation of vegetative hyphae usually containing food reserves. As their name implies, they are hard structures. When mature they may persist for long periods as independent bodies. De Bary (1887) and Lohwag (1941) restricted the term to resting vegetative bodies that germinate to form external reproductive structures or clearly related forms. All the remaining similar bodies they called sclerotioid structures. A separation of this type has not been attempted in this review and sclerotioid structures, such as microsclerotia and bulbils, are included with those classed by de Bary and Lohwag as true sclerotia. Regarding the family Sclerotiniaceae, from which many examples must be taken, I have accepted Whetzel’s (1945) definition of the ‘sclerotial stroma’ for the sclerotium. He described the sclero- tial stroma as ‘ a strictly hyphal structure under the natural conditions of its develop- ment. While elements of the substrate may be embedded in its medulla, they occur there only incidentally and do not constitute a part of the reserve food supply.’ Although I have accepted Whetzel’s general definition, I have not attempted to use the terms he suggested for different types of sclerotia. These terms only add further confusion to an already difficult characterization. According to Whetzel the stromata of the brown rot fungi (Sclerotinia (Monilinia) spp.) are true sclerotia, but some workers (Aderhold & Ruhland, 1905; Honey, 1928) have described them as ‘substrata1 stromata’ or ‘ pseudosclerotia’ because of the occurrence of host cells in the medulla. I agree with Whetzel that the mummified cells within the medulla do not function as reserve material and that their presence is purely incidental. Therefore the stromata of these fungi are used as examples of sclerotia in many places in the text.

111. LONGEVITY OF SCLEROTIA

Most of the work on the longevity of fungal structures has been carried out under laboratory or glasshouse conditions and information gained in this way may be mis- leading. Sometimes the conditions under which the materials are kept are more extreme than would be encountered in nature, while other factors of the normal environment may not be operating. Thus, results often appear to be contradictory. For example,

Survival of fungal sclerotia under adverse conditions 389 Last (1960) found that the germination of spores of Botrytis fabae was reduced from 100% to 20% after 65 days, but Bagga (1967) demonstrated that spores of Botrytis cinerea and some other fungi may survive for as long as 40 months when kept under dry storage conditions. However, such constant conditions would rarely be experienced in nature.

It is generally accepted that sclerotia are able to survive conditions that are too severe for ordinary vegetative hyphae and spores. Blakeman & Hornby (1966) studied the survival of conidia and sclerotia of Colletotrichum coccodes and Mycosphaerella ligulicola both in air-dried agar cultures and in greenhouse tests in sand and soils. There was a rapid loss of viability of the conidia of both fungi within a few days of burial in natural soil and none survived for 3 weeks. The sclerotia of C. coccodes retained 53 yo viability after 83 weeks in natural soil that was watered regularly, but those of M . ligulicola survived for less than 30 weeks. Maas (1969) found that the survival of conidia and sclerotia of Botrytis convoluta was favoured by low temperatures (his lowest limit was -70' C.) but that at temperatures above 25' C. conidia became rapidly inactivated, while sclerotia tended to lose viability at about 30° C. Sclerotia kept in moist, non-sterile soil lost viability rapidly at temperatures above 15' C., presumably as a result of the activities of antagonistic soil micro-organisms. Curl & Hansen (1958) found that many micro-organisms isolated from the stolons of white clover were antagonistic to Sclerotium rolfsi in culture. Some of these isolates sup- pressed the production of sclerotia. Halkilahti (1962) observed that sclerotia of Sclerotinia trifoliorum, when placed in soil, were sometimes destroyed within several months by the activities of micro-organisms while others survived for 4+ years. Different strains varied in their susceptibility to these organisms. Similar results were obtained from further work on S. trifoliorum and also Sclerotinia sclerotiorum (Ervio, Halkilahti & Pohjakallio, 1964). Severe infection of the sclerotia by both fungal and bacterial pathogens was normally favoured by high temperatures. Nevertheless severe infection by Mucor hiemalis was obtained over a temperature range of 3-23" C.

There are numerous records of sclerotia surviving in soils for many years. Verti- cillium dahliae persisted in soil for 14 years and Verticilliurn albo-atrum for about as long (Wilhelm, 1955), while Pollock (1918) found that stromatized plums produced apothecia of Sclerotinia fructicola after 10 years in the soil. My own observations on fruit mummified by this fungus are that in untreated garden soil, kept permanently moist at temperatures ranging from 10-20' C., well-developed peach and nectarine mummies disintegrated within 5 months and seedlings developed from the stones around which the stromata had formed (Willetts, unpublished data). In aseptic laboratory tests viability of such stromata was maintained for at least 3 years, and, as this material is still being maintained, possibly much longer. Sussman (1968) has tabulated many records of the longevity of sclerotia with the relevant references.

The numbers of sclerotia in soils may be increased by the formation of secondary sclerotia (Pape, 1937; Drbal, 1961; Halkilahti, 1962). Williams & Western (1965) observed the development of secondary sclerotia of Sclerotinia trifolimum and Sckotinia sclerotiorum and determined that the primary sclerotia have the ability to absorb and grow, either by direct intake of food materials or by the development of an

390 H. J. WILLETTS adhering system of secondary sclerotia. This increases the powers of survival of these fungi and also gives them a means of multiplication and rejuvenation. However, the development of apothecia results in these sclerotia becoming more susceptible to decay. Another important factor that increased degradation was an increase in soil moisture. Phymatotrichum omnivorum, which produces a mycelial state in the soil, is also able to rejuvenate and increase the numbers of sclerotia in the soil in the absence of the cotton host (King, Loomis & Hope, 1931). In uncropped soil, microsclerotia of Verticillium dahlia may germinate and sporulate over an extended period of time or they may only produce a few new sclerotia for a short time (Menzies & Griebel, 1967). The energy required appears to come from the reserves in the microsclerotia, rather than from organic substances in the soil. The depletion of nutrients makes the sclerotia less resistant to air-drying. These reports indicate that sclerotia are not always fully dormant structures but may continue to grow, sporulate and even multiply, using either endogenous or exogenous nutrients as the source of energy.

To summarize, it is clear from the available data that sclerotia are able to survive in soils, sometimes for long periods, but that survival is a complicated phenomenon and involves more than just complete dormancy of the structure. Viability appears to be retained more effectively at low and sub-zero temperatures, while it is progressively reduced at temperatures above about I~OC. Dry conditions and lack of competition from other organisms favour survival. Depletion of food reserves results in loss of resistance to environmental factors.

IV. STRUCTURE OF MATURE SCLEROTIUM

As early as 1853 Tulasne had realized from his work on Claoiceps that the sclerotium is only a stage in the life-history of fungi, and by 1887 the structures of many mature sclerotia had been studied. De Bary (1887) noted that the parenchyma-like tissues that form a high proportion of the structure are actually produced from closely interwoven hyphae. He distinguished between an outer layer of thick-walled, often pigmented cells that form a rind over the pseudoparenchymatous centre or medulla. In the majority of sclerotia, differentiation tends to produce concentric zones, but this is extremely variable. Only two layers, the rind and medulla, may be apparent in sclerotia of Aspergillus alliaceus (Rudolph, 1962), Pyronemu domesticum (Moore, 1962) and Clavicepspurpurea (de Bary, 1887). In other species the rind and/or medulla may consist of more than one region. Butler (1966) has given a clear review of fungal vegetative structures, in which she included details of the development and structure of sclerotia.

(I) Rind A rind is not present in some sclerotia such as those of Rhizoctonia solani (Townsend

& Willetts, 1954) and in most bulbils, e.g. Papulospura (Hotson, 1917). In some in- stances the outer cells of bulbils are empty. Gordee & Porter (1961) found from light- microscope studies that the microsclerotia of Vmticillium albo-atrum are composed of thick and thin-walled cells and the ultrastructural studies of Nadakavukaren (1963) and Brown & Wyllie (1970) confirmed these observations. The last-named workers

Survival of fungal sclerotia under adverse conditions 391 observed that many peripheral cells of the microsclerotia of V . albo-atrum lose their contents early in development, leaving degenerate hyaline cells among pigmented ones that contain organelles and are able to initiate new growth. The whole micro- sclerotium is enveloped in a pigmented mucilaginous matrix. Isaac (1949) distinguished between different isolates of Verticillium by the formation of microsclerotia consisting of thick-walled and darkened ‘cells’ or by the production of mycelial knots by the intertwining of torulose hyphae.

The time of development of the rind appears to be related to the way in which the sclerotium develops (Townsend & Willetts, 1954). Three different methods of development of sclerotia were described by these workers.

(i) The ‘loose ’ type, where sclerotial initials develop by increased branching and septation of ordinary hyphae, e.g. Rhizoctonia solani which forms a sclerotium consisting of loosely arranged brown, barrel-shaped cells with dense contents. Mycosphaerella ligulicola (Blakeman & Hornby, I 966) and possibly the microsclerotium of Verticillium albo-atrum (Pyke, 1961) are other examples.

(ii) The ‘terminal’ type, found in Botrytis c imea, Botrytis allii and Sclerotium cepiworum where development is by terminal branching of a hyphal tip followed by increased septation and fusion of the branches. Sclerotia of Py~one?t~~ domesticum (Moore, 1962) and Coprinus stercorarius (Brefeld, 1877) are also produced in this way.

(iii) The ‘strand’ type, as shown by Sclerotium rolfiii and Sclerotinia gladioli where branching and coalescence of mycelial strands give rise to the sclerotia. Other examples are Aspergillus alliaceus (Rudolph, 1962), Verticillium dahlia (Isaac, 1949), Colletotri- chum coccodes (Blakeman & Hornby, 1966).

The development of most sclerotia appears to fit into one of these three types. However, Hotson (1917) observed both terminal and intercalary development of bulbils in one species of Papulospora, and two methods of formation were found in morphogenetic studies of sclerotia in isolates of Sclerotinia sclerotiorum from different hosts (Willetts & Wong, 1971).

Where terminal development occurs, the rind forms relatively late and no further increase in size of the sclerotium takes place. The rinds of Typhula spp., Sclerotinia gladioli and Sclerotium rolfsii form during the continued growth of the sclerotium and the cells of the limiting layers are stretched tangentially. In Typhula there are two distinct types of rind (Corner, 1950). A thick cuticle-like deposit develops over the surface of the peripheral cells of sclerotia of Typhula intermedia, while in Typhula gyrans layers of thick-walled hyphae wind around the medulla to give an ‘epidermis’ of several layers and these may or may not be agglutinized. The sclerotial rinds of several fungi may have undifferentiated crusts of dried-up hyphae over their outer surfaces. Such a layer has been observed in Typhula sp. (Remsburg, 1g40), S. rolfiii (Townsend & Willetts, 1954) and, depending upon the conditions under which sclerotial development has taken place, Sclerotinia fimticola and related species (Willetts, 19683).

The Stereoscan electron microscope is a useful instrument for studying the surfaces of such structures as sclerotia since the dark pigmentation of the peripheral cells does not interfere with observations and the nature of the cells is such that they can be

392 H. J. WILLETTS viewed without collapsing. I studied (1969) the surfaces of sclerotia of Rhizoctoniu solani, Botrytis cinerea and Sclerotium rolfsi with this instrument. The skin over the surface of the sclerotia of S. rolfiii is almost continuous and formed from dried-up, pigmented hyphae, whose structure remains discernible. Presumably these hyphae dry out at an early stage of sclerotium development. The surface of the sclerotia of B. cinerea is made up of many closely packed hyphal tips which project outwards from the centre of the structure. These tips are thickened and retain their shape even in the high vacuum necessary for the preparation of the material for viewing and during the operation of the Stereoscan electron microscope. Ordinary vegetative hyphae and spores subjected to these conditions completely collapsed. Between the hyphae and over the more exposed areas of the surface a thin film was observed which was thought to be a layer of melanin or a layer of mucilaginous material in which melanin was incorporated. The peripheral hyphae of R. solani collapsed when being prepared for viewing, suggesting that the hyphae are thinner walled than those forming the surface layer of sclerotia of B. cinerea. The surfaces of the stromata of the brown rot fungi of fruits (Sclerotinia spp.) are similar in appearance to those of B. cinerea (Willetts, 1968~) . Both in culture, where the structures are often discrete, and on fruits these stromata may have an almost continuous skin or crust of dried-up aerial hyphae which becomes darkly pigmented. Gaps in this crust reveal thickened tips of rind cells beneath. Transmission electron-microscope studies showed that the walls of these cells are often greatly thickened but the cells retained some cytoplasmic contents (Willetts & Calonge, 1969b). Chet, Henis & Kislev (1969) found that ultra-thin sections of the sclerotia of S. rolfsii showed cell-wall residues, apparently part of the sclerotial skin, attached to the melanin-rich, thick-walled, and empty rind cells.

Whetzel (1945) attributed the dark colour of the rind cells of sclerotia to impreg- nated oxidation products of the dead protoplasmic contents. I suggested (19683) that a dark rind forms on all exposed parts of the stromata of the brown rot fungi in contact with air following drying or autolysis of the surface hyphae, and that the colour is due to the deposition and accumulation of dark, melanin-like pigments in and around the walls of the outer hyphae. If the rind is physically removed after differentiation, another quickly forms on most sclerotia under normal conditions. There are some exceptions to this, e.g. Typhula gyrans and Typhula sclerotioides (Corner, 1950). In culture, no pigmented rind develops against the glass side of a flask or Petri-dish. The brown pigment formed in the rind of sclerotia of Sclerotium rolfii was extracted by Bloomfield & Alexander (1967) and they found it to have the properties of a typical melanin. Chet, Henis & Mitchell (1967) concluded that sclerotial walls of S. rolfiii contain a melanin-like pigment which is absent from the walls of vegetative hyphae.

( 2 ) Cortex and medulla The tissue enclosed within the rind may be homogeneous or it may be further

divided into zones, the two main ones being the cortex and the medulla. I have not attempted to describe these separately as the distinction between them is not always clear.

The sclerotia of Botrytis allii have a rind of six to eight layers of rounded thick-walled

Survival of fungal sclerotia under adverse conditions 393 cells, a cortex, 3-4 cells thick, in which the pseudoparenchymatous cells are thin- walled and have dense contents, and a large central medulla where the filamentous nature of the hyphae can be clearly seen (Townsend & Willetts, 1954). The sclerotia of Botrytis cinerea have a similar structure, although the cortex is broader and the rind narrower.

Observations on the structure of mature sclerotia of Sclerotium rolfsii, with a light microscope, reveal a ‘skin’ and rind enclosing a cortex of thin-walled cells containing densely staining cytoplasm, and a medulla of loosely arranged, ordinary filamentous hyphae also filled with dense contents (Townsend & Willetts, 1954). Chet etal. (1969) studied the ultrastructure of the different regions in the sclerotium of this fungus and compared the fine structure of sclerotial and vegetative hyphae. They found that ribosomes and mitochondria are more abundant in vegetative cells whose walls are also thinner and less optically dense than those of sclerotial cells. The cells forming the cortex have thinner walls than the rind cells and contain many vesicles full of reserve food materials. Between the cortex and the medulla these authors distinguished an intermediate layer in which the cells had thicker walls than those of the cortex, and their cytoplasm was full of electron-dense granules, perhaps indicating polysaccharides, and membrane-bound electron-dense bodies. The inner layer (medulla) was composed of cells with extremely thick walls, thinner-walled cells full of reserve materials, and empty cells.

Willetts & Calonge (1969b) found three different types of hyphae in the medullary regions of stromata of Sclerotinia fructigena, Sclerotinia laxa and Sclerotinia laxa forma mali. Most of the hyphae are 4 7 p in diameter and contain food reserves in large vacuoles and lipid bodies, but, interspersed amongst these hyphae, is a network of smaller ones (1-2 p in diameter) with well-defined organelles. Thick-walled cells with some cell contents were thought to serve a protective function. Ultrastructural studies of vegetative hyphae of these fungi showed them to have thinner walls, more numerous mitochondria and more endoplasmic reticulum than sclerotial hyphae (Willetts & Calonge, 1 9 6 9 4

Scurti & Converso (1965), in their studies of the fine-structure of sclerotia of Typhula sp., observed two cell types - metabolically active cells and some that were thicker-walled and contained abundant food reserves. Wyllie & Brown (1970) did not find any tissue differentiation within sclerotia of Macrophomina phaseoli, but they noted the presence of a heavily pigmented, gelatinous matrix which bound the indivi- dual cells into a very compact structure. Other workers, e.g. de Bary (I 887) and Whetzel (1945), have described gelatinous matrices in which medullary hyphae are embedded. The matrix is probably of ecological and morphogenetic significance and will be considered in more detail in a later section.

V. STRUCTURE AND PHYSIOLOGY IN RELATION TO SURVIVAL

It would not be unreasonable to expect that, under uniform conditions, sclerotia with terminal and strand types of development, where a compact structure is formed, would be more resistant to deleterious agents than the loose type. Some evidence to

394 H. J. WILLETTS support this has been produced by Blakeman & Hornby (1966), who found that the strand-type sclerotia of Colletotrichum coccodes were much more persistant under the conditions of their experiments than the loose type of Mycosphaerella ligulicola. However, the morphological complexity and degree of differentiation does not neces- sarily relate to persistance in soils. The work on the longevity of sclerotia of Verticillium albo-atrum (Wilhelm, 1955) and Rhizoctunia solani (Pitt, 1964), both of which fit into the loose category of sclerotial development, refute this. However, the data available on work of this kind are limited, and no worth-while conclusions can be drawn from comparisons between only a few species.

Speculation may be made on the role in survival of the many common structural and some of the physiological features of sclerotia. Some of these features are possessed by other fungal structures, particularly mycelial strands, rhizomorphs and certain fruit bodies, and presumably serve the same functions in these structures as in sclerotia. Also, a particular structural or physiological feature may be effective against a number of different environmental factors and the factors themselves interact closely. Thus some repetition is inevitable in a discussion of this kind, but it is probably minimized and the information and arguments can be presented more clearly by dealing with each deleterious agent separately.

(I) Desiccation Garrett (1956) pointed out that conditions of extreme desiccation are seldom found

in soil where the sclerotia of many fungi develop and remain. Sclerotia formed on aerial parts of plants normally fall to the ground and are then protected by soil and litter or shaded by vegetation. In any one locality variations in microclimate provide conditions of varying degrees of severity, in some of which the organism can more readily survive and complete its life-cycle than in others. The position where fruits that have been mummified by Sclerotinia spp. fall in an orchard has a definite correla- tion with the subsequent formation of apothecia from them (Norton, Ezekiel & Jehle, 1923 ; Harrison, 1922).

Most seeds of higher plants can be stored in the air-dried state at temperatures between 15' and 2oOC. for periods ranging from a few months to several years. Generally the same applies to resistant fungal vegetative structures (Sussman & Halvorsen, 1966). On occasions, fungi may be subjected to extreme desiccation caused by dry winds and/or high temperatures. Also tissues may dry out as the result of frosts. If excessive water is removed from the hyphae, viability is reduced or lost completely. Therefore the ability to reduce water loss or to retain viability at low moisture content of the cell is of great ecological significance for the survival of the individual and thus of the species.

The rind serves the same function in the sclerotium as the epidermis in the plant. The close arrangement of the cells and the deposition of melanin and/or mucilaginous material in the walls, between the cells and sometimes as a layer over them, provide an effective barrier to the movement of water and substances into and out of the sclerotium. Several workers have indicated the need to rupture the rind before water can be taken up and germination proceed. Scott (1954) showed that abrasion of the

Survival of fungal sclerotia under adverse conditions 395 rind of Sclerotium cepivorum brought about germination of the sclerotium and Coley- Smith (1959) came to the same conclusion. Chet (1969) observed that the sclerotium of Sclerotium roljiii germinated more rapidly when the rind was punctured. The retention of the shape of rind cells when they are subjected to a high vacuum and the associated excessive desiccation indicates their resistance to drying. The extreme thickening of the walls of these cells, in addition to their close arrangement and covering ‘cuticle’, is probably also of importance in impeding desiccation.

The crusts or skins of dried-up hyphae that are found over the surface of some sclerotia must also reduce water loss. In nature, the outer surfaces of sclerotia are often protected by host tissue. Thus, the skin of fruits mummified by the brown rot fungi (Sclerotinia spp.) often remain intact (Willetts, 196th); Ciborinia forms a sclerotium by the digestion of the tissues between the lower and upper cuticles of the infected leaf, replacing them with closely interwoven hyphae (Whetzel, 1945) ; the sclerotium of the ergot (Claviceps purpurea) is partially embedded in the grain of rye or other grasses (de Bary, 1887); and in Cordyceps the cuticle of the infected host takes the place of a fungal rind in the resistant structure.

Apart from the presence of a protective layer on the outside of sclerotia, the compact nature of the cortex and medulla must also help to reduce the amount of physical damage and desiccation of the individual hyphae, particularly those in the centre of the structure. However, on occasions the amount of inter-hyphal moisture must be low and then resistance to further desiccation becomes the property of the individual cells. The majority of medullary hyphae have walls that are at least as thick as those of macroconidia. Also, the chemical composition of sclerotial hyphae is different from that of vegetative hyphae (Chet et al. 1967) in that they contain melanin which, like lignin, possibly reduces water loss.

Within the cells some water is bound osmotically and some is present simply as inclusion water in the three-dimensional network of peptide chains. Bound water is more difficult to remove from cells than is water of hydration and probably most of the latter is removed by slow air-drying. Only under extreme conditions is the bound water withdrawn from the cell, and when this happens the viability is reduced or lost. Desiccation of the protoplast must greatly increase the concentrations of the sub- stances dissolved in the aqueous phase within the hyphae, these substances having been already concentrated in the storage cells. It will therefore become even more difficult to withdraw any of the remaining water.

(2) Extremes of temperature The problems associated with desiccation and with resistance to high and low

temperatures are very similar. As with desiccation, the extremes of temperature that most fungal plant pathogens have to endure in the field are normally much less than those to which they are subjected when tested under experimental conditions. Unlike spores, which can be disseminated by wind and air currents, the large size of sclerotia tends to confine them to the locality where they develop. Where crops are cultivated, conditions may be adverse for several months, but during the growing season reason- able temperatures, moisture and light intensities normally prevail. Sclerotium-forming

25 B R E 46

396 H. J. WILLETTS fungi are found in most land habitats, with the possible exception of deserts, so that they may be subjected to low and/or high temperatures. Sclerotia appear to be better adapted to survive under cold rather than under hot conditions. Corner (1950) tabulated the temperature-relations of sixteen species of Typhula and concluded that low optimal temperatures characterize the genus; the species of the Sclerotiniaceae are mainly confined to temperate regions, especially the cooler parts of the north-temperate zone (Whetzel, 1945). There, low or subzero temperatures, down to -20' C. or even lower, may exist for periods of a few days to several months. In hot regions the air temperatures are probably never in excess of 50' C. and such high temperatures prevail for only a limited period each day. The temperatures in the soil, where most sclerotia are found, are considerably lower and the occurrence of microclimates must also be recognized.

(a ) Resistance to low and subzero temperatures Most sclerotia seem to be able to survive at subzero temperatures and some even

require a period of chilling to activate apothecial formation. Claviceps purpurea (Kirchoff, 1929; Mitchell & Cooke, 1968) and Sclerotinia fructicola (Ezekiel, 1921 ; Wormald, 1954) produce apothecia more readily after a period at a low temperature. Other sclerotia, such as those of Sclerotinia sclerotiorum, produce apothecia without any preconditioning, provided the sclerotia are mature.

Mazur (1968) reviewed the availabIe data on the survival of fungi after freezing and desiccation and concluded that the resistance of fungi to low temperatures depends on (i) the prevention of intracellular ice formation and (ii) their ability to resist dehydration. Slow air-drying is claimed to be a very effective method of dehydrating spores so that they retain viability at low and high temperatures (Mazur, 1968). Most of the water remaining in the spores may be bound water and therefore not available for the forma- tion of intracellular ice. Slow air-drying is a feature of sclerotia formed in culture and on aerial parts of plants, and the moisture content of those formed in the soil is probably reduced by the active exudation of water and drying out of the soil. How- ever, once the rind is fully differentiated, any water remaining in the interhyphal spaces should be trapped within the sclerotium if the rind functions efficiently. Temporary fluctuations in the moisture content of the soil or atmosphere should not substantially affect the water content of the sclerotium unless the structure is partly or completely immersed in water, when the rind probably becomes 'unsealed' as the result of imbibition of water.

Other mechanisms, apart from slow air-drying, must, however, operate in resistance to subzero temperatures. There are many instances of sclerotia that have high water contents surviving subzero temperatures for long periods. Sclerotia of Sclerotinia sclerotiorum and Sclerotinia fructicola were soaked in water until they were completely saturated and then kept for 5 months at - 14' C. When they were returned to room temperature, abundant conidia of S. fructicola were formed in 3 days and the sclerotia of S. sclerotiorum produced numerous clusters of microconidia in sterile distilled water, and mycelium on agar plates (Willetts, unpublished).

High concentrations of solutes within the cell depress the freezing-point and reduce

Survival of fungal sclerotia under adverse conditions 397 the formation of intracellular ice crystals to some extent. Mazur (1970) discussed the importance of certain protective additives in survival under cold conditions. The protective effect of glycerol was discovered by Polge, Smith & Parkes (1949); Lovelock & Bishop (1959) demonstrated protection by dimethyl sulphoxide. Other solutes, chiefly sugars, particularly sucrose (Meynell, 1958) and inositol (Webb, Cormack & Morrison, 1964), and some macromolecules also protect cells under certain conditions (Doebbler, 1966). Mazur (1970) concluded that ‘the ability of a cell to survive freezing may depend more on protection of the cell surface than on protection of the cell interior. Perhaps the cell interior is protected by the high concentrations of macro- molecules normally within it.’

Roberts (1969), in a review of the cold-hardening of higher plants, outlined the main problems that plants have to overcome when the temperature drops. He advanced an hypothesis that in cold-hardened plants there is a substitution of modified forms of proteins for the proteins that carry out particular functions at higher temper- atures. These isozymic substitutions could change the metabolism and, at low temper- atures, help the plant to withstand the accumulation of high concentrations of meta- bolites, reduce protein sensitivity to cold, increase the water-holding capacity of the proteins and induce the formation of protective substances. Although this hypothesis relates to higher plants, many of his examples were taken from micro-organisms and poikilothenns. The basic mechanisms of cold resistance and adaptation are probably similar in many different types of organisms.

(b) Resistance to h&h temperatures It has been well established that the heat resistance of microbial cells increases with

decreasing humidity. Precht, Christophersen & Hensel(1955) demonstrated this when they showed that superheated steam, which acts like dry air, has less killing effect on bacterial cells at 140-15oOC. than wet steam at IOOOC. Small changes in water content may have a considerable influence on survival. The available evidence suggests that proteins are more stable in a dry state and that the effect of water on the heat resistance of micro-organisms may be explained by its effect on the stability of proteins (Hansen & Riemann, 1963). The death of a cell results from a physical change in the structure of essential macromolecules when the water bound to the molecules is removed. Some chemicals that are capable of forming hydrogen bonds with proteins, similar to those formed with water, can replace the bound water and retain the integrity of the macromolecule. Inositol was found to be an efficient stabilizer against desiccation, probably because of its symmetry and structural similarity to water (Webb, Cormack & Morrison, 1964). Glycerol and sucrose were found to protect proteins in aitro from denaturation by heat (Jarabak, Adams, Williams- Ashman & Talalay, 1962; Yasumatsu, Ohno, Matsumura & Shimazono, 1965) and also in vivo (Molotkovskii & Zhesthova, 1964). Proteins in the medium appear to increase the stability of enzymes and other proteins to heat by combination brought about by electrostatic attraction (Hansen & Riemann, 1963). This was demonstrated by Precht et al. (1955), who found that gelatine protects pectinase and penicillinase against high temperatures. Also a heat-stable complex may be formed between an

25-2

398 H. J. WILLETTS enzyme and its substrate. As with resistance to cold, protein protection appears to be a surface phenomenon, but similar protective reactions may also take place between proteins inside the cell (Hansen & Riemann, 1963).

(3) Physiological features that injuence survival against several different factors

It is now appropriate to discuss certain physiological features of sclerotia that could be of significance in respect to survival at high and low temperatures, under conditions of desiccation and alsu against harmful radiations. These are: (i) the active exudation of water from the surface of sclerotia as they mature and a rind begins to differentiate; (ii) the types of carbohydrate that accumulate in sclerotia during their development and the secretion of carbohydrates into the medium; (iii) the formation of a gelatinous matrix in which medullary hyphae are embedded and which accumulates over the surfaces of some sclerotia.

(a) Exudation of water Water is generally considered to be extruded in various biological reactions (Lewin,

1970). It can be extruded as a result of a rise in temperature; variation of pH of the medium towards the isoelectric point of the proteins (proteins contain the minimum amount of water at their isoelectric points) ; in the case of a solid, by a lowering of the relative humidity; and by an increase in ionic strength. Hydrophobic groups in aqueous solutions are surrounded by particular orientations of the neighbouring water molecules, and if these groups associate the water molecules may be squeezed out. Lewin used the example of the association of phenylalanine with tyrosine or leucine and the resulting extrusion of significant amounts of water.

There are many early references to the extrusion of water from fungal tissues (Wilson, 1948). Everyone who has studied sclerotium formation will have observed the accumulation of drops of moisture on their outer surface. This starts soon after the sclerotia begin to increase in size. Cooke (1969) found that water-loss was most rapid during the early part of the exudation period, particularly in the first 3 days, and that after 13-16 days the sclerotia contained about 60% water. His further observations showed that the amount remained reasonably constant at this level and presumably the rind was fully differentiated by this time. The remaining interhyphal moisture probably forms a thin layer on the outer surface of the medullary hyphae and contains sugars such as mannitol, inositol, glucose and trehalose. Thus the medullary hyphae will be partly suspended in a solution that could stabilize proteins against desiccation and the damaging effects of extreme temperatures.

This active exudation of water lowers the moisture content of tissues and, as already stated, even a small change in the moisture may have a very significant effect on survival. Also, it is associated with the secretion of sugars, which is discussed below.

Survival of fungal sclerotia under adverse conditions 399

(b) Soluble carbohydrates - accumulation and secretion The available data on the carbohydrates present in sclerotia indicate that a number

of soluble sugars accumulate during sclerotial development (Ergle, I 948 ; Kitahara, Igsoa, b ; Le Tourneau, 1966). Cooke (1969) made a detailed study of the concentra- tions of soluble carbohydrates at different stages of development of sclerotia of Sclerotinia sclerotiorum and Sclerotinia trifoliorum and of the reduction of the level of hydration by the active and selective exudation of water. The water first accumulated as small or large drops on the surface from which it then evaporated or leaked away. At all stages these sclerotia contained trehalose, mannitol, a small amount of glucose and traces of inositol. The exudates always contained trehalose, inositol and traces of glucose and, in addition, those of S. sclerotiorum also contained mannitol. Throughout sclerotium development, total sugars slowly increased. Kybal (1964) suggested that excretion of ' honeydew' by Claviceps is a mechanism for losing carbohydrates from the ergot to maintain a favourable internal C : N ratio. Thus, the active and selective exudation of water and sugars helps to keep osmotic balance within the sclerotial cells and prevents disruption of metabolism. Also a gradient is maintained so that sugars continue to move into the developing sclerotium.

There are other records of fungi being able to secrete reducing sugars into the medium. Birkinshaw, Charles, Hetherington & Raistrick (193 I) found that some Aspergillus species may dispose of 50% of the consumed glucose into the medium as mannitol. Lewis & Smith (1967) considered that mannitol, in addition to functioning as a means of carbohydrate storage in fungi, may also be of importance in maintaining high osmotic pressures in the mycelium during periods of environmental water stress.

The process of secretion of soluble sugars, particularly those reported by Cooke (1969), is probably of very great significance in survival under several environmental conditions. Also these sugars are present within the cells where they may serve the same function. Trunova (1963) found that only those sugars that enter plant cells and are subsequently metabolized are effective in frost resistance. This may indicate that they do not function directly by lowering the freezing-point or as protein protectants. Obviously a great deal of further work is required on the relationship of sugars to survival.

(c ) Gelatinous matrix Mucilages are found in higher plants, algae and fungi. In water they swell to form

gels. There are numerous reports on the mucilages of higher plants, but they have been given very little attention in the fungi. Whetzel(1945) in his key to the genera of the Sclerotiniaceae used as a diagnostic feature the presence or absence of a gelatinous matrix. The sclerotia of such forms as Botrytis cinerea have a medulla made up of loosely interwoven hyphae ' embedded in a hyaline flexible to gelatinous matrix, there being no interhyphal spaces'. This was clearly illustrated by de Bary (1887).

The first complete study of the ontogeny of gel tissues of selected fungi was made by Moore (1965). She found that they are formed either by hyphal disintegration or by

400 H. J. WILLETTS direct secretion of mucilage from the protoplasm through small pores in the walls. With the absorption of water the mucilage becomes gelatinous, and if produced in small amounts the gel ensheaths the hyphae and some may collect between the hyphae. The hyphae may be loosely arranged or form a compact and organized tissue. The function of the mucilage is not known, but it is thought to serve as a water reserve (Wolf, 1958; Ingold, 1959).

Moore (1965) suggested that gels may be present in many more fungi than has been assumed. The sclerotia and mycelia of Sclerotinia fructicola and Sclerotinia sclerotiorum are not considered to possess significant amounts of mucilage, but in recent work in our laboratories large amounts of mucilage have been obtained from these fungi (A. L. Wong & H. J. Willetts, unpublished). The mucilage was obtained during attempts to extract proteins from vegetative and sclerotial material for electro- phoretic and antigenic studies. Preliminary analyses suggest that the slimy materials are mucopolysaccharides. Such substances may have a protective role against de- hydration (Webb, 1965). Comer (1950) described different degrees of agglutination of the sclerotia of Typhula spp. and he concluded that this indicates an evolutionary process giving better adaptation to xerophytic and low-temperature conditions.

Evidence of mucilage was found in electron microscope studies of stromatal hyphae of the brown rot fungi (Sclerotinia spp.) (Willetts & Calonge, 1969a). A thin electron- dense layer was found on the more exposed stromatal hyphae, which was similar to that found on the lateral walls of separated macroconidia and aerial hyphae. The absence of an outer electron-dense layer on hyphae growing submerged in solid media, in liquid cultures and in the medulla of sclerotia where desiccation was not significant, may indicate that the electron-dense layer is formed by the collapse and drying out of an outer, probably mucilaginous layer. The rind walls of the sclerotia of Sclerotinia sclerotiorum also have a rough outer electron-dense layer which was not observed on the outer surfaces of walls of medullary hyphae (Jones, 1970). Turian (1966) found a similar layer on the outside of conidia of Neurospora crassa which he thought was mainly of phospholipid associated with proteins and seemed to be connected with the hydrophobic properties of the conidia.

The agglutination found in some sclerotia and the thin, electron dense layer around individual hyphae may have a protective role against dehydration caused by drought or extreme temperatures. This could protect the surface of the hyphae in a manner similar to that suggested by Hansen & Riemann (1963). Mucilage may also be abund- ant within the hyphae, where it could have a stabilizing effect on intracellular proteins.

Apart from its protective role, mucilage could also be of importance in the rapid uptake and retention of water immediately before the germination of sclerotia. Further work on fungal mucilages could provide interesting data on survival, germina- tion and also on the genesis of complex fungal structures.

(4) Radiations Most of the work on the effect of radiations on fungi has been carried out in the

laboratory and little is known of the chronic effects of solar radiations that reach the surface of the earth. In nature, fungi are probably continually exposed to varying

Survival of fungal sclerotia under adverse conditions 401 levels of ionizing, ultraviolet and infra-red radiations. High levels of radiations may affect sclerotia formed on aerial parts of plants and could be lethal, but the majority of sclerotia are formed in the soil, where they obtain considerable protection. Those on the surfaces of the ground are, again, generally protected by vegetation and plant debris.

Many of the features already discussed in other sections will also be effective in helping sclerotia to overcome damage by harmful radiations. Melanized fungal spores are more resistant to ultraviolet radiations than are hyaline ones and work on this topic has been reviewed by Sussman (1968). The structure and composition of the rind, the active exudation of water, air-drying of tissues, and possibly mucilage, may all play their parts in providing resistance to harmful radiations. However, this is yet another aspect of survival that requires further investigation.

( 5 ) Starvation Sclerotia are normally only formed on well-nourished mycelia (Hawker, 19573) and

there is a considerable build-up of food reserves in the medullary cells. Ergle (1948) found that polysaccharides accumulated during the development of Phymatotrichum omnivorum and that these consisted mainly of glucose polymers. The major insoluble carbohydrate reserve of Sclerotinia libertiana appears to consist of a glucan - probably glycogen (Kitahara, 195ob), and during germination this is probably utilized, after conversion to soluble carbohydrate, together with trehalose and mannitol (Le Tourneau, 1966). Cooke’s work (1969) on soluble carbohydrates in sclerotia has already been described. Lipids are often a reserve material in sclerotia and several studies have been made on Clavicepspurpurea (Kybal, 1964; Howell & Fergus, 1964; Mitchell & Cooke, 1968). The first-named worker determined that 31-46% of the air-dry weight of sclerotia of this fungus may consist of lipid materials.

The reserves of food stored in medullary cells have been clearly observed in electron- microscope studies of sclerotia of Typhula sp. (Scurti & Converso, 1965), Sclerotium roljsii (Chet et al. 1969)~ Sclerotinia fructigena and Sclerotinia Iaxa (Willetts & Calonge, 1969 b), Verticillium albo-atrum (Brown & Wyllie, 1970) and Macrophomina phaseoli (Wyllie & Brown, 1970). The cells are often so packed with lipid bodies and vacuoles that mitochondria, endoplasmic reticulum and nuclei cannot be seen, though they must be present because new growth is initiated from these cells. Probably there is limited growth at most times following maturation and there are ample food reserves to support this type of growth for long periods. When conditions are optimal and/or a particular stimulus is provided, more extensive growth may take place, resulting in the formation of apothecia, large numbers of spores or mycelium.

(6) Resistance to toxic chemicals and enzymic activities of other organisms Chemicals, toxic to fungi, may be present in the natural environment, either

introduced by man or produced by other organisms.

(a) Fungicides Many data on fungicides have accumulated and information relevant to the present

consideration has been reviewed by Byrde (1965). Anti-fungal compounds usually

402 H. J. WILLETTS enter the cell and inhibit the activity of one or more enzymes. The protectant type of fungicide is actively taken up and accumulates in fungal cells at concentrations far in excess of those in the environment. Before penetration into the cell can take place the chemical has to possess some degree of lipid solubility. However, some compounds with low lipid solubility are toxic because they act at the cell surface or cause inactivation of extracellular enzymes, which play such a vital role in the metabolism of fungi. The structure and chemical composition of the cells of the rind must provide an excellent impermeable barrier and prevent the medullary hyphae from direct contact with toxic chemicals. Several references to the effectiveness of the rind as a means of preventing water uptake have already been given, and Chet (1969)~ using radioactive isotopes, demonstrated that only immature sclerotia, i.e. before complete differentiation of the rind, are capable of obtaining metabolites from the surrounding medium. He con- cluded that the rind is a mechanical barrier preventing the uptake of materials from the medium. Even if the peripheral cells are killed by toxic substances, the layer of which they form a part may still form an effective barrier so that the inner hyphae remain undamaged.

(b) Enzymes Survival in some instances may depend upon resistance to lytic factors such as

antibiotics and enzymes (Lloyd, Noveroske & Lockwood, 1965) that are produced by other organisms. Reference has already been made to some work dealing with this, but there are numerous reports that illustrate the antagonistic effects of other organisms on fungi. Some fungi are particularly susceptible to lysis by other micro-organisms while others are resistant to microbial degradation. Chlamydospores and sclerotia are particularly resistant (Potgieter & Alexander, 1966) as are the pigmented hyphae of Helminthosporium sativum, Rhizoctonia solani and Alternaria solani (Lockwood, 1960). Much evidence indicates that melanin or related substances may have great ecological significance in reducing damage by the lytic action of micro-organisms. Chet et al. (1967) found that sclerotial walls of Sclerotium rolfii contain a melanin-like pigment which is absent from hyphal walls and they suggested that this pigment is important in providing resistance to biological and chemical degradation. Support for this has been given by Potgieter & Alexander (1966) using Fusarium solani, Neurospma crassa and R . solani, Bloomfield & Alexander (1967) with S. rolfii and Aspergillus phoenicis and Kuo & Alexander (1967) with melanin-free and melanin-containing strains of Aspergillus nidulans.

The question arises, how does melanin prevent or reduce lytic break-down? Two mechanisms have been suggested.

(i) The chitin and probably also the glucans and other components of the hyphal wall are protected by the melanin secreted by the protoplast, either as a deposit on the outer surface of the hyphae and/or by the formation of a complex, particularly with chitin, in the cell wall.

(ii) The melanin acts as an inhibitor of the enzymes, mainly chitinases and glucan- ases, associated with biological degradation.

Bull (1970~) obtained a crude lytic enzyme complex of highlv purified 8-1,3-

Survival of fungal sclerotia under adverse conditions 403 glucanase and chitinase produced by a soil streptomycete and used this to study the degradation of hyaline and melanized cell walls of Aspergillus nidulans. Although he used an in vitro system he was careful to simulate as far as possible in vivo conditions. He found that the degree of lysis depended on the extent of melanization of the cell walls and all the enzymes in the lytic preparation were sensitive to native fungal melanin, but chitinase was inhibited to a greater degree than any of the others. The removal of the layer of extracellular material did not affect the susceptibility of hyphae to lytic enzymes and was thought to be unimportant as an anti-lytic barrier. There appeared to be a non-specific binding of the enzymes associated with wall degradation with melanin and therefore non-competitive inhibition. He concluded that there is electrostatic attraction between the enzymes and melanin. Further support for this has been given by a detailed investigation of a cellulase-melanin model system (Bull, 197ob).

There are examples from higher plants for both enzyme inhibition and substrate complexing as the means of protection against biological degradation (Bull, 1 9 7 0 ~ ) : organic substrates complex with tannins to resist enzymic breakdown (Lewis & Starkey, 1968) ; lignin protects cellulose from cellulase (Fuller & Norman, 1943) ; lignin-protein complexes are much more resistant to microbial attack than lignin- protein mixtures (Lynch & Lynch, 1958). Bull (1g7ob) concluded that the protection of protein by lignin is a physical affect and not by chemical bonding.

Thus the available evidence indicates that melanin is important in overcoming chemical and biological degradation, but the means by which this is achieved have not yet been resolved.

VI. CONCLUSIONS

Although there is an extensive literature on survival of sclerotia under adverse conditions many of the mechanisms are still not well understood. Usually, conditions remain constant for only a short time and the interaction between factors adds further complications to studies of this type. Most of the data have been collected from laboratory studies and many of the new techniques of electron microscopy, histo- chemistry and biochemistry are contributing greatly to the overall understanding of survival. However, this information needs to be substantiated by observations and trials carried out under natural conditions.

It is obvious from survival studies that sclerotia are highly adapted to resist conditions that are too severe for the mycelium and spores. Despite this, it must be expected that, of the many sclerotia formed on a host plant or in soil, some or, on occasions, many will lose their viability under certain conditions. However, the inoculum potential of only a few such comparatively large bodies that are richly supplied with food reserves is very great, particularly if they germinate to give rise to fruiting bodies and spores. The sclerotium is a very formidable means of perpetuating a fungal species.

404 H. J. WILLETTS

VII. SUMMARY

I. Sclerotia have been found to survive under adverse environmental conditions for long periods. Their viability is retained at low and subzero temperatures, while it is progressively reduced at temperatures above about 15' C. Dry conditions and lack of competition from other organisms favour survival. Depletion of food reserves results in loss of resistance to environmental factors. 2. The majority of sclerotia consist of an outer layer of thickened, close-fitting, often

pigmented cells that form a rind over a tissue of closely interwoven hyphae. A cortex and medulla is often discernible.

3. Features of sclerotia that are important in overcoming the harmful effects of desiccation and extremes of temperature include : the presence of a rind and sometimes an additional covering of either fungal or host tissue; the compact nature of the sclerotium; miscellaneous protectants on the surface of and in the hyphae; melanized hyphal walls ; high intracellular osmotic concentrations; slow air-drying to lower moisture content. Also the buffering action of the soil and the protection afforded by vegetation and plant debris are important.

4. The active exudation of water, the accumulation and secretion of soluble carbo- hydrates during sclerotium development and maturation, and the formation of large amounts of mucilage are of significance in resistance to desiccation, extremes of temperature and radiations.

5. The nature and the pigmentation of the rind together with the site of sclerotium development may give protection against harmful radiations.

6. The loss of sclerotium viability caused by toxic chemicals in the soil and atmos- phere or by the enzymic activities of other organisms is reduced by the rind and the melanization of hyphal walls.

7. Survival under starvation conditions is achieved by the accumulation of abundant food reserves (lipids and/or carbohydrates) and a low level of metabolic activity. In the presence of a compatible host exogenous sources of energy are sometimes used.

8. Although the sclerotium is highly adapted to survive adverse conditions for long periods, sometimes micro-environmental conditions may be so severe that only a few sclerotia can retain their viability. However, even a few such comparatively large bodies, rich in food reserves, have a considerable inoculum potential.

I am grateful to several colleagues for reading the manuscript, particularly Professor L. E. Hawker and Dr N. J. Hannon.

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