Bowling Green State University Library C

BIOLOGICAL STUDIES ON THE BASIDIOMYCETE PANUS TIGRINUS:

EFFECT OF LIGHT ON PILEUS FORMATION AND ANALYSIS

OF SPORE WALL COMPOSITION

Thomas Floyd Bobbitt

A Dissertation

Submitted to the Graduate School of Bowling Green State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

August 1975

Approved by Doctoral Committee

BOWLING GREEN STATE UNIVERSITY LIBRARY C .

fi 781028 C

ABSTRACT

Continuous fluorescent light resulted in increased numbers of abnormal fruiting bodies in the Basidiomycete, Panus tigrinus. Increased duration of the dark period prior to the time of continuous light decreased abnormal fruits. The number of normal fruiting bodies remained relatively constant regardless of light/dark treatment. Under total darkness no fruiting occurred. Light induced melanin- mediated changes which directly influenced fruiting. Studies of the development of varieties of P. tigrinus and their light-induced abnormal forms were made with scanning electron microscopy (SEM). Previous reports of var. tigrinus being pseudoangiocarpic were not confirmed. Both var. tigrinus. and var. squamulosus and their abnormal forms all developed gymnocarpically. Regardless of light conditions, fertile basidia and spores were produced on primordia before cap. expansion. Abnormal primordia of both varieties developed pockets of concentrated basidia desig nated as "niduli”. Basidiospores developed by the expansion of the wall of the external half of the swelling (1 * apophyse) situated at the tip of sterigmata. Uneven growth of a developing spore brought about a shift of approximately 4-5° of the spore apex, causing the spore to reposition in relation to the sterigmata. Germination of spores occurred by swelling and sloughing of the outer walls. The germ tube formed from a bulging of the outward-facing spore wall. The spore wall in both tigrinus varieties was com posed of five layers. Specific chemical tests were con ducted to determine their composition. The innermost layer, or endosporium, consisted of chitin microfibrils and was surrounded by an electron-dense mesosporium as a sublayer of the episporium. The perisporium layer was probably com posed of hexuronic acid. The outermost zone was a thin electron-dense ectosporium layer composed of lipid and pro tein. Plasma membrane invaginations along the spore wall were postulated to be collection sites for precursor materials. iii ACKNOWLEDGEMENTS

I would like to express my gratitude to Dr. R. E. Crang, my major advisor, for his guidance and patience throughout the period of this study. Thanks is also extended to Dr. Martin A. Rosinski for his encouragement and helpful ideas. The assistance of Mr. Patrick Ashbaugh in the prepara tion of TEM and SEM samples is also deeply appreciated. Sincerest thanks is extended to the National Sigma Xi Com mittee on Awards for their Grant-in-Aid of Research which made possible the financing of portions of this study. A special thank you is extended to my parents whose long years of sacrifice and encouragement made possible my graduate education. I only regret that my father did not live long enough to see my work completed. iv

TABLE OF CONTENTS Page ACKNOWLEDGEMENTS...... Ui

LIST OF TABLES...... V

LIST OF FIGURES...... vi

INTRODUCTION ...... 1

LITERATURE REVIEW ...... 4 Historical background ...... 4 Morphology of basidiocarp development ...... 6 Factors affecting fruiting ...... 8 pH...... 8 Gases...... « 8 Humidity and nutrition ...... 9 Light...... 10 Basidiospores ...... 16 Development of the sterigmata and spores .... 18 Spore germination ...... 19 Fungal cell walls ...... 21 Spore walls ...... 22 Basidiospore walls ...... 24

LIST OF REFERENCES...... 29

ARTICLE 1» Light effects on fruiting in Panus tigrinus var. tigr inus ...... • 38

ARTICLE 2« Basidiocarp development of the two varieties of Panus tigrinus and their light-induced abnormal forms ...... 43

ARTICLE 3» Basidiospore formation and germination of Panus tigrinus as studied by scanning electron microscopy ...... 50

ARTICLE 4î Structural and microchemical analysis of Panus tigrinus spore walls ...... 68 V LIST OF TABLES Table Page

LITERATURE REVIEW I. Summary of effects of light on fruiting of some Hymenomycetes as reported in the literature...... 11 II. Average time taken for development and ripening of individual spores ...... 20 ARTICLE 1 I. Observations of 20 cultures from six light and dark treatments...... 40 ARTICLE 4 I. The composition of P. tigrinus basidiospores as determined by various staining techniques • . 84 vi LIST OF FIGURES Figure Page LITERATURE REVIEW 1, Schematic diagram of the wall layers of a hypothetical smooth, hyaline basidiospore with ectosporium (EGT.), perisporium (PER.), episporium (EPI.) and mesosporium (MES.), and an endosporium (END.). (After Perreau- Bertrand, 1967)...... 38 ARTICLE 1 1. Thin, smoothly appressed mycelial growth characteristic of P. tigrinus receiving continuous light or alternating light and dark treatments ...... 41 2. Rough, cottony mycelial growth typical of cultures receiving pretreatment of two to three weeks darkness ...... 41 3. Primordia from culture at time of removal from two or three weeks dark treatment . . . . 41 4. Normal appearance of basidiocarp showing typical umbilicate morphology ...... 41 5. Morphological appearance of abnormal basidiocarps with anastomosing gills covering pileus ...... 41 6. Total number of normal and abnormal, and both types of fruiting bodies plotted against increasing light exposure ...... 40 ARTICLE 2 1. Var. tigrinus» basidiocarp primordium with highlighted area showing region where basidia are located...... 46 2. Var, tigrinus t basidium with its basidiospores from high-lighted area shown in Fig, 1 . 46 3. Var. squamulosus» young cap with outfoldings representing early gill formation ...... 46 Var. tigrinust young cap with gill outfold ings extending upward ...... 46 VI1

Figure Page 5. Var. tigrinus: normal fruiting body showing mature gills...... 48 6. Var. squamulosus: normal fruiting body with lateral development of hyphal membrane over maturing gills (arrow)...... 48 7. Mature abnormal fruiting body of var. tigrinus with interlacing gills ...... 48 8. Early abnormal cap region of var. tigrinus with niduli (arrows). Above the indicated niduli are developing interlacing gills .... 48 9. Var. tigrinus: abnormal cap showing detail of nidulum with cluster of basidia...... 48 ARTICLE 3 1. Basidium with flattened apex and four sterigmata protrusions...... 59 2. Basidium with developing sterigmata ...... 59 3. Later stage of sterigmata formation ...... 59 4. Basidium with the l'apophvse developed at tips of sterigmata...... 59 5. Maximum elongation of sterigmata and early enlargement of the spore ...... 61 6. Ballooning of the external half of the 1* apophyse...... 61 7. Further development of the new spores ..... 61 8. Basidiospores near maturity, after their repositioning to the same axis as that of the sterigmata ...... 61 9* Panus tigrinus (interpretive drawing). The directional shift of the apex of a developing basid iospore...... 63 10. Spore prior to germination...... 65 11. Spore approx. 25 hrs after being placed in germination medium. Outer wall layers have begun to slough ...... 65 • • * Vili

Figure Page 12. Outer wall layers detached from the spore ... 65 13. Spores in liquid medium for approx. 55 hrs. A bulge has formed on the outward-facing wall ...... 65 14. Approx. 72 hrs in germination medium the germ tube has formed...... 65 15. Germinating spore (approx. 72 hrs) with part of outer wall attached at the right side ... 65 ARTICLE 4 1. Spore showing wall microfibrils with dense region...... 92 2. Enlarged area of dense region showing fibrils superimposed over the region...... 92 3. Spore appearance after either cellulase or acetate buffer treatment ...... 92 4. Chitinase treated microfibrils showing com plete destruction of wall fibrils...... 92 5. Whole, untreated spores ...... 94 6. Basidospores treated with commercial Clorox. The outer walls are swollen and broken .... 94 7. Spore treated for lipid extraction showing roughening of outer wall...... 94 8. Spores treated with trypsin with outer walls of one removed and seen at lower left ..... 94 9. Spore treated first to remove lipids and then treated with trypsin showing roughened inner wall and the removal of the outer wall .... 94 10. Longitudinal section of spore showing nuclear region (N), lipid bodies (L), and mitochondrin (M). Arrows point to invaginations of the plasma membrane...... 96 11. Coiled structure located within lipid bodies...... 9° ix Figure Page 12. Hilar region of spore (H) with dense osmophilic bodies and a condensed structure which appears in contact with an osmophilic body ...... 96 13. Cross section of.spore showing invagination of the plasma membrane ...... 98 14. Detail of an oblique section through an invagination ...... 98 15. Section through the spore wall showing the five layers, ectosporium (EOT.), perisporium (PER.), espisporium (EPI.) and mesosporium (MES.), and an endosporium (END.) ...... 98 16. Section of a spore treated with chitinase. The plasma membrane and endosporium have been disrupted...... 98 17. Section through the wall of a spore treated with chitinase. The perisporium (second layer) is reduced in thickness, and the densities of the episporium and mesosporium (third and fourth layers) are reduced ...... 100 18. Section of spore treated with cellulase. Arrow points to separation of ectosporium (outer wall) layer ...... 100 19. Section through spore wall treated with lipid solvents showing decline in density of the two middle layers (episporium and mesosporium) ...... 100 20. Shadowed spore wall showing three layers, outer layer (A), middle layer (B), and inner layer (C)...... 102 21. Shadowed spore treated with chitinase, show ing lack of microfibrils ...... 102 22. Shadowed spore treated to remove lipid material. All layers remain ...... 102 23, SEM micrograph of spore wall material with no chemical treatment ...... 102 I

INTRODUCTION

Mushrooms of the genus Panus are Homobasidiomycetes belonging to the order Agaricales and traditionally placed in the family Agaricaseae, but more recently Singer (1962) has placed the genus in the Polyporaceae. Panus belongs to the tribe Lentineae and is closely related both to the genera Pleurotus and Lentinus. According to Singer (1962) the genus Panus differs from the other two genera in that there is an absence of marked reduction of the subhymenium, and the hymenophore trama is irregular. Both Pleurotus and Lentinus have a distinct subhymenium. In addition, Pleurotus has a pronounced irregular hymenophore trama. Panus is cos mopolitan, being distributed throughout the tropical and temperate regions of the world. Singer (1949) lists eight species in the genus and suggests there may be as many as twenty. The species Panus tigrinus (Bull, ex Fr.) Singer, is now recognized as consisting of two varieties: the former species Lentinus tigrinus (Bull.) ITies, and Lentodium squamulosum Morgan (Rosinski and Robinson, 1968). Both varie ties are widely distributed in eastern North America. Martin (1956) reported that Lentodium had been collected from Massa chusetts to Iowa, and in eastern Iowa was commonly found grow ing on fallen willow and soft maple logs in river bottoms. Graham (1970) stated that var. tigrinus was found growing on 2 dead hardwood from Minnesota to New England and as far south as Tennessee. The squamulosus variety is found only in North America and is the most common form of the genus (Miller, 1972). The growth habit for both varieties is often sub- gregarious or caespitose (Smith, 1908). Distinction between the two varieties can be readily made by the fact that P. tigrinus var. tigrinus produces typical agaric morphology with distinct gills, while var. squamulosus produces gills which anastomose early in develop ment and become covered by a hyphal membrane. The anastomosed gills form pockets which are lined with a hymenium (Bobbitt, 1965). Mature fruiting bodies are fleshy in fresh specimens of both varieties, but upon drying the bodies become leathery to woody. Stems are thin and flared toward the cap and some times divide into several branches, each supporting a cap. The caps are umbilicate to semiorbicular and are light tan to cream colored. The dorsal surface of the pileus is covered with dark brown hair-like scales, especially toward the center. The flesh is white to cream colored. In var. tigrinus the gills are white, narrow and decurrent with the edges eroded to serrate (Graham, 1970)« Light is known to play an important role in the regu lation of fruiting in many fungi, and preliminary studies have suggested that light may affect the morphological appearance of P. tigrinus basidiocarps. The present study was undertaken to elucidate the effects of light on basidio- 3 carp development of P. tigrinus. Associated with the develop ment of the fruiting body is the formation of basidia and basidiospores. A second objective was to study the basidiospore, its development on the sterigma, the morphological changes it undergoes during germination, and the morphologi cal and chemical composition of the spore wall. This dissertation is organized into five divisions. The first division is a review of the literature concerning« 1) factors influencing fruiting, 2) development of the basidio carp, 3) basidiospore formation, 4) basidiospore germination and, 5) basidiospore wall composition. The second division is a published manuscript describing the effects of light/dark treatments on fruiting of P. tigrinus var. tigrinus. The third division is a published manuscript dealing with the development of both varieties of P. tigrinus and their light- induced abnormal forms as studied with scanning electron microscopy. The fourth division is a manuscript on the development of the basidiospore and germination of the basidiospore of var. tigrinus. The fifth division is a morphological and chemical analysis of the spore wall of var. squamulosus. LITERATURE REVIEW

Historical Background The earliest report of P. tigrinus var. tigrinus was by Bulliard (1791) who gave it the species name tigrinus because of the tiger-like markings on the pileus, and placed it in the genus Agaricus. Persoon (1801) and Fries (1821) both referred to this mushroom as a member of the genus Agaricus. Later, Fries (1838) reevaluated the species and transferred it to the genus Lentinus, considering it to be closely related to L. lepideus Fr., the type species for the genus Lentinus. Singer (1962) restudied the genera Lentinus and Panus, and concluded that L. tigrinus was more closely related to members of the genus Panus. He stated» The sterile tissue of the lamellae has a different structure in these species, and the affinities of these species are with quite different groups. P. tigrinus is so similar to P. crinitus that it is often almost impossible to"~tell the two species apart when they grow together in the American sub tropics because P. crinitus sometimes becomes glabrous in age under the influence of heavy rains, etc. L. lepideus, on the other hand, does not resemble any species of Panus. Lyman (190?) and Martin (1956) have reviewed the literature concerning var. squamulosus. These authors indi cate that the first reported collection of var. squamulosus (Lentodium) was made by Lea around 1845» He collected speci mens in Ohio and sent them to the Reverend M. J. Berkeley in England. Both men considered the specimens to be an abnormality of Panus tigrinus (Lentinus tigrinus). Morgan 5 (1895), however, considered var. squamulosus to be a distinct agaric and established it as Lentodium squamulosum—a new genus and species. He recognized that L. squamulosum was similar to P. tigrinus (Lentinus tigrinus). and placed these two genera close together in the Agaricales. Lyman (1907) studied the mode of development of L. squamulosum in pure culture and supported Morgan's idea that the fungus was not an aberrant form. Singer (1949) considered L. squamulosum simply as a mutation of P. tigrinus. He had only seen sterile forms but did state that others had seen fertile forms. Martin (1956) supported the concept that var. squamulosus was a mutant form of var. tigrinus but, because of common occur rence in nature and the consistancy of form in both nature and pure culture, he retained the genus Lentodium. Rosinski and Robinson (1968) were able to demonstrate the true relationship between P. tigrinus and L. squamulosum. They crossed monokaryotic isolates of both forms and obtained complete intercompatibility. On the basis of their findings they reduced Lentodium squamulosum from a genus and species to a variety of Panus tigrinus, and designated it P. tigrinus var. squamulosus as distinct from P. tigrinus var. tigrinus. In addition, these authors observed that the morphology of the fruiting body of var. tigrinus was dominate to the fruit ing body type of var. squamulosus. Both varieties of P. tigrinus have been shown to be tetrapolar heterothallic (Quintanilha, et al, 1941j Robinson 6 and Rosinski, 1966). Rosinski and Faro (1968) were able to carry out crosses between var. squamulosus and var. tigrinus, followed by backcrosses to the recessive parent, and obtained results which strongly suggested that the difference in hymenophore morphology was controlled by a single pair of alleles.

Morphology of Basidiocarp Development The basidiocarp is the most conspicuous feature in the life cycle of the higher basidiomycetes and it is within this structure that karyogamy, meiosis and spore formation takes place. The mode of development of the fruiting body has been used to distinguish major groups of Basidiomycetes (Patouillard, 1887) but, at least in the modern interpreta tion of many of the groups, this does not hold true (Singer, 1949» 1962). Within genera there may be more than one type of development (Singer, 1962). In the Agaricales there are three main types of basidiocarp development based on the position of the hymenia (Singer, 1962« Bessey, 1950)« Those fruiting bodies which form their hymenia on the outside early and remain exposed throughout development are called gymnocarpous. An example of this type of development would be Omphalia chrysophyIla (Blizzard, 1917). A second method of development would be pseudoangiocarpous, in which the hymenium is initially naked but with development becomes enclosed by tissue originating from the stipe and/or cap. At maturity the hymenium becomes 7 exposed by the expansion of the cap. Members of the Rus- sulaceae and Boletaceae which produce a veil are of this type (Singer, 1962). The third type of development occurs in which the hymenium is produced internally and becomes exposed only at maturity. This mode of development is com mon among the dark-spored agarics, for example Agaricus compestris (Atkinson, 1906), and is called hemiangiocarpous. MeKnight (1955) observed an intergradation between agaricoid and gasteroid forms of the normally hemiangio carpous Psilocybe mutans grown in culture. He showed that genetic factors were not involved but found that light would bring about differences in morphology. Cultures grown in light produced more agaricoid forms and fewer gasteroid forms. Those cultures grown in darkness or reduced light produced gasteroid and other abnormal forms. Developmental studies of the two umbilicate varieties of P» tigrinus conducted with light microscopy have been reported by Lyman (1907), Kuhner (1925), and Bobbitt (1965). Lyman (1907) carried out an extensive study of var. squamulosus which he grew on sterilized sticks of wood with sugar and beef peptone added to the water. Bobbitt (1965) confirmed the observations of Lyman by growing the fungus on oatflake agar. Both concluded that development was gymnocarpic with a mem brane covering originating from interlacing elements of the trama. Lyman referred to the membrane as a "false veil". However, Bobbitt questioned the term "false veil", concluding 8 that it was misleading. He found that the enclosing membrane was not a distinct and sterile tissue, as occurs in hemi- angiocarpic or pseudoangiocarpic Basidiomycetes, but was an integral part of the trama. With age the membrane developed basidia and spores. Kuhner (1925) studied the development of var. tigrinus in specimens collected from the wild and interpreted the development to be pseudoangiocarpic. He reported that the hymenium was first exposed but with age became enclosed by tissue originating from the stipe and cap and then at matur ity became reexposed.

Factors Affecting Fruiting Few studies concerning environmental factors affect ing fruiting body formation of P. tigrinus have been reported in the literature.

PH Faro (1972) reported the optimal pH for fruiting occurred between pH 3.5 and 4.0, and for vegetative growth the optimal pH was between 3.5 and 4.5.

Gases Bobbitt (1965) reported that cultures growing in tubes with metal caps produced primordia with only rudimentary pilei which lacked a hymenium and spores. These rudimentary pilei turned brown and died without further development. It was suggested that metal caps retarded the free exchange of 9 gases and allowed a buildup of carbon dioxide. This idea supported the findings of Lambert (1933) working with Agaricus campestres, Plunkett (1956) working with Flammulina (Collybia) velutipes, and more recently Long (1966) working with Flam mulina velutipes, Niederpreum (1963) working with Schizo- ph.yllum commune, and McLaughlin (1970) working with Boletus rubinellus. In each case concentrations of carbon dioxide between 2-5% either affected primordium formation or pileus expansion. In some species a very narrow range was found in which small amounts of carbon dioxide were needed, while larger amounts became toxic. Tschierpe and Sinden (1964) found that Agaricus bisporus produced normal fruiting bodies when the atmosphere contained 0.05-0.06% carbon dioxide, but at levels of 0.1-0.18% fruiting body formation was retarded. The mechanism by which carbon dioxide affects basidiocarp development is unknown but Wehrli and Rast (1967) suggested that in Agaricus bisporus carbon dioxide may affect fatty acid and lipid metabolism. Schwantes and Hagemann (1965) found that in P. tigrinus oxygen was necessary in order for fruiting body formation.

Humidity and Nutrition The literature contains no studies on the effects of humidity on P. tigrinus, although in other fungi it has been found to be an important factor affecting fruiting (Buller, 1909« Plunkett, 1956). Nutritional studies have been limited primarily to those concerned with obtaining fruiting bodies .10 in culture (Lyman, 1907» Martin, 1956; Rosinski and Robinson, 19681 and Faro, 1972). Faro (1972) established that a small amount of calcium was needed in order for fruiting to take place. In most cases calcium present as a contaminate of the agar in the growth medium was enough to permit fruiting.

Light The need for light in the fruiting process of P. tigrinus was first suggested by Bobbitt (1965), who found that expanded caps did not occur in tubes which were main tained in the dark. In fungi the effects of light on fruit ing have been reviewed by Cochrane (1958), Carlile (1965), and Taber (1966). Light is often involved in one or more phases of basidiocarp development. Of the several thousand Basidiomycetes which produce hymenophores, less than twenty have been studied relative to the effect of light on develop ment (Table I). Light may be needed to initiate primordial formation, to bring about pileus development, both, or neither. In Agaricus bisporus light inhibits fruiting (Koch, 1958), on the other hand Favolus (Polyporus) arcularius requires light for both primordial and pileus development (Gibson and Trap- nell, 1957; Kitamoto, et al, 1968). In the case of Gyathus. a Gastromycete belonging to the family Nidulariaceae, 1700 ft-c of light is required to initiate primordia formation, but no further light is required to bring about mature basidiocarps (Lu, 1965). In order for formation of primordia to take place in Coprinus lagopus a small exposure to light (1/2 min TABLE I Summary of effects of light on fruiting of some Hymenomycetes as reported in the literature

______Light______Primordium Pileus Genus and Species Family Development Development Reference

Agaricus bisporus Agaricaceae* not required not required Koch (1958) Flammulina velutipes Tr ic holomatac eae* not required required Plunkett (1958) Goprinus lagopus Coprinaceae* minimal required required Lu (1974) Coprinus macrorhizus Coprinaceae* not required required Uno, et al (1974K Goprinus congregatus Coprinaceae* minimal required required Manachere (1971) Boletus rubinellus Boletaceae* not required required McLaughlin (WO) Favolus arcularius Polyporaceae* required required Gibson and Trapnell (1957) Polyporus squamosus Polyporaceae* not required required Buller (1909) Polyporus versicolor Polyporaceae* — required Koch (1958) Polyporus brumalis Polyporaceae* not required required Plunkett (1956) Lentinus lepideus Polyporaceae* not required required Buller (1905) TABLE I (Continued)

Lighlc Primordium Pileus Genus and Species Family Development Development Reference

Lentinus tuber-regium Polyporaceae* not required required Galleyraore (1949J Schizophyllum commune Polyporaceae* — required Raper and Krongelb (1958) Pleurotus ostreatus Polyporaceae* a* required Koch (1958) Pleurotos sp. Polyporaceae* required required Eger (1970) Cyathus stercoreus Nidulariaceae** required not required Lu (1965) Sphaerobolus stellatus Sphaerobolaceae*** «» M» required Alasoadura (1963) Poria ambigua Polyporaceae** not required required Robbins and Hervey (i960)

* Based on Singer (1962). ** Based on Bessey (195°). *** Based on Alexopolus (1962). 13 at 0.3 ft-c) was needed. After stipe elongation a second exposure to light was needed for pileus development (Lu, 1974). Similar observations have been made in Coprinus congregatus (Manachere, 1971). In other Basidiomycetes primordia initiation may take place in the dark, although at a reduced rate, and require light only for pileus develop ment and expansion (Buller, 1905; Galleymore, 1949; Plunkett, 1956; Robbins and Hervey, 1958; and McLaughlin, 1970). Polyporus versicolor and Pleurotus ostreatus (Koch, 1958) and Sphaerobolus stellatus (Alasoadura, 1963) are known to require light in order to fruit but at present it is not known if light is required for primordial initiation. The action of light on fruiting is not known but several theories have been suggested. Cochrane (1958) pre sented the idea that light inhibits vegetative growth which in turn triggers events which lead to reproduction. Bobbitt (1965) observed that cultures of P. tigrinus var. squamulosus incubated in the dark had a more luxurious growth than those grown in light. Lu (1965) postulated a second theory; in Cyathus a photoreceptive precursor was produced by the mature mycelium as the substrate became exhausted. Similarly, in Coprinus lagopus. a photoreceptive precursor was produced after exposure to light when the medium became exhausted and the precursor changed to an effector which could bring about initiation of primordia (Lu, 1974). At that point there was believed to be a dichotomy of pathways; if the primordia 14 received additional light a cap would differentiate, if not the stipe would elongate with no cap or hymenium. The precise location of the photoreceptor in fungi is uncertain. Controversy exists regarding the chemical iden tity of the receptor (Page, 1965). Mohr (1962) postulated that the effective photoreceptor was a flavoprotein. On the other hand, Schneiderhohn (1955) found in the yellow-pig mented Basidiomycete Coprinus lagopus. no evidence of the pigment being riboflavine. In addition, the light receptor did not appear to be carotene. Aschan-Aberg (1960b) did not detect either carotene or riboflavine in the basidiocarps of Collybia velutipes. although only yellow strains were capable of fruiting. In all reported cases of hymenomycete Basidio- mycetes, only the short wavelengths of visible light ("blue light"), and ultraviolet light were effective in stimulating fruiting (Mohr, 1962î Perkins and Gordon, 1969). The effects of light stimulus on the mycelium of Coprinus lagopus was found by Madelin (1956) to be restricted to irradiated parts of the culture. Similar findings were reported for Favolus arcularius by Kitamoto, et al (1968). The cells of the mycelium which were sensitive to light were concluded to be those actively dividing cells. The effects of the light lasted for about one day. However, Robbins and Hervey (i960) found that Poria ambigua mycelium was able to retain the effects of the light stimulus and produce fruiting bodies from a mycelium which had been grown in the dark. 15 A mycelium will not respond to light until it has reached a certain physiological age. Madelin (1956) found the light sensitive system of Coprinus lagopus was estab lished some time between the seventh and thirteenth day post inoculation. In Favolus arcularius the mycelium became light sensitive 2.5 days after inoculation (Kitamoto, et al. 1968), and in Schlzophyllum commune the culture was sensitive after 60 hrs of growth (Perkins, 1969). In Coprinus macrorhizus Uno, et al (1974) found the mycelium was sufficiently sensi tive after four days to produce malformed fruiting bodies. At 10 days after inoculation the illuminated mycelium was able to form normal basidiocarps. There appears to be a close relationship between melanin formation and fruiting in fungi (Leonard and Dick, 1968« Wilson and Baker, 1969« and Faro, 1972). In Schizo- phyllum commune, Leonard and Dick (1968) found a brown pig ment which developed in the region of fruiting body forma tion, they suggested the deposition of melanin-like pigments was an early stage of morphogenesis. Faro (1972) demon strated the brown pigment formed before P. tigrinus fruiting was melanin. Melanin synthesis inhibitors, such as phenyl- thiocarbamide, blocked the melanin synthesis which in turn prohibited fruiting. Leonard and Dick (1968) isolated a fruit-inducing sub stance (FIS) which stimulated fruiting body formation in mono« karyotic strains of Schizoph.yllum commune. A similar 16 substance was isolated and purified from Coprinus macrorhizus by Uno and Ishikawa (1972) who identified the active com ponents as adenosine-3*-monophosphate, adenosine 3*,5'-cyclic monophosphate (cyclic AMP), and a protein bound with the cyclic AMP. Uno, et al (1974) found in illuminated monokaryotic mycelia of C. macrorhizus the accumulation of cyclic AMP which began before formation of the fruiting bodies. The cyclic AMP reached a maximum level seven days after inocula tion, and then rapidly declined until the tenth day after inoculation at which time there was only 10% of the cyclic AMP found in seven-day old cultures. In addition, the adenylate cyclase activity of illuminated mycelia reached a maximum level after six days followed by a decline to about 20% of maximum 14 days after inoculation. The phosphodiester ase reached a maximum level on day seven and then declined. Light-grown mycelia showed higher cyclic AMP binding activity than the dark-grown mycelia. Uno, et al (1974) suggested light could induce synthesis of the cyclic AMP-binding pro tein. Cultures which were maintained in the dark produced low levels of cyclic AMP, adenylate cyclase and phosphodiester ase. The production of cyclic AMP and the increase in the cyclic AMP-binding activity was suggested to be responsible for fruiting body formation.

Basidiospores The chief characteristic of the Homobasidiomycetes is the production of basidia by the hymenial layer of the fruiting 17 body, Basidia are club-shaped and originate as terminal cells of a binucleate hyphae (Alexopoulos, 1962). Within a young basidium the two nuclei fuse and thè zygote nucleus undergoes meiosis, giving rise to four haploid nuclei which each migrate into one of the four developing basidiospores. The majority of the members of the Agaricales produce basidia with four basidiospores but there are exceptions in which one, two, or three spores are produced. The common cultivated mushroom, Agaricus bisporus, was found to be an example which produces only two spores (Buller, 1922). Typical Basidiomy cete spores are asymmetrically perched on the tips of curved, tapering, conical-shaped sterigmata which project from the basidia. The sterigmata and basidiospores are symmetrically spaced about the terminal end of the basidum, with all four spores equal distance apart (Buller, 1922). Basidiospores are usually unicellular and vary in shape from globose, oval, elongate, or curved. The spores vary in color from pink, yellow-brown, brown, purple-brown to black, although some may be hyaline (Bessey, 1950). The spores may have smooth -, spined, worty, ridged, reticulated or punctated surfaces (Singer, 1962), and their size may vary in length from two to 40 microns depending upon the species (Alexopoulos, 1962). The spores of both varieties of P. tigrinus are smooth and hyaline but produce white spore prints. The spores are nonamyloid in Melzer's solution (Miller, 1972). 18 The spore size for var. squamulosus has been reported to be 4.4-7.3 x 2.5-3.2 microns (Martin, 1956), and 6.0 x 3.0 microns for var. tigrinus (Graham, 1970). The number of basidiospores produced by a single large basidiocarp may be enormous. Buller (1909) estimated basidiospores were shed at the rate of a million spores a minute for as long as 50 hrs in some species. He also estim ated not more than one spore in 20 billion survived in nature to give rise to another basidiocarp.

Development of the Sterigmata and Spores Sequential events in the development of sterigmata and spores studied by light microscopy have been reported by Buller (1922), Malengon (1942), and Perreau-Bertrand (1967). Sterigmata were found to begin as small protrusions on the surface of the basidia and then to elongate rapidly. As elongation took place the sterigmata curved outward and became conical. At the tip of the sterigmata spherical bodies developed which eventually became part of the hilum. The external half of the spherical body grew out and upwards, thus forming the asymmetrical spore. Buller (1922) reported the fully grown spore to have a long axis inclined at an angle of about 45° to the long axis of the sterigma, but Malen^on (1942) reported the spore was first tilted away from the axis of the sterigma, but repositioned itself during growth parallel with the axis of the sterigma. The length of time required for development and 19 ripening of individual spores of several species were studied by Buller (1922) who found great variation in the length of time spores remained on the sterigma (Table II).

Spore Germination The process whereby dormant spores are transformed from a state of low metabolic activity to one of high activity may be considered spore germination. In such spores the forma tion of germ tubes becomes the visible evidence that meta bolic changeovers have been completed (Gottlieb, 1964), The process of fungal spore germination has been reviewed by Cochrane (1958). Environmental factors such as temperature, pH, moisture, oxygen and nutrition have been studied and have been shown to influence germination (Duggar, 1901; Baden, 1915; Webb, 1919; Hein, 1930b; Styler, 1930; Buller, 1931; McCallan and Wilcoxon, 1932; Kauffman, 1934; Smith and Brodie, 1935; and Kneebone, 1950). Recently, the utilization of scan ning electron microscopy has been employed in order to study morphological changes in spores during germination (Hiratsuka, 1970; Jones, 1971; and Wergin, et al, 1972). Hiratsuka (1970) observed that aeciospore germ tubes of Cronartium coleosporioides emerged through an irregular slit in the spore wall, whereas no germ pore region could be detected on the surface of Uromyces dianthi urediospores by Jones (1971)» Germination studies of pycnidiospores of Botryodiplodia theobromae by Wergin, et al (1972) revealed the outer wall of the spore to be fractured by the emerging germ tube. 20

TABLE II Average time taken for development and ripening of individual spores*

Organism Hours Minutes

Flammulina velutipes 0 47 Collybia fusipes 1 3 Marasmius oreades 1 5 Collybia radicata 1 30 Armillaria mellea 1 30

Pluteus cervinus 3 25 Russula cyanoxantha 5 10 Agaricus bisporus 8 0 Coprinus sterquilinus 32 0

* After Buller (1922). 21 Based on the manner of germ tube emergence from the spore, there are three modes of germination (Hawker, 1968). (1) In those fungi which produce zoospores with a single wall layer, the wall bulges in order to form the germ tube. (2) In spores which have walls composed of more than one layer, such as conidia, ascospores and some basidiospores, the outer layer ruptures, allowing the inner layers to protrude. The protruding inner layers become the germ tube. (3) Many fungi which produce thick walled spores have germ pores, which are weak areas on the wall and are the sites of germ tube emergence. The literature contains no studies on germination for P. tigrinus.

Fungal Cell Walls The cell walls of filamentous fungi and yeasts have been studied as reviewed by Cochrane (1958), Salton (i960), Northcote (I963), and Aronson (1965). The fungal cell wall is a multilayered structure of differing composition (Coch rane, 1958). Wall material from various groups of fungi have been reported to contain carbohydrates, lipids, pro teins, purine-pyrimidines and minerals (Kreger, 1954? Bart- nicki-Garcia and Nickerson, 1962j and Crook and Johnston, 1962). Chitin and cellulose comprised the major components of many cell wall types. However, Wisselingh (1898) and Young (1958), both stated that chitin and cellulose never exist together in the walls of fungi. Chitin has been found to be the major cell wall constituent in the Zygomycetes, 22 Ascomycetes, and Basidiomycetes, whereas the Oomycetes have been found to contain cellulose as their main constituent (Cochrane, 1958). Additional investigations have demonstrated in two species of Aspergillus (Farr, 1954), at least one species of Rhizidiomyces (Fuller and Bar shad, I960), and Ceratocystis ulmi (Rosinski and Campana, 1964), both chitin and cellulose co-existed in the cell walls. The amount of chitin present in wall material varied ten-fold from 2.6 to 26.2% of the dry weight of the mycelium depending upon the species assayed (Blumenthal and Roseman, 1957). Electron microscopy studies of Allomyces macrogynus showed chitin microfibrils in the tips of chemically cleaned rhizoids arranged longitudinally, whereas in older rhizoidal walls the microfibrils exhibited a crossed orientation (Aronson and Preston, I960). Dodge and Lawes (1969) observed with transmission electron microscopy chemically cleaned microfibrillar components of the walls of zoosporangia and resistant sporangia of Allomyces arbusculus. The microfibrils in the zoosporangia wall were randomly arranged. In the resistant sporangia microfibrils were also randomly arranged, but the layer was perforated by numerous pores 0.25 microns in diameter and one micron apart. The microfibrils tended to be concentrically arranged around the pores.

Spore Walls Few studies have been performed on the chemical and structural composition of fungal spore walls, and fewer 23 studies have been concerned with the walls of basidiospores. Prentice and Cuendent (1954) were able to demonstrate that uredospores of Puccinia graminis contained 2% D-glucose, 19% D-mannose, and 3% D-arabitol on a dry-weight basis. In a transmission electron microscopy study, Williams and Leding ham (1964) found the uredospore wall of P. graminis to be composed of three distinct layers. The outer layer was a thin continuous dark line which the authors suggested to be the site of hydrophobic properties of the wall. Inside the outer layer was a very narrow electron-transparent zone which was swollen after treatment with potassium permanganate. The innermost layer was composed of electron-dense microfibrils. The utilization of various dyes in order to dis tinguish layers of the spore wall have been attempted, but the usefulness of dyes is limited because they tend to lack specificity except for broad groups of chemical compounds (Perreau-Bertrand, 196?). In addition, different wall layers are difficult to resolve because of the small size of the spores. The teliospore wall of Tilletia contraversa was studied by Graham (1957) who treated the spores with dyes, enzymes and reagents. The author concluded the wall was com posed of four distinct layers. The outer layer was composed of a pectin-lipoid complex. The reticulum, or second layer from the outside, was composed of a bipectin-protein-hemicellulose-chitin complex. The third layer from the outside was composed of chitin while the innermost layer was composed 24 of hemicellulose-chitin-pectin. The ascospore wall layers of Neurospora tetrasperma were studied by Lowry and Sussman (1958). These investiga tors found three distinct layers. An outer ribbed layer could be removed without affecting germination by treatment with 20% Clorox (approximately 1% sodium hypochlorite) for 30 minutes. Beneath this outer layer was an intermediate layer which was melanized and rigid. By the application of pressure to the treated spore, the intermediate layer could be ruptured to reveal an inner layer. It was believed the two innermost layers provided support and rigidity to the spore in order to maintain its shape. The composition of the three layers was not determined, but Clorox reacted with the outer wall layer as an oxidizing agent. Horikoshi and Iida (1964) have studied physically dis rupted spore wall material of Aspergillus oryzae and found the wall contained glucosamine, amino acids, phosphate, ash, nucleic acids and polysaccharides. Electron microscopy of the isolated spore material revealed fibrous and porous struc tures, but the authors did not elaborate on the chemical nature of these structures.

Basidiospore Walls Mature, ungerminated basidiospores are difficult to study using electron microscopy because of difficulty in fixative penetration, and difficulty in cutting embedded material (Stock and Hess, 1970» Vogel and Weaver, 1972). In 25 addition, as has been the case with fungal wall material (Cochrane, 1958)» no pure spore wall material free of extraneous cytoplasmic material, and/or which has not suf fered from loss of material during purification has been obtained. The basidiospore wall is a multi-layered structure. Its chemical composition is not well known (Alexopoulos, 1962). Although the composition of the layers is unknown, chitin has been eliminated as a wall component in the basidiospores of Ustilago zeae (Gottlieb, 1964). The early literature concerning the identification of the various layers which comprise the basidiospore wall has been reviewed by Singer (1962) and Perreau-Bertrand (1967). Their termi nology was based on earlier work of two French mycologists, M. Locquin and R. Heim. Many of the wall layers have been observed with light microscopy but since the development of the transmission electron microscope, the morphology of the layers has been better characterized. The terminology and wall layer descriptions used by Singer (1962) and Perreau-Bertrand (1967) follows. Basidio spores may have as many as six distinct layers defined pri marily by their relative position. Within a given species the spore wall may not have all six layers (Fig. 1). The external thin spore layer has been designated the ectosporium. When viewed with light microscopy the layer was very diffi cult or impossible to perceive. Viewed with transmission 26 electron microscopy the layer appears as a simple dark line surrounding the spore. Immediately beneath the ectosporium was a clear, electron-transparent layer called the perisporium. Next was a thin layer not found in all spores called the exosporium. Internal to the exosporium was the episporium. This layer was the framework of the spore, giving the spore its characteristic shape. As viewed with light microscopy the layer appeared as a colored membrane because of light refraction. When viewed with the electron micro scope the layer appears electron-dense. The layer was not homogenous. The next layer was the mesosporium appearing as a thin granular irregular zone, and as viewed with the electron microscope as a densely stained layer. This layer may be a portion of the episporium. The most internal layer lying next to the protoplasmic membrane was the endosporium. As viewed with the transmission electron microscope this layer was not electron-dense 27

Fig. 1 Schematic diagram of the wall layers of a hypo thetical smooth, hyaline basidiospore with ectosporium (EOT.), perisporium (PER.), episporium (EPI.) and meso sporium (MES.), and an endosporium (END.). (After Perreau- Bertrand, 1967.) 28

ECT

.PER«

.EPI« & MES

E N D Vi

LIST OF REFERENCES FOR LITERATURE REVIEW 30 REFERENCES

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LIGHT EFFECTS ON FRUITING IN PANUS TIGRINUS VAR. TIGRINUS

Thomas F. Bobbitt and Richard E. Crang

Appears Ini Canad ian Journal of Botany Volume 52, Number 1, 1974 B'éìmpressionTdu lEgjfiBjS« IjMfijmîaircffl HSSHI

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Light effects on fruiting in Panus tigrinus var. tigrinus

Thomas F. Bobbitt and Richard E. Crang Department of Biology, Bowling Green State University, Bowling Green, Ohio 43403 Received July 27, 1973

Bobbitt, T. F., and R. E. Crang. 1974. Light effects on fruiting in Panus tigrinus var. tigrinus. Can. J. Bot. 52: 255-257. In the Basidiomycete Panus tigrinus var. tigrinus, continuous fluorescent light results in increased production of numbers of abnormal fruiting bodies. With progressively longer dark periods before continuous light there is a decrease of abnormal fruits. The total number of fruiting bodies remains relatively constant regardless of light /dark treatment. However, no fruiting occurs under conditions of total darkness. Differentiation between normal and abnormal fruiting bodies begins after light exposure, and the normal primordia develop a dark pigmentation near the tip. Such pigmentation is believed to be significant in inducing normal basidiocarps.

Bobbitt, T. F., et R. E. Crang. 1974. Light effects on fruiting in Panus tigrinus var. tigrinus. Can. J. Bot. 52: 255-257. Chez le Basidiomycète Panus tigrinus var. tigrinus, la lumière fluorescente continue résulte en une production accrue du nombre de fructifications anormales. Avec des périodes obscures progressivement plus longues précédant l’illumination continue, il y a une diminution dans le nombre de fructifications anormales. Le nombre total de fructifications demeure relativement constant quel que soit le traitement lumière /obscurité. Cependant, aucune fructification n’est formée sous une obscurité totale. La différen ciation entre les fructifications normales et anormales commence après l’exposition à la lumière et une pigmentation foncée se développe près de l’extrémité du primordium normal. Les auteurs croient que cette pigmentation joue un rôle dans l’induction des basidiocarpes normaux. [Traduit par le journal]

Introduction in which flasks containing the fungal inoculum of Panus tigrinus var. tigrinus (Bull, ex Fr.) Singer were exposed to Many studies have shown that light, tempera various light and dark conditions. Linder each of the ture, humidity, pH, nutrition, and aeration all light treatments, 20 cultures in 125-ml flasks were tested. play a role in basidiocarp formation. These Each flask contained 25 ml of sterile synthetic medium (Faro 1972) solidified with 1.5% Difco Bacto agar and factors must be taken into account in any study inoculated with 1-cm2 section of dikaryotic mycelium of such organisms in culture. Key worth (1942) from stock cultures. Flasks were plugged with cotton and observed that abnormal fruiting bodies resulted kept at 23-24°C. from aeration of Coprinus ephemerus with un Twenty inoculated flasks were placed under continuous washed laboratory air. Plunkett (1956) also found direct Sylvania Cool White (F40 CW) fluorescent lights of between 80 and 85 ft-c, as measured by a Weston (Model abnormal fruiting bodies in Polyporus brumalis, 756) illumination meter. Another20inoculatedflasks were but could not attribute them to aeration, light, placed under continuous light for 1 week, and then or humidity. He suggested that the club-like placed in total darkness for 2 weeks before being returned abnormalities may have been the result of still- to continuous light. Sixty more inoculated flasks were air culture conditions where they were frequently placed in total darkness, then, at l-week intervals over a 3-week period, 20 flasks were removed from the dark and found. placed under the continuous light. An additional 20 Light is known to be required at different inoculated flasks were placed under alternating fluorescent stages of basidiocarp production in various light and dark conditions of 12 h each. Cultures were species as reviewed by Taber (1966). Among these monitored over a 5-week period and the number and type of fruiting bodies were tabulated per treatment. An studies, Gibson and Trapnell (1957) reported a R X C comparison of the G statistic with the critical chi- light requirement for fruiting body initiation in square value at 0.05 significance was carried out to Polyporus arcularius, whereas Plunkett’s obser determine independence among treatments according to vation showed light was only needed for pileus procedures outlined by Sokal and Rohlf (1969). expansion in P. brumalis and Collybia velutipes. Results Our study was undertaken to elucidate the effects of light on fruiting in Panus tigrinus. Mycelial growth differed according to light treatment, but in virtually all cases it covered the Materials and Methods surface of the nutrient medium. Those cultures To determine the effects of light and dark on basidio receiving continuous light, or alternating light carp production, a series of experiments were undertaken and darkness, produced a thin, smoothly ap- Mi

256 CAN. J. BOT. VOL. 52, 1974 pressed surface mycelium (Fig. 1). Cultures the surface of decurrent gills radiating from the exposed to dark treatments produced a thick, stipe. The pilei of abnormal fruiting bodies were rough, cottony mycelium (Fig. 2). Primordia and spherical and their surface covered with anasto fruiting bodies began forming both under mosing gills (Fig. 5). Differentiation between continuous light and alternating light and dark, normal and abnormal fruiting bodies began soon usually 9-14 days after inoculation. Cultures after exposure to light, and primordia which receiving 1-week dark treatment initiated fruiting developed normally had a dark-gray to dark- about 7 days after removal from the dark, brown pigmentation near the tip. Apices of whereas cultures receiving two or three dark primordia which developed into abnormal fruit treatments began forming fruits within 1 to 3 ing bodies remained white or light brown. days after exposure to continuous light. Cultures The observations from 20 flasks for the six receiving 2- or 3-week dark treatments often had treatments are reported in Table 1. The number primordia already formed (Fig. 3) when they of normal, abnormal, and total fruiting bodies were transferred into the light but never ex produced per treatment plotted against four panded pilei. By the time fruiting began in dark- light treatments of increasing duration are shown treated cultures the mycelium had become in Fig. 6. The number of normal fruits remained blotched with reddish-brown pigmentation. fairly constant between treatments, within a The basidiocarps were of two morphological range of six. In addition, cultures given 1-week types. Normal morphology was typical of umbili light pretreatment followed by 2 weeks of dark cate Agarics (Fig. 4) with the hymenial layer on ness and then exposed to continuous light showed virtually no abnormal fruiting bodies, but did show a slight increase in the number of normal fruiting bodies. When cultures were kept under 12-h alternating photoperiods of light and dark, an 84.6% increase in abnormal fruiting bodies was recorded at the end of 5 weeks, but the numbers of normal fruiting bodies did not differ significantly from cultures exposed to 1 week light followed by 2 weeks dark, and 2 weeks light. When an R X C test of independence using the G statistic was carried out on data for the number of abnormal and normal fruiting bodies, a significant G statistic was found between light WEEKS treatments. A marked decline in the number of Fig. 6. Total number of normal and abnormal, and both types of fruiting bodies plotted against increasing light abnormal fruiting bodies resulted from an in exposure. crease in the duration of the dark period.

TABLE 1 Observations of 20 cultures from six light and dark treatments

1 week light 5 weeks Continuous 1 week dark 2 weeks dark 3 weeks dark 2 weeks dark alternating light 4 weeks light 3 weeks light 2 weeks light 2 weeks light light/dark N A N A N A N A N A N A Range 0-9 0-12 0-17 0-10 0-6 0-4 0-8 0-2 0-6 0-2 0-6 0-7 Average 1.9 4.3 1.9 3.5 1.7 0.8 2.0 0.35 2.75 0.2 2.9 1.3 Total 38 86 38 70 34 16 40 7 55 4 58 26 Average number of total fruits 6.2 5.4 2.5 2.35 2.95 4.2

Note : N = normal and A = abnormal fruiting types. Range is the minimum and maximum numbers in the 20 cultures of each type of fruiting body. Average is the mean number of each fruiting type for the 20 cultures. Total number of each fruiting type per treatment is listed. In addition, the average number of total fruits including both normal and abnormal types is given per treatment. RXC test of independence for the six treatments gives a significant G statistic (G statistic = 119.7745 > x2 0.05 (6) = 11.070), indicating that in each case fruiting body type is dependent on light treatment. Plate 1

Fig. 1. Thin, smoothly appreseci mycelial growth characteristic of P. tigrinus receiving continuous light or alternating light and dark treatments. 3j X. Fig. 2. Rough, cottony mycelial growth typical of cultures receiving pretreatment of 2 to 3 weeks darkness. 1-J X. Fig. 3. Primordia from culture at time of removal from 2 or 3 weeks dark treatment. 3 ] X. Fig. 4. Normal appearance of basidiocarp showing typical umbilicate morphology. 3J X. Fig. 5. Morphological appearance of abnormal basidiocarps with anastomosing gills covering pileus. 3 J X . BOBBITT AND CRANG: LIGHT EFFECTS ON FRUITING 257

Discussion strated that fruiting was accelerated by light be Plunkett termed the abnormal fruiting bodies tween the 7th and 13th day postinoculation. he observed in still-air cultures of P. brumalis as Before this age either the light-sensitive system abortions which had taken place before cap was not functionally established, or the culture formation, but the abnormals observed in this was unable to retain the effects of light until the investigation were not abortive forms. They were fungus was able to respond. Cultures receiving functionally normal, producing viable spores 1 week light, 2 weeks dark, and then 2 weeks indistinguishable from those produced by normal light showed low numbers of abnormal fruiting fruiting bodies. However, true abortive forms bodies, thereby similar to cultures receiving a pre were found by Bobbitt (1965) when cultures of treatment of 3 weeks darkness and then continu P. tigrinus var. squamulosus {Lentodium squamu- ous light. This indicates that the light-sensitive losum) were grown using metal caps. Developing system for P. tigrinus is not formed until after the primordia produced rudimentary caps and lacked 7th day postinoculation. The results reported a hymenium with spores. It was suggested that here demonstrate that morphogenesis of the possibly a buildup of carbon dioxide around the developing basidiocarp of P. tigrinus is effected primordia was the cause of these forms since by light and dark conditions before primordia cultures stoppered with cotton plugs produced formation. only the normal forms. The mechanism by which fruiting in Basidio mycetes is brought about is incompletely known, Acknowledgment although factors effecting fruiting have been We thank Dr. M. A. Rosinski, formerly of the studied for a number of years. Light is one of the Botany Department at the University of Iowa, factors which has been shown to be involved with for making available P. tigrinus cultures. fruit formation in other fungi as well as in P. tigrinus. Primordia develop in darkness but Bobbitt, T. F. 1965. A developmental study of Lentodium pileus expansion requires light. Leonard and squamulosum Morgan. M.S. Thesis, University of Iowa, Dick (1968) found that a brown pigment ap Iowa City, Iowa. Faro, S. 1972. Physiological aspects of pigment produc peared in the region of fruiting in Schizophyllum tion in relation to morphogenesis in Panus tigrinus. commune and suggested that the deposition of Mycologia, 64: 375-387. melanin-like pigments was an early stage of Gibson, I. A. S., and J. Trapnell. 1957. Sporophore production by Polyporus arcularius. Trans. Br. Mycol. morphogenesis. Faro (1972) demonstrated that Soc. 40: 213-220. the brown pigment formed before fruiting in P. Keyworth, W. G. 1942. Occurrence of tremelloid out tigrinus was melanin. Thus, P. tigrinus melanin growth on Coprinus. Trans. Br. Mycol. Soc. 25: 307- 310. molecules may absorb light energy, either Leonard, T. J., and S. Dick. 1968. Chemical induction directly or indirectly, and serve to induce bio of haploid fruiting bodies in Schizophyllum commune. chemical intermediates to initiate basidiocarp Proc. Natl. Acad. Sci. U.S.A. 59: 745-751. Madelin, M. F. 1956. The influence of light and tempera formation. Uno and Ishikawa (1973) have re ture on fruiting of Coprinus lagopus Fr. in pure culture. ported the activity of cyclic adenosine mono Ann. Bot. (Lond.), 20: 467-480. Plunkett, B. E. 1956. The influence of factors of the phosphate (AMP) as one such biochemical fruit- aeration complex and light upon fruit body form in inducing compound found in a homocaryotic pure cultures of an Agaric and Polypore. Ann. Bot. strain of Coprinus lagopus. In studies of a black (Lond.), 20: 563-586. Schaeffer, P. 1953. A black mutant of Neurospora crassa. mutant of Neurospora crassa, Schaeffer (1953) Mode of action of the mutant allele and action of light found that visible light depressed melanogenesis on melanogenesis. Arch. Biochem. Biophys. 47: 359- activity during growth of the mycelium. If 379. Sokal, R. R., and F. J. Rohlf. 1969. Biometry. W. H. melanin is significant in the mediation of fruiting, Freeman and Co., San Francisco, California. then its reduction may bring about an abnormal Taber, W. A. 1966. Morphogenesis in Basidiomycetes. In The fungi. Vol. 2. Edited by G. C. Ainsworth and change in the appearance of developing fruiting A. S. Sussman. Academic Press, New York. pp. 387- bodies. This light influence may not only be a 412. function of intensity or duration but, as we have Uno, I., and T. Ishikawa. 1973. Metabolism of adenosine 3',5'-cyclic monophosphate and induction of fruiting demonstrated, its alternating periodicities. In bodies in Coprinus macrorhizus. J. Bacteriol. 113: cultures of C. lagopus Madelin (1956) demon 1249-1255. ARTICLE 2

BASIDIOCARP DEVELOPMENT OF THE TWO VARIETIES OF PANUS TIGRINUS AND THEIR LIGHT-INDUCED ABNORMAL FORMS

Thomas F. Bobbitt and Richard E. Crang

Appears In» Mycologia Volume 67» Number 1, 1975 Reprinted from Mycologia, Vol. LXVII, No. 1, pp. 182-187, Jan.-Feb., 1975 Printed in U. S. A.

BASIDIOCARP DEVELOPMENT OF THE TWO VARIETIES OF PANUS TIGRINUS AND THEIR LIGHT-INDUCED ABNORMAL FORMS

Thomas F. Bobbitt and Richard E. Crang Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403 182 Mycologia, Vol. 67, 1975

BASIDIOCARP DEVELOPMENT OF THE TWO VARIETIES OF PANUS TIGRINUS AND THEIR LIGHT-INDUCED ABNORMAL FORMS

Thomas F. Bobbitt and Richard E. Crang Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403

The basidiomycete Panus tigrinus (Bull, ex Fr.) Singer is now recognized as consisting of two varieties, the former species Lentinus tigrinus (Fr.) Fries and Lentodium squamulosum Morgan (Rosinski and Robinson, 1968). Distinction between the varieties can be readily made by the fact that P. tigrinus var. tigrinus produces typical agaric morphology with distinct gills, whereas P. tigrinus var. squamulosus produces gills which anastomose early in development to form a gastro- mycetelike basidiocarp. Developmental studies of the two umbilicate varieties conducted with light microscopy have been reported by Lyman (1907), Kuhner (1925) and Bobbitt (1965). Although both Lyman and Bobbitt found var. squamulosus to develop in a basically gymnocarpic fashion, Kuhner reported that var. tigrinus developed pseudoangiocarpically. For many years the two varieties have been suspected to be closely related. Singer (1949, 1962) and Martin (1956) both supported the concept that var. squamulosus was a mutant form of var. tigrinus, but because of its common occurrence in nature, Martin retained the genus Lento dium. Hybridization between the two varieties was reported by Ro sinski and Robinson (1968). Rosinski and Faro (1968) theorized that the difference in hymenophore morphology between the two was controlled by a single pair of alleles, with the allele yielding var. tigrinus being dominant. Bobbitt and Crang (1974) demonstrated that fluorescent light influenced hymenophore morphology in var. tigrinus. Increased amounts of light induced larger proportions of abnormal orbicular fruits with the cap surface being covered with interlacing gibs. Developmental studies using scanning electron microscopy (SEM) have not been reported for either of the two varieties or their abnormal forms. Observation of these two varieties was undertaken using SEM in order to help clarify the morphological differences in basidiocarp development between these two closely related varieties. Dikaryotic cultures of the two varieties were grown in cotton-plugged 125-ml flasks containing 25 ml of sterile synthetic medium solidified with Brief Articles 183

1.5% Difco Bacto agar (Faro, 1972) or sterile oat flake agar (Martin, 1956). Cultures were maintained at 24—25 C under one of three light conditions: total darkness; 12 hr alternating light and dark; or con tinuous light. The light employed consisted of Sylvania Cool White (F40 CW) fluorescent light of between 75 and 85 ft-c (800-900 lux) as measured by a Weston (model 756) illumination meter. Stages of developing basidiocarps were collected, fixed with 3-5% glutaralde hyde in 0.1 m phosphate buffer (/>H 7.2), dehydrated through a graded alcohol series and dried by carbon dioxide critical point method accord ing to procedures outlined by Anderson (1951). Dried material was attached to aluminum stubs with electroconductive paint and coated with gold. Observations were made with an Hitachi HHS-2R scanning ■electron microscope at 15 or 20 kV. Evidence of fruiting in both normal umbilicate varieties, and their abnormal forms, could usually be observed 2-5 wk after inoculation, consisting of a mounding of the mycelium into one to several dome shaped elevations. The mounds elongated rapidly into white clavarioid structures representing basidiocarp primordia (Fig. 1). Close observa tion of elongating primordia as small as 5 mm in length revealed a region of scattered basidia with sterigmata and spores approximately 7, the distance from the tip. Basidiospores produced along the stipe were identical to those on typical hymenophores (Fig. 2), and readily germinated on cultural media. The production of these spores was not found to be influenced by either light conditions or media employed. Miller (1971) observed similar production of fertile basidiospores over the apices of primordia in species of Lentinellus maintained at tem peratures from 18 to 23 C. Both his observations of Lentinellus and those of this report have shown that as clavarioid forms they were able to complete their life cycle without the production of gills. Early development of the normal forms of both varieties was similar. As primordia elongated, hyphae at the apex began to grow outward, initiating cap formation and giving the tip a flattened appearance. With cap expansion, outfoldings immediately beneath and along the stipe developed which represented early stages in the development of the: gills (Fig. 3) and which grew outward and upward until they fused with the expanding cap (Fig. 4). Additional series of outgrowths formed between the gill plates, and in turn developed into mature gills. In var. tigrinus the gill plates remained fully exposed (Fig. 5), whereas in var. squamulosus hyphae at gill edges later grew laterally creating a membrane which covered spaces between adjacent gills (Fig. 6). As cap maturation continued, the gill tissues grew outward 184 Mycologia, Vol. 67, 1975

Figs. 1-4. Panus tigrinus. Normal fruiting structures grown under alternat ing light and dark conditions. 1. Var. tigrinus: basidiocarp primordium with high lighted area showing region where basidia are located. X 35. 2. Var. tigrinus: Brief Articles 185

with additional bridges forming between gills. In both varieties, hymenial basidiospores appeared first at the base of the gills adjacent to the stipe and, with maturity, development of basidia extended over the entire surface of the gills. Since observations of Rosinski and Robin son (1968), and Rosinski and Faro (1968) showed only a subtle genetic difference existing between the two varieties, such a close relationship would appear to rule out major differences in the mode of develop ment of their basidiocarps. From observations of the two varieties using SEM it is apparent that Kuhner (1925) was in error in describing var. tigrinus as pseudoangiocarpic, a condition where the hymenial region develops within a cavity produced by the downward growth of the edge of the cap so as to become loosely associated with the stipe. At no time during this study was the hymenial region of either variety enclosed by hyphal material originating either from the cap or the stipe. The hymenial region of var. squamulosus was found to be enclosed by hyphal material originating from the gill tissue. Bobbitt (1965) demonstrated that this hyphal covering was not sterile but had a fertile inner surface lined with basidia and spores. The current study has shown that the mode of development for both varieties is gymnocarpic. The difference which separates these two varieties is apparently a genetic control governing laterally oriented growth of gill tissue. The gymnocarpic mode of development was observed in the abnor mal forms of both varieties and differed in their development from the normal forms inasmuch as the orbicular caps were comprised of masses of gill material (Fig. 7). The abnormal forms developed iso lated pockets of basidia on the surface of their primordial tips (Figs. 8, 9). Hyphal material surrounding these pockets continued to grow outward forming an interlacing gill network (Fig. 8). In addition, the abnormal orbicular form of var. squamulosus developed hyphal bridges between the interlacing gills and an outer hyphal membrane over the gills similar to that of normal forms. At maturity, basidial formation extended outward from the pockets over the surface of the gills becoming a hymenium. It is proposed that the pockets of con centrated basidia be called niduli (Sing, nidulum; L. w'dMi=nest). The establishment of niduli occurred in conjunction with early cap forma tion in abnormal forms of both varieties and were produced only in

basidium with its basidiospores from highlighted area shown in Fig. 1. X 5,400. 3. Var. squamulosus: young cap with outfoldings representing early gill formation. X 35. 4. Var. tigrinus: young cap with gill outfoldings extending upward. X 140. 186 Mycologia, Vol. 67, 1975

Figs. 5-9. Panus tigrinus. Normal and abnormal fruiting bodies grown in alternating light and dark. 5. Var. tigrinus: normal fruiting body showing ma turing gills. X 15. 6. Var. squamulosus: normal fruiting body with lateral de velopment of hyphal membrane over maturing gills (arrow). X 60. 7. Mature abnormal fruiting body of var. tigrinus with interlacing gills. X 10. 8. Early abnormal cap region of var. tigrinus with niduli (arrows). Above the indicated niduli are developing interlacing gills. X 85. 9. Var. tigrinus: abnormal, cap showing detail of nidulum with cluster of basidia. X 1,000. Brief Articles 187

the presence of light. Although this study has indicated that the production of basidia is not necessarily light dependent, normal cap expansion does require light as previously reported (Bobbitt and Crang, 1974). Further investigations into the environmental and biochemical aspects of niduli and of hymenial differentiation are needed in order to further elucidate the control mechanisms of sporulation. This study was supported in part by an equipment grant from the Kresge Foundation.

LITERATURE CITED

Anderson, T. F. 1951. Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. New York Acad. Sci. 13: 130-134. Bobbitt, T. F. 1965. A developmental study of Lentodium squamulosum Morgan. M. S. Thesis. University of Iowa, Iowa City, Iowa. 44 p. ----- , and R. E. Crang.; 1974. Light effects on fruiting in Panus tigrinus var. tigrinus. Canad. J. Bot. 52: 255-257. Faro, S. 1972. Physiological aspects of pigment production in relation to mor phogenesis in Panus tigrinus. Mycologia 64 : 375-387. Kuhner, R. 1925. Le développement du Lentinus tigrinus Bull. Compt. Rend. Hebd. Séances Acad. Sci. 181: 137-139. Lyman, G. R. 1907. Culture studies on polymorphism of Hymenomycetes. Proc. Boston Soc. Nat. Hist. 33: 125-209. Martin, G. W. 1956. On Lentodium squamulosum. Proc. Iozva Acad. Sci. 63: 280-286. Miller, O. K. 1971. The relationship of cultural characters to the taxonomy of the Agarics, p. 197-215. In R. H. Petersen [ed.], Evolution in the higher Basidiomcyetes. University of Tennessee Press, Knoxville. Rosinski, M. A., and S. Faro. 1968. The genetic basis of hymenophore mor phology in Panus tigrinus (Bull, ex Fr.) Singer. Amer. J. Bot. 35: 720. ----- , and A. D. Robison. 1968. Hybridization of Panus tigrinus and Lentodium squamulosum. Amer. J. Bot. 55: 242-246. Singer, R. 1949. The agaricales in modern taxonomy. Lillola 22: 1-832. ----- . 1962. The Agaricales in modern taxonomy. 2nd ed. J. Cramer, Weinheim. 915 p. ARTICLE 3

FORMATION AND GERMINATION OF PANUS TIGRINUS BASIDIOSPORES AS STUDIED BY SCANNING ELECTRON MICROSCOPY

Thomas F. Bobbitt and Richard E. Crang

To Be Submitted to: Transactions of the American Microscopical Society 51 Formation and Germination of Panus Tigrinus Basidiospores as Studied by Scanning Electron Microscopy

Thomas F. Bobbitt and Richard E. Crang Department of Biological Sciences« Bowling Green State University« Bowling Green, Ohio 43403

Since the mid 1960’s the scanning electron microscope (SEM) has increasingly been demonstrated to be a valuable tool for morphological studies of mycological specimens (Ellis, et al, 1970« Rousseau, et al, 1972» Wergin, et al, 1973» and Fennell, et al, 1974). The SEM allows structures to be observed in what appears to be three-dimensions because of the great depth of field and magnification. Most SEM studies have been performed using air-dried specimens. How ever, specimens such as basidiospores and hyphal material are extremely fragile, and upon air-drying or in the vacuum of the microscope column they tend to collapse and/or become distorted. Anderson (1951) first demonstrated the preserva tion of biological structures by the use of a critical point drying method. Bigelow and Rowley (1968) suggested the cri tical point technique could solve problems of distortion, although they did not use the techniqtie'in their study in which they replicated spaces of fleshy fungi. Only recently have studies been reported which utilize the critical point method for Basidiomycete material (Pegler, 1974» Bobbitt and 52 Crang, 1975). The sequential events in the development of the basi diospore and the germination of the basidiospore of Panus tigrinus are presented in this study. Previous work revealed events involved in the development of Hymenomycete Basidio- mycetes using light microscopy (Malençon, 1942» Corner, 1948» and Perreau-Bertrand, 1967), but no studies have been reported using SEM. All previous studies of spore formation used interpretive drawings rather than actual micrographs. Two studies have been carried out using SEM to study spore germina tion and germ tube formation in fungi (Jones, 197 It Wergin, et al, 1972) but in both cases the spore and germ tubes had been air dried. In order to determine the sequential events of basidium formation and spore germination, 125 ml flasks containing 25- 50 ml of sterilized Faro’s media solidified with 1.5% Difco agar (Faro, 1972) were inoculated with a dikaryotic mycelium of P. tigrinus var. tigrinus (ATCC 28757). Flasks were stoppered with cotton plugs. Cultures were maintained at 24- 25 G and illuminated for 12 hrs each day with Sylvania Cool White (F40 CW) fluorescent lights. Light intensities were between 75 and 85 ft-c (800-900 lux) as measured by a Weston (model 756) illumination meter. Basidiocarp formation began approximately two wks after inoculation. Developing fruiting bodies were aseptically removed from the flasks, fixed with 3-5% glutaraldehyde in 0,1 M phosphate buffer (pH 7.2), 53 dehydrated through a graded ethanol series, brought to 100% amyl acetate, and dried by the carbon dioxide critical point method (Anderson, 1951). Dried basidiocarps were attached to aluminum stubs with electro-conductive carbon paint and the specimens coated with gold for 5-6 min at 1,5 kV, 8 mA, and at a working distance of 6 cm in a cold sputtering appara tus, Observations were made with an Hitachi HHS-2R SEM operating at 15 or 20 kV, and at a working distance of 15 mm and a 45° specimen tilt.

Mature basidiospores were collected by aseptically placing basidiocarps in sterilized distilled water. Gentle agitation dislodged spores which were centrifuged at approximately lOOOxg in an Adams Sero-fuge (Model 0511) for two min. The supernatant was discarded and the spore pellet transferred to a 250 ml flask containing 100 ml of sterilized liquid Faro's medium (Faro, 1972) stoppered with a foam rubber plug through which a tube had been inserted. The spore sus pension was bubbled with filtered air in order to keep the spores in suspension and provide adequate oxygen. At six hr intervals, one ml samples were aseptically removed, fixed and dehydrated. Spores dried by critical point method on 0.6 um pore size Nuclepore membrane filters (Nuclepore Co., Pleasanton, Ca.) were attached to aluminum stubs with electro-conductive carbon paint and gold coated as described for the basidiocarps. Observations with SEM were made at a working distance of five mm. 54 With SEM the entire gill surface of maturing basidio carps could be observed. Basidium formation began on the stipe between the gill plates and then expanded over the gill surface. From one fruiting body several stages of basidia and spore formation were observed. The surface of the develop ing hymenium was covered with club-shaped hyphal tips. The development of basidia and basidiospores of P. tigrinus was essentially the same as that reported by Malen£on (1942) using light microscopy. The first evidence of hyphal differentiation into a basidium appeared when the tip of the hypha flattened and four protrusions developed (Fig. 1). Developmentally, the protrusions elongated (Figs. 2, 3), and eventually became the sterigmata. Before the end of the elongation period, spherical bodies from 0,5 to 1.0 um diameter were formed on the ends of each sterigmata (Fig. 4). Malen£on (1942) referred to these bodies as 1 *apophyse sporogene or in shortened term 1'apophyse. The l1apophyse were spore-produc ing outgrowths from which new spores developed. As the sterigmata completed their elongation, they bowed outward (Figs. 5» 6). The external half of the 1*apophyse began to balloon outward while the inner surface remained unchanged (Figs. 5, 6, 7). The half of the 1*apophyse which ballooned represented the developing spore. At that stage, the apex of the young spore fell on a line running from the inner base of the 1*apophyse to the position of greatest expansion of the 55 spore. At first the outward expansion was relatively uni form, giving the appearance of a bubble (Figs. 6, 7), but shortly thereafter the inner face of the spore slowed in expansion, while the outer surface continued to swell. The uneven growth caused a shift in the symmetry of the spore, giving the spore a bilateral symmetry (Fig. 8). With an uneven growth rate, the apex of the spore shifted approxi mately 45° and caused the spore to reposition itself along

the same axis as the sterigma (Fig. 9). At maturity, the portion of the 1*apophyse which did not expand during spore formation, became the spore hilum. The base of the hilum was the site where the mature spore separated from the sterigma. The surface of mature P. tigrinus basidiospores appeared to be smooth at the level of obtainable SEM resolu tion (Fig. 10), The first 24-30 hrs after the spores were placed in liquid medium no external morphological changes could be seen. At the end of 36-48 hrs the outer wall layer (or layers) of the spores began to slough (Fig. 11) leaving the outer wall as an empty shell (Fig. 12). The surface of the spore was rough in appearance due to patches of outer wall materials which remained. By 48-60 hrs a pronounced bulge could be seen in the outward-facing or convex spore wall. The bulge represented early development of the germ tube (Fig. 13). Between 70-72 hrs the germ tube elongated (Figs. 14, 15) and developed into the primary mycelium. 56 Hawker (1968) described three modes of spore germina tion based on the manner of germ tube emergence from spores. Those spores in which the wall was composed of a single layer bulged in one or more places to form a germ tube. Spores which had walls composed of more than one layer showed a rup turing of the outer layer which allowed the inner layers to protrude as a germ tube. Some thick-walled spores have a germ pore, or thin area on the wall which may be dissolved or pushed outward allowing the emergence of a germ tube. Earlier SEM studies of germinating spores dealt with spores whose mode of germination was of the germ pore type (Jones, 1971« Wergin, et al. 1972). The mode of germination of P. tigrinus spores as found in the present study closely follows the second type of germination described by Hawker (1968). The rupture of the outer spore.wall of P. tigrinus could be the result of water uptake, which caused the wall to swell. The portion which sloughs may be one or more layers thick but could not be determined by SEM. This outer coat may cor respond to the layer which Williams and Ledingham (1964) described by means of transmission electron microscopy as swelling extensively in uredospores after potassium permangan ate fixation. Presumably in nature the outer wall is not com pletely removed, but instead tears, allowing extrusion of the developing germ tube. Certain problems were encountered in this study. Electron beam penetration at higher kV’s was a problem, 57 resulting in "ghost" images. In addition, basidiospores perched on the tips of sterigmata tended to shift due to heating of the specimen. However, the use of glutaraldehyde to stabilize structures, coupled with the critical point dry ing method provides the best preservation for SEM studies. 58

Figs. 1-4. Panus tigrinus. Development of sterigmata and basidiospores. 1. Basidium with flattened apex and four sterigmata protrusions. 10,000 X. 2. Basid ium with developing sterigmata. l4,000X. 3. Later stage of sterigmata formation. 11,000 X. 4. Basidium with the 1*apophyse developed at tips of sterigmata. 8,000 X. 59 60

Figs, 5-8. Panus tigrinus. Development of basidiospore on sterigmata. 5. Maximum elongation of sterigmata and early enlargement of the spore. 10,000 X. 6, Balloon ing of the external half of the 1*apophyse. 6,000 X. 7. Further development of the new spores. 8,000 X. 8. Basidiospores near maturity, after their reposition ing to the same axis as that of the sterigmata. 10,000 X. 61 62

Fig. 9. Panus tigrinus (interpretive drawing). The directional shift of the apex of a developing basidiospore 63 64

Figs. 10-15. Panus tigrinus. Stages of germinating spores, 10. Spore prior to germination. 16,000 X. 11. Spore approx, 25 hrs after being placed in germina tion medium. Outer wall layers have begun to slough. 14,000 X. 12. Outer wall layers detached from the spore. 9,000 X. 13. Spores in liquid medium for approx. 55 hrs. A bulge has formed on the outward-facing wall. 11,000 X. 14. Approx. 72 hrs in germination medium the germ tube has formed, 10,000 X. 15. Germinating spore (approx. 72 hrs) with part of outer wall attached at the right side. 10,000 X. 65 66 REFERENCES

Anderson, T. F. 1951. Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N. Y. Acad. Sci. 13« 130-134. Bigelow, H. E., and J. R. Rowley. 1968. Surface replicas of the spores of fleshy fungi. Mycologia 60« 869-887. Bobbitt, T. F., and R. E. Crang. 1975« Basidiocarp develop ment of the two varieties of Panus tigrinus and their light-induced abnormal forms. Mycologia 6^7» 182-187. Corner, E. J. H. 1948. Studies in the basidum. I. The ampoule effect, with a note on nomenclature. New Phytol. 4£j 22-51. Ellis, J. J., L. A. Bulla, G. St, Julian, and C. W. Hessel- tine. I97O. Scanning electron microscopy of fungal and bacterial spores. In: Scanning Electron Microscopy/ 1970. 0. Johari (Ed.) IITRI, Chicago, Ill. 147-152. Faro, S. 1972. Physiological aspects of pigment production in relation to morphogenesis in Panus tigrinus. Mycologia 64« 375-387. Fennell, D. I., G. St. Julian, L. A. Bulla, Jr., and F. L. Baker. 1974. Scanning electron microscopy of conidio- spore ornamentations in Aspergillus species. In« Scanning Electron Microscopy/1974. 0. Johari and I. Corvin (Eds.) IITRI, Chicago, Ill. 414-420. Gottlieb, D. 1964. Germination of fungus spores. Endeavour. 2J: 85-89. Hawker, I. E. 1968» Physiology of Fungi. Reprinted by Yerlag Von J. Cramer, N. Y. 360. Jones, D. R. 1971. Surface structure of germinating Uromyces dianthi urediospores as observed by scanning electron microscope. Can. J. Bot. 4-9: 224-3. Malençon, M. G. 1942. La sporegenese chez les basidiomycetes. Comptes Rendus Seances 215« 584-586. Pegles, D. N. 1974. Critical point technique for scanning studies of basidiomycete hymenia. Trans. Br. Mycol. Soc. 6js 175-211. 67 Perreau-Bertrand, J. 1967. Recherces sur la différencia tion et la structure de la paroi sporale chez les Homobasidiomycetes a spores orness. Ann. Sci. Nat., Bot. 12: 639-746. Rousseau, R., H. 0. Halvorson, L. A. Bulla, Jr,, and G. St. Julian. 1972. Germination and outgrowth of single spores of Saccharomyces cerevisiae viewed by scanning electron and phase contrast microscopy. J. Bacteriol. 109» 1232-1238. Wergin, W. P., L. D. Dunkle, J. L. Van Etten, G. St. Julian, and L. A. Bulla, Jr. 1973. Microscopic observations of germination and septum formation in pycnidiospores of Botryodiplodia theobromae. Developmental Biol. .22» 1-14. " a

ARTICLE 4

Structural and Microchemical Analysis of Panus Tigrinus Spore Walls 69 ABSTRACT

The basidiospore wall of Panus tigrinus was found to be composed of five layers. The innermost layer, or endo sporium, consisted of chitin microfibrils. Surrounding the endosporium layer was an electron-dense mesosporium which was probably a sublayer of the episporium. The mesosporium and episporium layers were apparently composed of lipid- protein-polysaccharide material. Outside the episporium was an electron-transparent perisporium layer which was probably composed of hexuronic acid. The outermost layer was a thin electron-dense ectosporium layer composed of lipid and pro tein. Invaginations of the plasma membrane into the cyto plasm along the spore wall were observed. These structures were postulated to be collection sites for spore wall pre cursor materials prior to their transport into the spore wall and assembly into established wall substances. 70 INTRODUCTION

Transmission electron microscopy (TEM) has revealed the basidiospore walls were composed of various layers. Descriptions of the layers have been reported by Singer (1962), Perreau-Bertrand (1967), and Pegler and Young (1971). Those species of Basidiomycetes producing spores with smooth surfaces have walls composed of five layers (Perreau-Bertrand, 1967). The chemical composition of wall layers is not well known (Alexopoulos, 1962). Only a few attempts have been made to determine the chemical composition of spores or spore walls from Basidiomy cetes. Prentice and Cuendet (1954) demonstrated whole uredo spores of Puccinia graminis contained 2% D-glucose, 19% D- mannose and 3% D-arabitiol on a dry-weight basis. The basidiospores of Ustilago zeae were found not to contain chitin (Gottlieb, 1964). On the other hand, Graham (i960) reported the teliospores of Tilletia contraversa possessed chitin as well as lipid, pectin, protein, and hemicellulose. The present investigation was undertaken in order to characterize the structure and basic chemical composition of the wall layers of the hyaline, smooth-walled, basidiospores of Panus tigrinus. 71 MATERIALS AND METHODS

The dikaryotic culture of Panus tigrinus var. squamulosus used in this study was originally obtained from Dr. M. A. Rosinski, formerly at the Department of Botany at the University of Iowa. A subculture of the strain has recently been deposited by the author with the American Type Culture Collection, Rockville, Maryland (ATCC number 28757). Basidiospores were obtained from basidiocarps grown in 125 nil flasks containing 25-50 ml of sterilized synthetic media (Faro, 1972) solidified with 1.5% Difco Bacto agar. The medium consisted of the following 1 glucose, 3° g; NH^-tar- trate, 1 g; KHgPO^, 1 g; MgSO^»7^0, 2.88 mg; FeSO^07H20,

2.78 mg; CuS0^«5H20, 0.74 mg; MnS0^“4H'20, O.67 mg; ZnSO^^HgO, 2.88 mg; thiamine-HCl, 120 ug; CaCl2, 25 mg; in one liter of distilled water. Flasks of the medium were plugged with cotton and autoclaved 15 min at 15 psi. The flasks were inocu- o lated with one cm sections of dikaryotic mycelium and main tained under a 12 hr photoperiod at 23-24 C. Light was obtained from Sylvania Cool White (F40 CW) fluorescent bulbs at an intensity of 80-85 ft-c as measured by a Weston (Model 756) illumination meter. Fruiting usually began two-three wks after inoculation. Basidiospores were collected aseptically by removing mature fruiting bodies and placing them in 7 1/2 X 1 cm test tubes containing three or four ml distilled water. Gentle pressure was applied to the basidiocarps with a stainless steel spatula, causing the release of basidiospores from the fruiting structure. The spore suspension was transferred to a clean test tube with a Pasture pipette and centrifuged at 3400 rpm (lOOOxg) with an Adams Sero-fuge (Model 0511) for two min. The supernatant was discarded. Routinely, three washings were carried out on each group of fruiting bodies, and the spores pooled. This method gave large numbers of spores free from extraneous mycelial material.

Light Microscopy Preliminary attempts to characterize the composition of the basidiospore wall were made by the use of semi-selective stains. Although the stains employed were not highly specific, they were used in order to determine the presence or absence of major groups of chemical compounds. A positive or negative result was determined by comparison of stained spores with stained substances of known composition serving as standards.

Lipids The presence or absence of lipids was determined by the use of Sudan IV stain (Johansen, 1940). Basidiospores were placed for one hr in a solution containing 0.5 g Sudan IV (Allied Chemical Co., New York, N.Y.) stain dissolved in 100 ml 70% ethanol and then rinsed in 50% ethanol followed by centrifugation. Fats and oils stained red. 73 Callose Callose was determined by the technique reported by Johansen (1940). Basidiospores were placed in a 0.005% solu tion of Aniline Blue (Matheson, Coleman and Bell, Cincinnati, Oh.) in 50% ethanol for eight hrs, and then rinsed in water by centrifugation and observed. Callose, when present, stained blue under bright-field light microscopy.

Lignin Determination of lignin was by the procedure of Siegel (1953). Spores were placed in fresh commercial Clorox (Clorox Co., Oakland, Ca.} 5.25% sodium hypochlorite) solu tion for five min, then placed in cold 1% solution of sodium sulfite. A bright red color developed within a few minutes if lignin was present,

Chitin The test for chitin was based on the technique reported by Roelofsen and Hoette (1951). Spores were auto claved at 15 psi and 121 C in 60% aqueous KOH for three hrs. The action of KOH converted the chitin to chitosan by the removal of the n-acetylate from the chitin molecule (Forster and Webber, I960). Spores were washed by centrifugation in distilled water. Half the sample was placed on a slide with a few drops of IKI in 1% sulfuric acid. Chitosan stained violet. The remaining half of the spore material was treated with 2% acetic acid solution for 30 min before applying IKI 74 in 1% sulfuric acid. The weak acetic acid solution dissolved the chitosan and the spore material did not stain positive with IKI.

Cellulose Cellulose was determined by the technique reported by Johansen (1940), Basidiospores were placed in IKI solution for 15-30 min. The spores were then centrifuged and mounted under glass coverslips with IKI, then a drop of 65% sulfuric acid was added to the side of the coverslip and allowed to diffuse into the spore film. Walls containing cellulose stained "steel" blue.

Pectic Compounds Two methods were used to test for pectic substances. 1. The first method was based on the technique reported by Johansen (1940). Basidiospores were placed in I15OOO aqueous ruthenium red solution (Electron Microscopy Sciences, Ft. Washington, Pa.) for one hr. Pectic substances appeared pink to red. 2. A second method was based on Gee, et al (1959). Spores were treated with an alkaline hydroxylamine solution (14 g NaOH in 100 ml of 60% ethanol, and hydroxylamine HC1 14 g in 100 ml of 60% ethanol, in a total ratio of 1»1) for one hr. An equal volume of a solution containing one part concentrated HC1 and two parts 95% ethanol was added to acidify the reaction mixture. The excess solution was 75 removed from the spores by centrifugation, and the spores flooded with a 10% solution of ferric chloride in 0.1 N HG1 in 60% ethanol. The spores were left in the solution for one hr. After the esterification reaction was complete, the spores were washed by centrifugation and allowed to remain for one hr in 60% ethanol which facilitated removal of uncom- plexed ferric ions. Esterified pectins appeared red.

Chemically Cleaned Spore Wall Material In order to study the microfibrillar components of the walls of basidiospores, amorphous wall materials and cellular cytoplasm were chemically digested by a modification of the method described by Dodge and Lawes (1969). A spore pellet containing approximately one g wet weight of spores collected as described previously, was brought to a volume of 51 ml with distilled water. A one ml aliquot of the spore suspen sion was withdrawn and diluted 1:50,000 with distilled water and used to determine spores/ml with a hemocytometer. The dry weight per ml of the spore suspension was determined by placing 5 ml of spore suspension in a tared aluminum pan, followed by drying at 90 C for 48 hrs and then weighing with an electric balance. Both spore counts and dry weight determination were carried out in triplicate. The remaining 35 ml of spore suspension was concen trated by centrifugation and subjected to 10 ml of 2% KOH in a 90 C oven for one hr. The spores were washed two or three times with distilled water by centrifugation. Washed 76 spores were macerated by suspending them in 10 ml of a mix ture of equal volumes glacial acetic acid and 3°% hydrogen peroxide at 70 C for one hr. The spores were then centri fuged and the supernatant discarded. Ten ml of 2% sulfuric acid were then added, the spore suspension cooled, washed twice with distilled water, and centrifuged. A final wash consisted of absolute methanol to remove residual lipids. Cleaned wall material was then brought to 35 ml with dis tilled water and divided into seven, five-ml aliquots. Three of the aliquots were used to determine the dry weight of the cleaned spore wall material, as described above, and the per cent of the dry weight of a whole spore which was made up of microfibrils was calculated. One aliquot was treated with chitinase (Tracey, 1955). The enzyme (Calbiochem, La Jolla, Ca.j lot 73217) was prepared by dissolving 100 mg chitinase in 100 ml 0.05 M acetate buffer (pH 5»°) (Merchant, Kahn, and Murphyj 1964) solution for six hrs at 40 C. An qliquot of spore material was treated with cellulase as described by Crang and Hein (1971). The enzyme (Calbiochem, La Jolla, Ca.i lot 72410) was prepared by dissolving 100 mg cellulase in 100 ml of 0.05 M acetate buffer (pH 4,7). The spore material was concentrated as before and added to 10 ml of cellulase for six hrs at 40 C. An aliquot was concentrated and subjected to the action of acetate buffer (pH 4.7) for six hrs at 40 C and served as a control. A final aliquot was not treated and served as a control. 77 Each of the four aliquots was washed and concentrated in one ml of distilled water by centrifugation and prepared for metallic vacuum-evaporation and subsequent observation with TEM. Samples from each aliquot were deposited on collodion-coated 300-mesh electron microscope grids and dired. Coated grids were shadowed at a 20° angle in a Denton Vacuum evaporator (DV-502) with gold-palladium (60»40) and examined in an Hitachi HS-8F-1 transmission electron micro scope operating at 50 kV.

Scanning Electron Microscopy Scanning electron microscopy (SEM) was employed in order to study the morphology of both treated and unreated basidiospores. Untreated spores were fixed with 4.0% glutaraldehyde in 0.1 M Sorenson’s phosphate buffer (pH 7.2), dehydrated through a graded ethanol series and then brought to 100% amyl acetate. Solution changes were made by centri fugation as described above. Spores in 100% amyl acetate were pipetted onto 13 mm Nuclepore membrane filters which had a 0.6 um pore size (Nuclepore Co., Pleasanton, Ca.) and excess amyl acetate was drained leaving a large number of spores attached to the filters. Filters and spores were dried by the carbon dioxide critical point method according to procedures outlined by Anderson (1951). Dried filters with spores were attached to aluminum stubs with electro- conductive carbon paint and coated with pure metallic gold for five to six min at 1.5 kV, 8 mA and at a working distance 78 of six cm in a cold sputtering apparatus. Observations were made with an Hitachi HHS-2R scanning electron microscope operating at 20 kV, and at a working distance of 15 mm and a 45° specimen tilt. Lowry and Sussman (1958) reported, at least in the ascospores of Neurospora tetrasperma, a 1% solution of sodium hypochlorite (20% Clorox) removed the outer spore wall layers Basidiospores were treated with Glorox in order to determine if similar results could be achieved. Five ml of a 20% aqueous solution of Clorox was added to approximately 4 mg (wet weight) basidiospores and incubated for 60 min at room temperature. Basidiospores were washed by centrifugation in three or four changes of distilled water, followed by glutaraldehyde fixation, dehydration and prepared for drying by the critical point method previously described. Attempts were also made to remove the outer wall layer of the spore by means of solvents and enzymes. Removal of lipids was carried out by the use of a mixture of acetone, ether, and methanol (1»1:1). Approximately four mg (wet weight) basidiospores were incubated in 10 ml of the solvent mixture for 60 min at room temperature. The spores were washed by centrifugation in three or four changes of dis tilled water, fixed with glutaraldehyde, and dried by the carbon dioxide critical point method. Attempts were also made to remove proteins from the spore walls. Approximately four mg of spores were suspended in 10 ml of 0.1 M Sorenson’s 79 phosphate buffer (pH 7.2) to which five mg crystalline tryp sin (Sigma, St. Louis, Mo.» lot 118B-1840) had been added. Digestion was carried out for three hr at 37 C, spores were washed twice by centrifugation in distilled water, fixed, and dried. A third treatment consisted of pretreating the spores to remove lipids and then subjecting the spores to trypsin digestion before fixing and drying by the critical point method.

Transmission Electron Microscopy The ultrastructure of basidiospore walls as well as the cytoplasm was studied with TEM. Preliminary results indicated the best method for fixing basidiospores for TEM was with a modified paraformaldehyde-glutaraldehyde fixa tion procedure (Karnovsky, 1965). The fixative was prepared by adding 15 ml of freshly prepared 8% paraformaldehyde to 10 ml of 8% glutaraldehyde and 15 ml of 0.1 M Sorenson’s phosphate buffer (pH 7.2). The paraformaldehyde was prepared by dissolving four g paraformaldehyde in 46 ml distilled water at 65 C. The solution was neutralized and cleared by adding a few drops of 20% NaOH and then filtered to remove any white precipitate, prior to cooling to room temperature. Approximately six mg (wet weight) basidiospores was added to 10 ml fixative and after ten min at room temperature the fixative and spores were placed in a 4 C refrigerator for two hr. After fixation, the spores were washed three times by centrifugation (each wash time was at least 15 min) with cold 80 buffer-water (1:1, v:v). Postfixation was performed in 2% osmium tetroxide buffered to pH 7.2 with 0.1 M Sorenson’s phosphate buffer &r two hr at 4 C. The basidiospores were dehydrated in a graded ethanol series and embedded in Spurr low-viscosity embedding media according to the method of Spurr (1969). Curing was for eight hr in a 70 C oven. Sections approximately 70 nm thick were cut with glass knives on an LKB Ultrotome III (LKB Produckter AB, Bromma, Sweden), collected on 300-mesh unsupported grids, and post- stained with 2% aqueous uranyl acetate (Pease, 1964) for 20 min followed by lead citrate (Reynolds, 1963) for five min. Observations were made with an Hitachi HS-8F-1 transmission electron microscope operating at 50 kV. Micrographs were taken on Kodak electron microscope film type 4489, and developed with Kodak D-19 for four min. Basidiospores were treated enzymatically and/or with selective solvents as previously described to remove cellu lose, chitin, and lipid and then prepared for TEM observation. In addition, a sample of spores was treated to extract pro teins. Spores were added to 10 ml of 0.1 M Sorenson’s phos phate buffer (pH 7.2) containing 0.2 g pronase (Calbiochem, La Jolla, Ca.; lot 400325) and incubated at 37 C or 48 hr. Spores were incubated in 0.05 M acetate buffer (pH 5«0) for eight hr at 40 C and served as a control for the cellulase and chitinase treatments. 81 technically Ruptured Spore Wall Material Basidiospore wall material free from cytoplasmic material was obtained by treating the spores through a modi fication of the method described by Lamanna and Mallette (1954). A 20 ml spore suspension containing approximately 90 mg of spores was placed in a prechilled Eberbach stainless steel semi-micro blending assembly (Scientific Products, Chicago, Ill.) containing 20 g 0.25-0.30 mm glass beads (Matheson Scientific, Inc., Cleveland, 0.). Spores and beads were blended for five min, then chilled and blended for an additional five min. Broken spores were transferred to a 150 ml beaker. The blender cup was washed with 25 ml dis tilled water and the wash water added to the beaker contain ing the beads and broken spores. The beads in the 150 ml beaker were allowed to settle and the supernatant containing spore wall material was removed and centrifuged at 3400 rpm for 10 min. Wall material was washed twice by centrifugation with distilled water. Mechnically ruptured wall material was divided into six equal amounts and retained for further treatments. One sample was not treated, another sample was subjected to 0.1 M Sorenson’s phosphate buffer (pH 7.2), and a third was treated with 0.05 M acetate buffer (pH 5*0) at 40 C for eight hr. The other samples were treated as described previously to extract protein (pronase), chitin, and lipid. All samples were washed three or four times with distilled water by 82 centrifugation and concentrated in one ml of distilled water. Samples were deposited on collodion-coated electron micro scope grids, dried, shadow cast with gold-palladium (60«40) at 20° angle prior to TEM observation. In addition, a por tion of the untreated wall material was deposited on Nucle- pore membrane filters, air dried, coated with pure metallic gold, and observed with SEM. 83 RESULTS AND OBSERVATIONS

Light Microscopy Table I contains the results of staining whole basi diospores with various stains. Negative results were obtained for lignin, callose, and cellulose. Ruthenium red caused the spores to appear bright red after treatment and was considered to be positive. Preston (1952) stated ruthen ium red was not specific for pectic compounds but would also stain oxidized cellulose and other cytoplasmic components, thus hydroxylamine chloride staining was used to verify the presence of pectic compounds. Using this technique the spores stained a brownish yellow color, which was considered to be negative for pectins. Sudan IV stained lipid droplets within the cytoplasm a deep red and also stained the outer edges of the spores a dark pink, suggesting the presence of lipids in the spore walls. Spores treated to convert chitin into chitosan, and then stained with IKI, turned a violet color which was positive for the test. Spores treated to dissolve chitosan with a weak acetic acid solution gave a negative response with IKI.

Chemically Cleaned Spore Wall Material

Dry Weight Measurements Air dried spores were found to have an average single spore weight of 4.6 X lO“1^ following drying at 90 C for -13 48 hr they had an average dry weight of 4.04 X 10 J g. TABLE I The composition of P. tigrinus basidiospores as determined by various staining techniques

Lipid + Lignin - Callose - Chitin Cellulose - Pectic Compounds Ruthenium Red 4* Hydroxylamine Chloride -

+ Positive Reaction - Negative Reaction 85 Water was calculated to be 12.1% of the spore weight.

Cleaned Spore Wall Material Basidiospores, treated by chemical digestion, con tained resistant microfibrils (Fig. 1). These microfibrils retained the general shape of the spore and consisted of a randomly oriented network. The microfibrils were long and had a diameter of approximately 20 nm. Each spore network of fibers had at least one dark area, usually centrally located. Increased magnif ication revealed these dark struc tures were internal to the microfibrils. Microfibrils could be seen transversing over the dark areas (Fig. 2). Microfibrils treated with cellulase or acetate buffer were little different in appearance from untreated micro fibrils (Fig. 3). On the other hand, spores treated with chitinase were completely destroyed, leaving only debris and irregularly-shaped bodies believed to be remnants of the dark bodies found within the microfibril network (Fig. 4). The dry weight of the chemically resistant micro fibrils was determined and compared to whole spore dry weights. The microfibril material was found to comprise approximately 3»5% of the dry weight of the whole spore.

Scanning Electron Microscopy Basidiospores of P. tigrinus observed with SEM were elongate and elipsoidal. The surface of the spores was smooth. The most prominate morphological structure associated 86 with the spore was the hilar appendage (hilum) which was the site of spore attachment to the sterigma of the basidium (Fig. 5). The outer wall of basidiospores treated with a 20% solution of Clorox was swollen and had begun to tear (Fig. 6). These results were similar to those reported by Lowry and Sussman (1958) for the outer ribbed spore wall of Neurospora tetrasperma. With removal of the outer layer, the basidio spore appeared to have a smooth inner wall surface, with a few areas of outer wall material remaining attached. The outer walls of basidiospores treated with solvents and enzymes differed from untreated spores. Extraction of lipids by solvents resulted in a slight roughening of the spore surface (Fig, 7)» but the outer wall layer was not freed from the spore. The action of the enzyme trypsin resulted in the removal of the outer wall layer, revealing a rather smooth inner spore wall layer (Fig. 8). There appeared to be some breakdown of the outer wall material by the enzyme action. Spores treated first with lipid solvents and then treated with trypsin had the outer wall removed and the sur face of the inner wall appeared rough (Fig. 9). This sug gested either portions of the outer wall had remained attached to the inner wall or the inner wall had begun to breakdown.

Transmission Electron Microscopy Figure 10 illustrates the ultrastructural composition of a typical P. tigrinus basidiospore. A typical eukaryotic 87 nucleus was usually centrally located in the spore. Mito chondria were scattered along the periphery of the spore cytoplasm with their cristae extending lengthwise. The cyto plasm of the spore contained large amounts of dark-steining granular material representing glycogen deposits. Lipid bodies not bound by a membrane were found scattered through out the cytoplasm and were only slightly electron-dense. Some lipid bodies located at the periphery of the spore cyto plasm contained coiled structures which stained darkly (Fig. 11). In addition, other electron-dense bodies approximately the same size as the lipid bodies were often seen in the cytoplasm of the spore (Fig. 10). Figure 12 shows a lipid body adjacent to two dark-staining bodies. The lipid body contained a dense core which appeared to be in contact with the two other dense bodies. Surrounding the cytoplasm, and inside the spore wall, was a thickened plasma membrane. Sections in a horizontal or longitudinal plane showed that the plasma membrane formed invaginations penetrating into the cytoplasm (Figs. 10, 13). The invaginations appeared to be made up of two unit mem branes which formed a cup-like structure (Fig. 14). Between the two unit membranes, electron-dense granules could be observed. The matrix of the invagination was similar to the background cytoplasm of the spore. The spore wall was found to consist of five layers (Fig. 15). Immediately outside the plasma membrane was an 88 electron-transparent layer of approximately 8-16 nm thick ness. The next layer was a dense zone of approximately 8 nm thickness. Then came a moderately dense, 16 nm layer which was flared in the region of the hilum and appeared to be com posed of several layers of fibrous materials. The next layer was a uniformly thick 12 nm electron-transparent zone. The outermost region was a narrow, electron-dense zone approxi mately 3-4 nm thick. Basidiospores treated with chitinase showed a marked degradation of the inner spore wall layer (Fig. 16). It is possible that the low pH of hydrolysis caused the disintegra tion of the plasma membrane. In addition to the breakdown of the inner wall layer, the second layer from the outside showed some degradation following chitinase treatment (Fig. 17). The outer layer was ruptured. The two electron-dense middle layers became slightly less electron-dense, but this could have resulted from degradation due to the low pH of the incu bation medium. Cellulase-treated spores showed no loss of wall layers, but the plasma membrane was disrupted. The outer thin layer tended to separate from the second layer (Fig. 18). Spores subjected to acetate buffer at 40 G lost the dense- staining properties in the innermost dense layer but in all other respects were similar to the non-treated spores. Both lipid extraction and pronase treatment caused a decline in density of the middle two dense layers (Fig. 19). 89 technically Ruptured Spore Wall Material Chemically-intact spore wall material was easily obtained by mechanically rupturing the spore walls. Virtu ally all spores were ruptured into pieces ranging from almost whole spore walls to small fragments of wall material. Mechnically-ruptured spores, which had not been treated to remove any components, possessed three distinct wall layers (Fig. 20). The outermost layer, an amorphous zone appearing as fragments on the remaining wall layers, appeared to be extremely fragile, since the action of the glass beads tended to break or remove it. The middle layer had a fine granular appearance. Microfibrils were embedded in the middle layer toward the interior of the wall material. Mechnical-rupturing of wall material followed by treatment with chitinase showed the microfibrils had been degraded and removed (Fig. 21). An outline of where the fibrils had been could be seen surround ing the spore wall material. Wall material treated with acetate buffer at 40 C had a middle layer which was smoother than was the case for untreated material. The internal por tion of the wall had fibrils embedded in the middle layer. Lipid-extracted wall materials showed no appreciable differ ence from untreated materials (Fig. 22). Ruptured spore walls treated with pronase formed a white jelly-like material which would not sediment in the centrifuge. Sonication of the material did not disrupt it and it was discarded. In Figurq 23 mechnically-ruptured spore wall material 90 prepared for SEM revealed three wall layers.* The outer wall layer appeared battered and partially broken. The two pos sible interior walls appeared folded but otherwise smooth.

Preliminary attempts at energy-dispersive x-ray micro- analysis coupled with SEM were performed on untreated, mechnically-ruptured wall material, but because of the limited amount of material and the lack of statistically significant results, the work was not included in this study. However, variance in the amounts of chlorine, potassium, and calcium suggested the wall material was composed of three layers. 91

Figs. 1-4. Panus tigrinus. Chemically digested spores. 1. Spore showing wall microfibrils with dense region. 36,000 X. 2. Enlarged area of dense region showing fibrils superimposed over the region. 54,000 X. 3. Spore appearance after either cellulase or acetate buffer treatment. 35,000 X. 4, Chitinase treated microfibrils showing complete destruction of wall fibrils. 30,000 X. 92 93

Figs. 5-9. Panus tigrinus. Spores observed by means of SEM. 5. Whole, untreated spores. 14,000 X. 6. Basidiospore treated with commercial Clorox. The outer walls are swollen and broken. 8,000 X. ?. Spore treated for lipid extraction showing roughening of outer wall. 12,000 X. 8. Spores treated with trypsin with outer walls of one removed and seen at lower left. 14,000 X. 9. Spore treated first to remove lipids and then treated with trypsin showing roughened inner wall and the removal of the outer wall. 14,000 X. 94 95

Figs. 10-12. Panus tigrinus. Spores observed by means of TEM. 10. Longitudinal section of spore showing nuclear region (N), lipid bodies (L), and Mitochondrion (M). Arrows point to invaginations of the plasma mem brane. 36,000 X. 11. Coiled structure located within lipid bodies. 4-7,000 X. 12. Hilar region of spore (H) with dense osmophilic bodies and a condensed structure which appears in contact with an osmophilic body. 54,000 X. 96 97

Figs. 13-16. Panus tigrinus. TEM sections of basidio spores. 13. Gross section of spore showing invagination of plasma membrane, 45,000 X. 14. Detail of an oblique section through an invagination. 72,000 X. 15. Section through the spore wall showing the five layers, ectospor ium (EOT.), perisporium (PER.), episporium (EPI.) and meso sporium (MES.), and an endosporium (END.). 110,000 X. 16. Section of a spore treated with chitinase. The plasma membrane and endosporium have been disrupted. 36,000 X. 98. 99

Figs. 17-19. Panus tigrinus. TEM sections of basidio spore wall. 17. Section through the wall of a spore treated with chitinase. The perisporium (second layer) is reduced in thickness, and the densities of the episporium and mesosporium (third and fourth layers) are reduced. 50»000 X. 18. Section of spore treated with cellulase. Arrow points to separation of ectosporium (outer wall) layer. 70,000 X. 19. Section through spore wall treated with lipid solvents showing decline in density of the two middle layers (episporium and meso sporium). 54,000 X. 100 101

Figs. 20-23. Panus tigrinus. Mechnically-ruptured spore wall material. 20. Shadowed spore wall showing three layers, outer layer (A), middle layer (B), and inner layer (G). 36,000 X. 21. Shadowed spore treated with chitinase, showing lack of microfibrils. 35»000 X. 22. Shadowed spore treated to remove lipid material. All layers remain. 45,000 X. 23. SEM micrograph of spore wall, material with no chemical treatment. 3*500 X, 102 103

DISCUSSION

The basidiospores of P. tigrinus were found to be similar in their ultrastructure to that reported for other fungi (Voelz and Niederpruem, 1964; Manocha, 1965» and Hyde and Walkinshaw, 1966). Osmiophilic bodies were found scattered throughout the spore but larger numbers tended to be in the region of the hilum. Hyde and Walkinshaw (1966) observed similar bodies in Lenzites saepiaria and referred to them as lipid bodies. It is possible in P. tigrinus spores that these darkly stained bodies are the result of the condensation of lipid material. The coiled bodies within lipid bodies of Fig. 11 may represent early stages in the condensation process, and Fig. 12 may represent a later stage in the process. Surrounding the cytoplasm of the spore was a plasma membrane which was observed in several sections to be invagin- ated. Shatkin and Tatum (1959) found invaginations of the hyphal plasma membranes of Neurospora crassa and proposed they were associated with cellular activity. Aggregates of vesicles between the cell wall and plasma membrane were found by Moore and McAlear (1961) in several different fungi which they called "lomasomes". In the nearly mature basidium of Schizophyllum commune, Wells (1965) found invaginations of the plasma membrane, which he labeled extracytoplasmic loculi. Since the invaginations were observed in maturing basidia, he postulated they may function either to increase the turgor 104 pressure within, the basidum or they were associated with cytoplasmic degeneration. Hyde and Walkinshaw (1966) found invatinations of the cell membrane occurring at irregular spacings along the spore and hyphal walls of Lenzites saepiaria. These authors suggested that loamsomes, vesicu lar bodies, and concentric complex membranes were different manifestations of the same structure and postulated the structures were a means of moving substrates or enzymes through the cytoplasm. Lomasomes and iomasome-like bodies were suggested to be involved in cell wall formation. Craw ley (1965) and Barton (1965) described cytoplasmic organelles associated with the cell walls of Chara and Nitella, and sug gested these organelles were involved in cell wall, formation. Further, the lomasomes of Penicillium vermiculatum have been demonstrated to be involved in formation of ascospore walls (Wilsenach and Kessel, 1965). Figure 10 shows two invaginations along the walls of a basidiospore of P. tigrinus. Between the two membranes granules may be seen. The invaginations may be sites where spore wall precursors accumulate before being transported into the wall for fibril formation. Ruiz-Herrera and Bart- nicki-Garcia (1974) reported isolating a soluble chitin synthetase from Mucor rouxii, and were able to synthize chitin microfibrils in vitro. Their work suggests the cell wall microfibrils are formed within the wall itself and not within the cytoplasm. As the basidiospores of P. tigrinus 105 develop and expand in size there exists the necessity for rapid formation of chitin microfibrils. These invaginations may represent sites which provide the material needed for rapid chitin formation within the wall. Since these struc tures were not found in all spores, they may be functional only in developing spores. When the spores have reached maturity the invaginations may disappear. Invaginations observed in this study may be reminants of lomasome-like bodies. The basidiospore wall of P. tigrinus as seen in TEM has five layers. These layers correspond to the endosporium, mesosporium, episporium, perisporium and ectosporium previ ously described by Singer (1962), Perreau-Bertrand (1967), and Pegler and Young (1971). The endosporium, mesosporium and episporium have been reported to be of sporal origin, whereas the perisporium and ectosporium are of basidial origin (Pegler and Young, 1971). The innermost layer of the spore, the endosporium, was observed to be an electron-clear zone and believed to be composed of chitin microfibrils. This conclusion was based on the fact that chitinase caused the complete disruption of the layer. Spores treated chemically to remove the amorphous wall material had only a net of randomly-oriented micro fibrils which were degraded by the action of chitinase. Additional support for the conclusion was found in mechani cally-ruptured spore wall material. The inner portion of 106 the walls was observed to be composed of microfibrils, which were also destroyed by chitinase. The chitin microfibrils were calculated to make up approximately 3*5% of the dry weight of the spore and corresponds closely with the 3.1% chitin reported for hyphal walls of Collybia sp. (Blumenthal and Rosemena, 1957) and the 3-5% for the hyphal walls of Schizophyllum commune (Wessels, 1965). Immediately surrounding the endosporium was a highly electron-dense layer interpreted as the mesosporium. The action of pronase and lipid solvents caused a marked decrease in electron-density but did not disrupt the layer. Shadow- cast preparations of spores which had been mechnically-ruptured did not reveal the mesosporium to be a separate layer and suggested that this layer extended into the spaces of the microfibrils of the endosporium. Since the layer was not disrupted by any of the enzymes or solvents employed, the layer may be composed of a lipid-protein-polysaccaride complex. The episporium layer (immediately outside the meso sporium) was found to be slightly less electron-dense then the mesosporium. Protein and lipid extraction reduced the density of the episporium in sections observed by TEM. No morphological distinction between the episporium and the mesosporium was seen in mechanically-ruptured spore wall material. The mesosporium probably represents an inner por tion of the episporium. The episporium may be composed of a mixture of lipids, proteins, and polysaccarides. The 107 hyphal walls of Polyporus betulinus (Duff, 1952), Poly- st ictus sanguineus (Crook and Johnston, 1962), and Schizo- phyllum commune (Wessel, 1965; Wang and Miles, 1966) have been reported to be primarily composed of glucans, with only trace amounts of other sugars such as mannose, xylose and fucose. Outside the episporium, was an electron-transparent zone, the perisporium, which corresponds to the perisporium layer of Neurospora tetrasperma ascospores which Lowry and Sussman (1958) found became swollen and lost following treat ment with Clorox. Williams and Ledingham (1964) found an electron-transparent zone in the uredospores of Puccinia graminus which swelled extensively after KMnO^ fixation. Sussman and Halvorson (1966) reported isolated perisporium material for Neurospora sp. ascospores was composed mainly of hexuronic acid residues. Hexuronic acid has reducing properties (Jellinck, 1963) and in conjunction with sodium hypochlorite the primary hydroxyl group would be oxidized to a carboxyl group (Lehninger, 1970). This would explain why the spores stained with ruthenium red. Staining with ruthen ium red appears to depend upon the presence of carboxyl groups which stain indiscrimately (McCready and Reeve, 1955). Luft (1964, 1966) reported ruthenium red had an affinity for highly polymerized acidic polysaccharides. In all probabil ity the perisporium of P. tigrinus was composed of some acidic polysaccharide such as hexuronic acid. 108 The outermost layer, or ectosporium, could only be seen with TEM. This layer remained intact after treatment with pronase and lipid solvents. 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