FACTORS AFFECTING THE GERMENATION 0? THE BASEDEOSPORES OF CALVATIA GEGANTEA Thesis For “19 Degree of DH. D. fiECHEGAN STATE UNEYERSETY Ronaid Wayne Wilson 1965 WWIWIHUIIIIWWIIJIJWII 1293 10729 5689 LIBRAR Y Michigan State University ' This is to certify that the thesis entitled FACTORS AFFECTING THE GERMINATION OF THE BASIDIOSPORES OF CALVATIA GIGANTEA presented by Ronald Wayne Wilson has been accepted towards fulfillment of the requirements for __Ph_._Q._ degree in Jillian;— /V "l/ , , . Mali) ”/ .._, i“ K“ ‘ , " (’4? 7’:f”c"(j > \xic'jf'.’ /ZL‘\ Major professor Datejeb ruary IO, 196L 0-169 fill MSU RETURNING MATERIALS: Place in book drop to remove this checkout from LIBRARIES ”— your record. FINES will ,flx/r" be charged if book is " returned after the date ,JJ“" stamped below. a. a a%%§ ABSTRACT FACTORS AFFECTING THE GERMINATION OF THE BASIDIOSPORES OF CALVATIA GIGANTEA by Ronald Wayne Wilson The purpose of this investigation was to study the germination of the basidiospores of Calvatia gigantea. At the start of this investigation, these Spores could only be germinated in malt extract agar in the presence of a culture of the yeast, Rhodotorula mucilaqinosa var. sanguinea; thus, the major aspect of the study was to elucidate the action of the yeast in stimulating the spores to germinate. The technique consisted of dispensing the spores into molten medium, and after solidification, the yeast was added at four points on the surface of the medium. Approximately 30 days were required before germination occurred. The maximum amount of germination, depending on the sporophore used, was between two hundred and six hundred spores per million. Gibberellic acid and furfural had no effect on ger- mination. Dinitrophenol had no effect at concentrations of -6 5.0x10 and lower; germination was inhibited at higher Ronald Wayne Wilson 2 concentrations. Light was slightly inhibitory to germina- tion. The spores germinated over a wide range of pH values, but 4.3—5.0 was optimum. A basal salt medium, to which various carbohydrate and nitrogen sources were added, supported a reduced amount of germination even though the B. mucilagjnosa was absent. Although many carbohydrates supported germination, maltose, soluble starch, and sucrose were best. The best nitrogen source was KNOB; amino acids and ammonium salts did not support germination unless the yeast was present. The addition of complex media to the basal medium was inhibitory unless the yeast was also added. Potato— dextrose agar supported germination nearly as well as malt extract. The yeast stimulated germination of the spores over a distance of 15 cm. Furthermore, the yeast was capable of producing its action on the spores even though separated from them by a dialysis membrane. The possibility that the yeast produces a volatile stimulator was ruled out. Various extracts of the yeast and filtrates of spent media were not capable of stimulating germination. Paper chromatOgraphic studies comparing media with and without the yeast failed to show Chemical differences due to the yeast. Ronald Wayne Wilson 3 The spores remained dormant until 23-30 days after the yeast was added. Furthermore, the stimulatory action of the yeast appeared to be continuous after 5 days of growth. Several species of Rhodotorula and an unidentified bacterium were qualitatively similar to the R. mucilaqinosa in stimulating germination. FACTORS AFFECTING THE GERMINATION OF THE BASIDIOSPORES OF CALVATIA GIGANTEA BY Ronald Wayne Wilson A THESIS Submitted to Michigan State Univer81ty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1965 To my parents fi ACKNOWLEDGEMENTS The writer wishes to express his sincere appreci- ation to Dr. E. S. Beneke whose interest, direction, and encouragement during the course of this work has made possible and facilitated its completion. The writer also wishes to express thanks to Drs. R. C. Belding, W. B. Drew, J. L. Lockwood, and C. J. Pollard for their many helpful suggestions, constructive criticisms, and much needed advice. This investigation was supported in part by a grant from the National Institutes of Health (CY-4105) and by a fellowship from the U.S. Public Health Service (l-Fl-GM-ZO, 737—01). TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . Collection and Storage of Sporophores Media Used Apparatus and Sterilization Procedures Basic Procedure Followed Determination of Results RESULTS AND OBSERVATIONS . . . . . . . . . . Physical and Chemical Stimulators of Germination Nutritional Aspects of Germination Manipulation of the Incubation Conditions and Characterization of the Action Provided by the R.m. Attempts To Find Active Fractions of the Malt Extract or the R.m. Chemical Analysis of the Incubation Media Time-Rate Studies on Germination Effects of Different Species of Yeast and Bacteria DISCUSSION 0 O O 0 O O 0 O 0 O O 0 O 0 O 0 0 iv 12 12 12 13 13 15 17 17 22 37 53 56 57 63 67 SUMMARY . . . . LITERATURE CITED APPENDIX . . . . Table II. III. IV. VI. VII. VIII. IX° XI. XII. XIII. XIV. LIST OF TABLES Effect of light on germination . . . . . . Effect of pH on germination . . . . . . . Preliminary investigation of carbohydrates utilized during germination . . . . . . . Variations of carbohydrates utilized during germination by sporophores #1601, #1605, and #1608 . . . . . . . . . Investigations on nitrogen sources utilized during germination . . . . . . . Effects of supplementing the basal medium Effects of various percentages of carbo- hydrates and malt extract . . . . . . . . Effect of the number of layers of medium . Effect of Petri plate orientation . . . . Effect of the number of R.m. colonies . . Quantitative results using dialysis membrane to separate the R.m. from the incubating spores . . . . . . . . . . Effects of using different species of RTIOdotoruJ-a O O O O O O O O O O O O 0 O 0 Effects of different species of Rhodotorula and different carbohydrates . . . . . . . Effects of a yellow bacterium . . . . . . vi Page 18 21 24 25 30 32 34 38 39 41 51 65 65 66 LIST OF FIGURES Figure Page I. Effect of distance from R.m. colony on germination . . . . . . . . . . . . 43 II. Effects of the time of addition of the R.m. on germination . . . . . . . . . . . 59 III. Effects of removal of the R.m. on germination . . . . . . . . . . . . . . . . 61 IV. Rate of R.m. growth . . . . . . . . . . . . . 62 LIST OF PLATES Plate Page I. A standard 4-point control plate after 35 days of incubation . . . . . . . . . . 16 II. Experimental apparatus for "agar bridge'l teStS O O o O O O O O 0 O O O O O O 0 O 44 III. An illustration of the attempt to transfer the postulated stimulator . . . . . . 47 IV. Colonies derived from germinated basidiospores after 9 weeks of incubation. The R.m. was retained within a dialysis membrane . . . . . . . . . . 49 V. A comparison of the amount of germination when a dialysis membrane was used to separate the basidiospores of g, gigantea from the yeast . . . . . . . . . . . . . . . . 50 VI. Colonies of mycelium derived from germinated basidiospores of Q. gigantea in malt extract broth . . . . . . . . . . . . . 52 viii INTRODUCTION The discovery of the tumor-inhibiting substance(s), calvacin, in the giant puffball, Calvatia gigantea (Persoon) Lloyd, by Lucas §£_§1, (1958) launched an exten— sive study into the physiology of this organism. One facet of this study was the determination of variations in cal- vacin production by different strains which were derived from germinated basidiospores. Bulmer, Beneke, and Stevens (1962) showed that different strains did indeed vary with respect to calvacin production. However, in order to ger- minate the basidiospores of Q, qiqantea, Bulmer and Beneke (1960, 1961, and 1962) found it necessary to use a living co-culture of the yeast, Rhodotorula mucilaqinosa var. sanguinea (Jorgensen) Harrison. This was a modification of a technique used by Fries (1941, and 1943). The purpose of this investigation was to study in detail the factors involved in the germination of the basidiospores of Q, gigantea. The major aspect was to 1Synonyms for Calvatia gigantea are: Lycoperdon gigan- teum, Batsch ex Pers., Bovista gigantea, (Batsch ex Pers.) Nees, Lycoperdon bovista, Fr., Calvatia maxima (Schaeff) Morgan, Calvatia bovista, Macbride, Calvatia primitiva, Lloyd. elucidate the nature of the action of the B, mucilaqinosa in stimulating the germination of the puffball basidiospores. LITERATURE REVIEW An excellent review of the scant literature on the germination of the basidiospores found in the series Gas- teromycetes was presented by Bulmer (1960), and Bulmer and Beneke (1961). These two papers contained most of the pertinent facts known up to that time on the germination of the basidiospores of organisms in the Gasteromycetes. Sig- nificantly, reports on the germination of the basidiospores of Q, gigantea were absent. Thus, it appears that the factors known about the germination of these spores are published by the above authors (1960, 1961, 1962). The first successful attempt these authors had at ger— minating the basidiospores of g, gigantea employed liquid barley extract medium.) This method required 7-9 weeks before colonies derived from germinated spores became visi- ble, and then, only 1 spore in 20 million was found to germinate. Their next and most important finding was based on a technique introduced by Fries. Fries (1941) was suc- cessful in germinating the basidiospores of several genera of Gasteromycetes by the following method: (1) basidiospores were seeded onto the surface of malt extract agar, and (2) a yeast, "Hefe X", which is now believed to be Rhodotorula mucilaginosa was inoculated at 8-10 points on the surface of the agar. Bulmer and Beneke (1961) modified this technique in the following manner: the spores were first seeded into malt extract agar onto which a top layer of the same medium was added, and secondly, the yeast, Rhodotorula mucilaqinosa var. sanguinea (Jorgensen) Harrison (=R.m.) was added at 4 equidistant points on the surface of the top layer (see Materials and Methods section of this text for greater detail). Using this method, Bulmer and Beneke (1960, and 1961) in- creased germination to approximately three hundred per million. The following are some of the most important findings of Bulmer and Beneke (1960, 1961, and 1962). The presence of the yeast was required in studying all of the variables. Chloromycetin at a concentration of 6 parts per million was found optimal for eliminating bacterial contamination with- out adversely affecting the germination percentages. The optimum temperature for germination was found to be between 24 and 26 C. The concentration of spores also affected germination. The percentage of spores germinating from different sporophores was different. Attempts to replace the growing yeast with the vitamins, biotin, thiamine, pyri- doxine, inositol, and riboflavin were unsuccessful. The presence of Q, gigantea co—cultures did not substitute for the yeast. Information on the highly involved relation- ship between storage temperatures and intervals was also presented. In addition, numerous spore treatments and various media were reported ineffective in stimulating germination. Bulmer and Beneke (1964) reported on the germination of the basidiospores from several species of puffballs in the general Lycoperdon and Scleroderma. They found that these spores were affected by the yeast in the same manner as those of Q, gigantea. Bulmer (1964) also reported success- ful germination of basidiospores of 42 additional species from 11 genera of Gasteromycetes using the yeast co-culture technique. He noted that these basidiospores were similar to those of g, gigantea in that the percentage of germination was extremely low, and that an incubation period of several weeks was required before colonies became visible. Dowding and Bulmer (1964) made cytological studies of Q, gigantea and 4 species of Lycoperdons. They found the basidiospore-forming mechanism in the puffballs studied to be similar to that already studied in mushrooms. Furthermore, no abnormalities occurred in the mechanism which might explain the low percentage of germination. Duggar (1901) investigated the germination of the basidiospores from several Boletus species and two species of Coprinus. He failed to germinate any of the spores on several media. He was, however, successful in germinating the basidiospores of Coprinus micaceus by using a bean de— coction. Ferguson (1902) reported that although the ba- sidiospores of Agaricus campestris germinated on many media, consistently high percentages of germination were obtained only if a living co-culture of A, campestris was present. Swartz (1928) and Kaufmann (1934) reported several un- successful attempts at germinating the basidiospores of several species of the puffballs in the genera Lycoperdon, Bovistella, and Calvatia. Brown (1946) published a review article on biostimulators of various germination processes that occur in nature. He cited several examples of pollens, seeds, and fungal spores which require the association of some other living organism for germination. He concluded that the activators are probably metabolites of the associated organism, and that several modes of action are possible and probable. Lange (1948 demonstrated that the basidiospores of Coprinus species were stimulated by the growing germ tubes of other germinating spores, or by the growing mycelium of the same species. He also reported that the spores germinated at a rate of 0.01-1.0 percent on horse dung agar. Heat treatments of the spores had little or no effect on germination. Kneebone (1950) investigated the germination of the basidiospores of approximately 175 species in the sub—class Hymenomycetes. The following are some of his conclusions and generalities. Those fungi with mycorrhizal relation- ships were ones whose spores were hardest to germinate. The amount of germination was low (usually less than 1 per- cent) even though most of the spores were viable as indicated by tests with 2,3,5, triphenyltetrazolium chloride. Light had little or no effect on germination. Some of the spores were stimulated by extracts of various organic substrata. There was, however, no correlation between the extracts which stimulated germination and the substrata on which the sporophores grew. He also reported that there was no evi- dence that furfural stimulated germination, and furthermore he observed that heat shocking, if anything, was detrimental . . 1 . . to germination . The presence of 11v1ng yeast co—cultures 1Sussman (1953) showed that furfural was very effective in stimulating the germination of Neurospgra ascospores; had no effect on germination. He felt that nutrition was not a critical factor, but that permeability was very im- portant in the germination process. Kneebone's conclusions were that spore metabolism was very slow, and that a critical enzyme or precursor was required to initiate the process. He seems to be of the opinion that germination may be stimu— lated by several different mechanisms since the factors found to be effective were so diverse. Mandels (1955), while studying the germination of Myrothecium verrucaria spores, observed that growth ceased shortly after germ tube emergence, and that a lag phase existed for some time. The presence of yeast extract, con- taminating bacteria and fungi, or biotin eliminated this lag period. Several lines of evidence were cited which indicated that the factor responsible for eliminating this lag period was biotin. Since this organism is autotrophic for biotin, it was suggested that possible alterations in permeability shortly after germination allowed biotin to diffuse out of the cells; growth only ensuing after it was established in a suitable concentration. This paper offered a philosophical heat shocking was also important in stimulating the germina- tion of these spores. point concerning the distinction between germination (germ tube emergence) and early growth of the mycelium. Good and Spanis (1958) studied several factors involved in the germination of the basidiospores of Fomes igniarus var. populinus. The spores were found to germinate between pH 3 and 7, but pH 4 was optimum. Of the contaminants that in- vaded the plates, none was found to stimulate germination. They reported that 8 percent malt extract was optimum for germination, although a few spores did germinate in a basal medium plus glucose or maltose. The spores also germinated well in extracts made from wounded portions of certain trees. Chromatographic studies revealed that small amounts of carbohydrates were present in these extracts. Judging from the fact that glucose and maltose supported germination, but did not augment germination when added to malt or tree extracts, they concluded that some other factor(s) must be present in the extracts which stimulated germination. Ex- tracts from aged wounds promoted more germination than those from fresh wounds; they attributed this to alterations in the substratum by saprophytes. t _a_1. (1964), and Hafiz and Niederpruem (1963), Ratts Niederpruem (1964) have presented papers concerned with the 10 metabolism and metabolic machinery of the germinating ba- sidiospores of Schizophyllum commune. The first paper in this series pointed out that the spores did not germinate in Petri plates which contained KOH in center wells. Thus, there was an indication that CO2 might be required for germination. The second paper in this series listed many enzymes of the Krebs cycle and the respiratory chain which were identified in cell-free extracts of S, commune basidio- spores. The last paper presented results from cytochemical and manometric studies. The cytochemical studies indicated the presence of stored carbohydrates and the absence of stored lipids in the basidiospores. A respiratory quotient of near one suggested that the spores metabolized carbo- hydrates. Several carbon sources were found to markedly increase the endogenous respiration. The use of several metabolic inhibitors indicated the presence of a functional cytochrome system in these spores. Two excellent review articles on the generalities of fungus spore germination were prepared by Gottlieb (1950, and 1964). For general information on the physiology of spore germination, the writer suggests: Cochrane (1958), Foster (1949), Lilly and Barnett (1951), and Gray (1959). 11 Additional knowledge concerning the physiology of Q, gigantea has been gained from studies directed mainly at calvacin production and is cited below. Roland _§_al, (1960) reported on the antitumor activity of calvacin against 24 different types of tumors. Information was also presented on screening problems involved with "scaling up" the calvacin production. Sedlmayr (1960), and Sedlmayr. Beneke, and Stevens (1961 a,b) studied certain aspects of the growth requirements of g, gigantea. The emphasis was on carbohydrate utilization and vitamin requirements. Many carbohydrates were utilized, and all strains required thia- mine. Cook (1960) showed that the variations in calvacin production by the same strains differed depending on the carbohydrate present in the growth medium. Beneke (1963), in his presidential address to the Mycological Society of America, summarized all the work done to that time on the various aspects of the g, gigantea studies. He also reported for the first time the presence of the dolipore-parenthesome type of septum in Q, gigantea. MATERIALS AND METHODS Collection and Storage of Sporophores The sporophores used for these experiments were collected, allowed to dry completely at room temperature, placed in- dividually into plastic bags, and finally stored in card- board boxes at room temperature. Collection dates and loca- tions of the puffballs can be found in the Appendix of this manuscript. The identification numbers for the sporophores used for each experiment will be cited with the data. Taxonomic characteristics and locations for finding the giant puffball, Calvatia gigantea, can be found in A. H. Smith's book (1951). Media Used All of the dehydrated media used was Difco brand, and was prepared according to the directions on the bottles. The medium giving the highest amount of germination, and hence used for the control was Difco Malt Extract to which 20 grams per liter of agar were added. The basal medium used was essentially that of Lindeberg's (1941). The com- position of the basal medium was as follows: P . KH2 O4 1 0 g M SO .7H 0 0.5 12 13 FeC13(sol. 1/500) 0.5 ml MnC12(SOl. 0.1 M) 0.5 m1 CaC12(sol. 0.1 M) 5.0 ml ZnSO4(sol. 1/500) 0.5 ml Distilled H20 994.0 m1 To the basal medium, various carbohydrates and nitrogen sources were added. When used, Chloromycetin at a concen- tration of 6 parts per million was added prior to autoclaving. Apparatus and Sterilization Procedures Waring blendors were used to prepare the spore suspen- sions, and the concentrations of spores in the suspensions were determined with an A—O haemacytometer. Pipettes were used to dispense the spores. Standard Pyrex Petri plates were used for most experiments, although some Falcon brand disposable plates were employed. Glassware was sterilized by dry air at 165-180 C for 3 hours. Most media were sterilized by autoclaving at 121 C, 15 psi for 15 minutes. Details on the sterilization of special preparations will be discussed later for the purpose of clarity. Basic Procedure Followed The basic procedure followed for these studies was a modification of Fries' technique (1941, and 1943) as described 14 by Bulmer (1960) and Bulmer and Beneke (1961). To prepare the inoculum, the basidiospores were first removed from the sporophore with sterile forceps a few mm below the peridium to avoid any contaminating agents present on the surface. A spore mass, approximately the size of a large pea, was then homogenized in a sterile blendor which contained 200 ml of sterile water for about 20 seconds. The spore concentration was determined after homogenization. The desired number of spores was pipetted into large test tubes which contained 25 ml of sterile molten medium, and then the contents poured into sterile Petri plates (entire process will be called "seeding"). At this point, the plates will be called "1 Layer" plates. In some cases, a second layer of medium was added after the first had solidified; such plates will be referred to as "2 Layer" plates. The next step was to inoculate the Rhodotorula mucilaqinosa (R.m.) at 4 points on the surface of the agar. Finally, the plates were sealed with rubber gaskets. Plastic plates were sealed in polyethylene bags. The plates were incubated in an inverted position in a constant temperature room at 25 C. This room was kept dark except when entered for the removal of materials. A plate with the R.m. and germinating spores is shown in Plate I. 15 Special techniques and modifications to the basic procedure will be cited in the text for purposes of clar- ity and brevity. Determination of Results The amount of germination was determined by counting the number of macroscopically visible colonies only, and spores germinating under the R.m. colonies were not counted. All results were calculated on the basis of the number of spores germinating per million. All data given are averages made from counts made of not less than seven plates. All experiments were repeated at least once. 16 PLATE I If! A standard 4-point control plate after 35 days incubation. The four pink colonies are the Rhodotorula mucilaqinosa, and the many small colonies are mycelium of g. gigantea which were derived from germinated basidiospores. RESULTS AND OBSERVATIONS Physical and Chemical Stimulators of Germination The purpose for using many of the following treatments known to be effective in the germination of other spores was twofold: (l) to attempt to increase the rate and/or amount of germination, and (2) to attempt to replace the growing yeast by physical or chemical stimulators of germination. Physical Treatments and Incubation Variables One of the first experiments was to determine how heat treatment would affect germination. To accomplish this, a spore suspension was prepared, and aliquots of this suspen— sion were removed and heated for 10 minutes at 45, 50, 55, 60, 65 and 70 C. Approximately 75 percent of the spores treated at 50 C and 100 percent of those treated at 55 C or higher were inactivated. There was no effect on ger— mination by treatment at 45 C. Plates without the R.m. showed no germination. To study the effects of light, a number of control plates were kept in a constant temperature room under the conditions described before. The seeded plates which were exposed to light were left at room temperature and exposed to the fluor— escent lights of the laboratory and the diffuse sun light 17 18 which entered through the windows. Other plates were incu— bated beside the light exposed plates, however, they were placed in light-proof metal boxes. The results shown in Table I suggest that light was slightly inhibitory to germination. Although there was no difference in the amount of growth or color of the R.m. colonies, the possibility still exists that the metabolism of the yeast was affected by the light. Table I Effect of Light on Germination Germination/Million Diameter #1601 #1608 of R.m. Incubated in Light . 242 219 18t2 mm Incubated in Dark 270 248 18£2 mm Control 277 234 18i2 mm Medium used: 1.0% malt extract; 1 layer of medium To determine if the size or nature of the Petri plates used was an important variable, the following experiments were conducted. Pyrex plates (lelOO mm, and 30x100 mm), and Falcon brand plastic plates (15x100) were seeded and inoculated in the usual manner. There was no difference in the amount of germination due to the types of plates with the two sporophores tested. 19 It is well known that heat, especially at autoclaving temperature, will affect media and subsequently the para- meters being measured. To determine if the means of ster- ilizing the media had an effect on germination, several sterilization methods were employed. Seitz filtered media were prepared by adding 12.5 ml of filtered media to 12.5 ml of sterile molten (48 C) 4 percent water-agar, and seeded. The 2 percent media which were autoclaved for 15, 30, and 45 minutes were cooled to 42 C, and seeded. After autoclaving for 15 minutes, the agar in some of the tubes was allowed to solidify, and then steamed for 10 minutes to re-melt the agar. The results showed that malt extract which was auto- claved and steamed supported approximately 20 percent less germination than if Seitz filtered. Germination on the defined media showed no variation due to the method of sterilizing. However, due to the greater amount of time required and the greater chance for contamination encoun- tered by the filtration method, it was decided to sterilize all other media by autoclaving. Sporophores #1601 and #1609 were used. Since it is possible that a germination inhibitor was present in the spores or spore wall, the spores were 20 washed (leached) five times with sterile distilled water. The results using malt extract showed that, even after washing, the presence of the yeast was necessary before germination would occur. When the defined medium was used, washed spores showed germination percentages simi- lar to those that had not been washed (whether the R.m. was present or not). Tween 80, a wetting agent, at concentrations of 0.25, 0.50, 1.0, and 2.0 percent had no effect on germination. To determine if the hydrogen ion concentration af- fected germination, the pH was adjusted with HCl and NaOH. A NaCl control was also run to detect any effect due to the additional sodium or chloride ions. The findings shown in Table II demonstrate that the spores germinated over a wide range of pH values, however, there was an optimum between pH values of 4.3 and 5.0. Effects of Chemical Stimulators The basidiospores from sporophores #1601 and #1609 were treated with the following concentrations of furfural; 1.0, 0.5, 0.1, 0.01, 0.001 and 0.0001 M. Some of the spores were treated by soaking them for periods of 24 hours, or for 7 days in the furfural solutions. The spores were then 21 seeded into malt extract which had the above concentra- tions of furfural in the medium. Table II Effect of pH on Germination pH Prior to pH at the Germination/Million Diameter Autoclaving Time of #1601 #1605 of R.m. Counting 3.5 - - — - 4.0 3.7 187 124 19i2 mm 4.5(control) 4.3 306 222 " 5.0 4.6 304 209 " 5.5 5.0 311 230 " 6.0 5.1 297 223 " 6. 5.4 259 196 " 7.0 5.6 189 102 " 7.7 6.2 107 98 " 4.5 (NaCl) 4.3 312 206 " Medium used: 1.0% malt extract; 1 layer of medium Seitz-filtered gibberellic acid solutions were added aseptically to molten malt extract agar to give the follow- ing concentrations; 1/200, 1/400, 1/1,600, 1/3,200, 1/6,400, and 1/12,800 (w/v). Two sporophores were used. Dinitrophenol was incorporated into the various media at concentrations of lxlO-4, 5x10-5, lxlO-S, 5xlO-6, and 1x10—6 M. The media used were malt extract, and the basal medium plus glucose and KNO Two sporophores were used. 3. Furfural and gibberellic acid had no effect on germina- tion in either rate or amount, nor did they replace the 22 growing R.m. Dinitrophenol at concentrations of 1x10-4, 5x10-5, and 1x10”5 inhibited germination completely, while the lower concentrations had no effect on the rate or amount of germination. None of these agents had a visible effect on the growth of the yeast. Nutritional Aspects of Germination The literature on fungus spore germination contains no simple key to the nutritional requirements for this process. The reason is that different fungus spores germinate over an entire spectrum of nutritional conditions, ranging from distilled water to complex infusions and extracts of natural products. Since the basidiospores of g, gigantea had pre- viously been germinated only in extracts, it was impossible to know what these media supplied. In addition, the presence of the growing yeast complicated the picture even more. Hence, it was thought that if a defined medium could be found which would support germination, a step would be made in resolving the question of whether simple nutrition alone was the key to the process or whether the malt extract con— tained a factor(s) which was stimulatory or necessary for germination. Fortunately, the first basal medium used 23 supported the germination of a small number of spores. The ingredients of the basal medium are given in the Materials and Methods section. Effects of Various Carbon Sources on Germination Table III relates the findings of a preliminary inves- tigation in which several carbohydrates were added to the basal medium. Although the R.m. growth was indicated with (+), it can be approximated from other data that each (+) would represent 2-3 mm. These results showed that the basidiospores of g, gigantea could be germinated without the yeast being present. Higher germination values were recorded, however, when the R.m. was present. The carbo- hydrates supporting the greatest amount of germination were maltose, soluble starch, and sucrose. Although the pH varied with different carbohydrates, there was no change in the pH due to the presence of the growing yeast. Citric acid, glucose-l-PO4, K—acetate, and glycerol were also tested as possible carbon sources, but none supported germination. These findings prompted the evaluation of the effects of various carbohydrates on different sporophores. Further- more, it could simultaneously be determined if the) germina- tion rate was different on the synthetic medium. 24 Table III Preliminary Investigation of Carbohydrates Utilized During Germination Carbohydrate Germination/106 pH R.m. R.m. No R.m. R.m. No R.m. Growth Arabinose 0.0 0.0 4.2 4.2 3+ Xylose 0.0 0.0 4.0 4.0 0 Glucose 0.0 0.0 4.1 4.1 3+ Fructose 0.0 0.0 4.5 4.5 3+ Galactose 0.0 0.0 4.2 4.2 2+ Rhamnose 0.3 0.0 4.7 4.7 1+ Sorbose 0.0 0.0 4.0 4.3 1+ Cellobiose 4.0 0.0 4.5 4.5 3+ Lactose 2.0 0.0 4.6 4.6 1+ Maltose 39.0 13.0 4.4 4.4 3+ Sucrose 15.0 7.0 4.4 4.2 3+ Threhelose 4.1 0.0 4.5 4.5 3+ Inulin 0.0 0.0 4.6 4.6 3+ Dextrin 4.3 5.0 4.7 4.7 2+ Glycogen 1.7 0.0 4.7 4.7 2+ Sol. Starch 19.0 14.0 4.5 4.5 2+ No CHO 0.0 0.0 4.9 4.9 0 Malt Extract 394.0 0.0 4.3 4.3 10+ Medium used: basal plus 2.0% carbohydrates and 0.5% NH4 tartrate; 2 layers of medium; sporophore #1601 ‘There were differences in the rates of germination when various sporophores were tested, as the results in Table IV show. Likewise, it appears that germination was higher and began earlier if the yeast was present. Data not presented showed that galactose supported the germination of approxi- mately 10 spores per million when the yeast was present and sporophores #1605 and #1608 were used. Dextrin supported 25 about the same amount of germination as galactose with #1608. Fructose did not support germination of any spores. The amount of R.m. growth for these three sugars was similar to that given for the other sugars in Table IV. Table IV Variations of Carbohydrates Utilized During Germination by Sporophores #1601, #1605, and #1608 Carbohydrate Days Germination/106 Diameter and after of R.m. Sporophore Seeding R.m. No R.m. Colony(mm) Glucose #1601 30 0.0 0.0 35 1.9 0.0 45 3.2 3.2 55 3.4 3.4 7i2 #1605 30 1.6 0.0 35 3.0 0.2 45 9.0 0.2 44 10.0 0.4 712 #1608 30 1.3 0.0 35 1.5 0.0 45 1.5 0.0 55 1.7 0.0 712 Sucrose #1601 30 6.2 2.4 35 12.0 9.0 45 17.0 12.0 55 19.0 13.0 7t2 #1605 30 3.0 0.2 35 12.0 0.8 45 15.0 0.9 55 16.0 0.9 7f2 In 26 Table IV (Continued) Carbohydrate Days Germination/106 Diameter and after of R.m. Sporophore Seeding R.m. No R.m. Colony(mm) #1608 30 25.0 2.4 35 41.0 5.0 45 50.0 7.0 55 51.0 7.0 712 Maltose #1601 30 21.0 0.0 35 44.0 1.9 45 51.0 12.0 55 53.0 18.0 7&2 #1605 30 2.3 0.0 35 8.0 0.0 45 23.0 6.1 55 26.0 8.0 7i2 #1608 30 0.8 0.0 35 7.0 0.3 45 18.0 0.4 55 19.0 0.4 712 Sol. Starch #1601 30 4.0 2.2 35 11.0 7.0 45 15.0 10.0 55 1s.0 12.0 712 #1605 30 2.1 1.0 35 9.0 4.0 45 20.0 7.9 55 21.0 13.0 712 #1608 30 27.0 18.0 35 33.0 19.0 45 46.0 27.0 55 48.0 34.0 7&2 27 Table IV (Continued) Carbohydrate Days Germination/106 Diameter and after of R.m. Sporophore Seeding R.m. No R.m. Colony(mm) Glycogen #1601 30 0.0 0.0 35 0.4 0.0 45 2.1 0.6 55 4.0 1.1 7i2 #1605 30 2.2 0.3 y 35 11.0 1.7 L 45 18.0 5.0 55 22.0 5.0 7i2 #1608 30 6.5 0.7 35 8.0 1.7 45 11.0 4.4 55 12.0 5.0 7&2 Malt Extract #1601 40 55.0 0.0 35 146.0 0.0 45 256.0 0.0 55 261.0 0.0 20i2 #1605 30 89.0 0.0 35 205.0 0.0 45 297.0 0.0 55 308.0 0.0 20t2 #1608 30 67.0 0.0 35 - 145.0 0.0 45 233.0 0.0 35 236.0 0.0 20i2 Medium used: basal plus 1.0% carbohydrates and 0.5% KNO3; 1.0% malt extract; 1 layer of medium; pH values were similar to those in Table III 28 Seven concentrations from 0.25 percent to 2.0 percent of glucose, soluble starch, and sucrose revealed that the maximum germination occurred between 0.75 and 1.25 percent. Using similar concentrations of malt extract, it was found that 1.0 percent of this medium gave the maximum amount of germination; germination decreased sharply between 3 and 6 percent. Germination was approximately eight-fold greater at 1.0 percent than at 6 percent. Two sporophores were used. Germination Versus Growth on Various Media Since it could be argued that the observed germination on a given carbohydrate was only a matter of selecting those strains which could grow on that particular carbo- hydrate, the following was done. Small portions of a single spore colony on a given carbohydrate were transferred to plates containing the same basal ingredients but different carbohydrates. The growth was measured every two days for a three week period. The carbohydrates used for the germina- tion media and transfer media were: glucose, maltose, sol- uble starch, and sucrose. The results showed that, disregarding the slight dif- ferences in the amount of mycelium used to make the transfers, 29 all transfers grew well and at the same rate regardless of the carbohydrate used for germinating the spores. This would indicate, at least for the colonies and sugars used, that the spores contain the metabolic machinery to utilize the carbon sources equally. Since all of the carbohydrates tested contained glucose, it might be argued that differences in osmotic pressure were producing an effect. This possibility should be considered in the light of two other experiments. Chromat- ographic studies (pageEY7) revealed that sucrose was hy- drolized to glucose and fructose soon after the R.m. was added. Hence, the sucrose medium would have had the same osmotic pressure as the medium containing an equal weight of glucose. The previous studies which showed that fruc- tose did not support germination; hence, there should have been no additive effect. Effects of Various Nitrogen Sources on Germination The information in Table V demonstrates that differences existed between the amount of germination and the nitrogen sources present in the medium. It appears that KNO3 was the best nitrogen source. Furthermore, there is a subtle suggestion that ammonium salts were inhibitory unless the 30 Table V Investigations on Nitrogen Sources Utilized During Germination Carbohydrate Germ./106 pH R.m. and Nitrogen R.m. No R.m. R.m. No R.m. Growth Glucose KNO3 4.5 0.7 4.2 4.4 3+ NH4C1 0.0 0.0 4.4 4.5 3+ NH4NO3 0.0 0.0 4.3 4.1 3+ (NH4)ZSO4 0.0 0.0 4.0 4.0 3+ NH4Tartrate 0.0 0.0 4.1 4.1 3+ Mg(NO3)2 6.0 4.0 - 4.1 3+ no Nitrogen 3.2 0.0 4.3 4.3 2+ Sucrose KNO3 11.0 4.0 4.0 4.1 3+ NH4C1 0.0 0.0 4.0 4.0 2+ (NH4)2504 0.0 0.0 4.1 4.1 3+ Mg(NO3)2 11.0 6.0 4.1 4.1 3+ NH4Tartrate 17.0 0.0 4.3 4.3 3+ NH4NO3 0.0 0.0 4.0 — 2+ no Nitrogen 4.0 1.1 4.3 4.3 2+ Maltose KNO3 47.0 20.0 4.4 4.5 3+ NH4C1 2.0 0.0 4.1 4.1 2+ NH4NO3 0.0 0.0 4.4 4.4 3+ (NH4)2504 0.0 0.0 4.3 4.3 3+ NH4Tartrate 43.0 18-0 4.5 4.5 3+ no Nitrogen 12.0 4.6 4.5 4.5 2+ Sol. Starch KNO3 18.0 6.0 4.5 4.5 2+ NH4C1 3.1 0.0 4.6 4.5 2+ NH4NO3 4.0 1.2 4.5 4.5 3+ (NH4)2SO4 0.0 0.0 4.4 4.5 2+ Mg(NO3)2 6.0 6.0 4.5 4.5 2+ NH4Tartrate 4.2 0.0 4.6 4.6 3+ Malt Extract (control) 264.0 0.0 4.3 4.3 10+ Medium used: basal plus 2.0% carbohydrates and 0.5% nitrogen sources; 1 layer of medium; sporophore #1601 31 yeast was present. Similar results were obtained with 2 other sporophores. The amino acids, cysteine, histidine, glutamine, gly- cine, alanine, arginine, and casamino acids were also used as nitrogen sources. Germination was only observed when maltose was the sugar and glycine or alanine were the nitrogen sources, and the yeast was present. The yeast growth was 7+8 mm with the amino acids and 12 mm with the casamino acids. The fact that some spores germinated in both experiments without a nitrogen source corroborates the work of Knaysi (1945). He found that the endospores of Bacillus subtilis did not require an exogenous nitrogen source to germinate. Judging from these data, it suggests that one possible action of the yeast is to remove inhibitors from the incubat- ing medium. Effects of Supplementing the Basal Medium Malt extract and peptone were added to the basal medium to determine: (1) if, in the absence of the R.m., chemically undefined media were inhibitory to germination, and (2) if the amount of R.m. growth, which is less on the defined medium, could be a limiting factor. 32 The data in Table VI demonstrate that, when malt extract was added to the basal media, germination was Table VI Effects of Supplementing the Basal Medium Medium Germination per Million Diameter #1601 #1605 #1609 of R.m. no no no Colony R.m. R.m. R.m. R.m. R.m. R.m. (mm) Glucose no N 8.1 1.5 12.0 4.0 5.1 0.8 712 KNO3 5.2 0.9 18.0 8.1 10.0 8.6 812 Peptone 0.0 0.0 ”0.0 0.0 0.0 0.0 1312 Malt Ex. 147.0 0.0 160.0 0.0 129.0 0.0 1512 Maltose no N 21.0 7.3 21.0 14.0 19.0 9.6 712 KNO3 64.0 38.0 21.0 11.0 73.0 23.0 812 Peptone 3.0 0.0 0.0 0.0 0.0 0.0 1312 Malt Ex. 166.0 0.0 187.0 0.0 116.0 0.0 1512 Sucrose no N 6.3 4.2 14.0 6.7 11.0 0.0 712 KNO3 12.0 9.8 31.0 21.0 23.0 10.0 812 Peptone 1.1 0.0 0.0 0.0 0.0 0.0 1312 Malt Ex. 151.0 0.0 149.0 0.0 114.0 0.0 1512 S. Starch. no N 10.0 11.0 15.0 8.0 12.0 5.5 712 KNO3 24.0 18.0 23.0 19.0 19.0 14.0 812 Peptone 0.0 0.0 0.0 0.0 0.0 0.0 1312 Malt Ex. 154.0 0.0 166.0 0.0 121.0 0.0 1512 Malt Ex. control 269.0 0.0 330.0 0.0 178.0 0.0 1912 Medium used: basal plus 1.0% carbohydrates and 0.5% 'KNO3, peptone, malt extract; 1.0% malt extract for controls; 1 layer of medium 33 inhibited if the R.m. was absent. If the R.m. was present, the addition of malt extract increased both the amount of germination and the growth of the R.m. The peptone, al— though it supported good growth of the R.m., inhibited germination in all cases but two (3.0 and 1.1 germinations per million) even when the R.m. was present. To examine these questions in greater detail, various percentages of malt extract and carbohydrates were mixed. The results are shown in Table VII. Two points can be ob- served here; 0.1 percent malt extract was inhibitory in half the cases when added to plates that did not have the yeast, and completely inhibitory when 0.3 percent or greater was used, and furthermore, the amount of yeast growth and subsequently the amount of germination became a function of the percentage of malt extract. Effects of Thiamine Since the amount of yeast growth was reduced on the basal medium, it was thought that by increasing its growth, germination might also be increased. Thus, thiamine was added to the medium. The results showed that the R.m. growth was increased (7 mm—17 mm) due to the added thiamine I however there was no increase in the amount of germination. Effects of Various Percentages of Carbohydrates and Malt Extract Table VII Germination/Million %Glucose %Malt "X” #1601 #1605 Dia. - No No of R.m. R.m. R.m. R.m. R.m. 1.0 0.0 2.1 0.4 11.0 4.1 712 0.9 0.1 28.0 0.0 54.0 0.2 1012 0.7 0.3 81.0 0.0 94.0 0.0 1312 0.5 0.5 134.0 0.0 197.0 0.0 1412 0.3 0.7 173.0 0.0 252.0 0.0 1512 0.1 0.9 248.0 0.0 298.0 0.0 1712 0.0 1.0 264.0 0.0 316.0 0.0 1912 %Sucrose %Malt "X" 1.0 0.0 14.0 4.0 16.0 3.4 712 0.9 0.1 32.0 0.0 56.0 0.0 1012 0.7 0.3 78 0 0.0 87.0 0.0 1312 0.5 0.5 139.0 0.0 201.0 0.0 1412 0.3 0.7 180.0 0.0 270.0 0.0 1512 0.1 0.9 241.0 0.0 300.0 0.0 1712 0.0 1.0 264.0 0.0 316.0 0.0 1912 %Sol Star. %Malt "‘" 1.0 0.0 17.0 14.0 26.0 28.0 712 0.9 0.1 45.0 0.1 94.0 1.2 1012 0.7 0.3 93.0 0.0 122.0 0.0 1412 0.5 0.5 143.0 0.0 187.0 0.0 1512 0.3 0.7 178.0 0.0 286.0 0.0 1612 0.1 0.9 252.0 0.0 309.0 0.0 1812 0.0 1.0 264.0 0.0 316.0 0.0 1912 %Malt "X" 0.1 24.0 0.0 57.0 0.0 1012 0.3 87.0 0.0 105.0 0.0 1412 0.5 153.0 0.0 191.0 0.0 1512 0.7 171.0 0.0 265.0 0.0 1612 0.9 257.0 0. 314.0 0.0 1912 Medium used: basal plus indicated amounts of carbo- 1 layer of hydrates and malt extract, and 0.5% KNO37 medium 35 When thiamine was added to the malt extract medium there was no effect. Sporophores #1601 and #1608 were used. This suggests that increased diametric growth of the yeast is not the sole factor in increasing germination. Miscellaneous Media used To Study Germination The basidiospores from sporophores #1601, #1605, and #1609 were used for the following experiments. The basal medium, to which 5 concentrations from 0.5 to 10.0 percent coconut milk had been added supported a very few ger- minations when the R.m. was present. Sabourauds-glucose agar did not support germination, however, the amount of R.m. growth was greater than on the malt extract. Corn meal agar to Which 0.5 percent yeast extract was added failed to support germination. The R.m. had the same diametric growth as the malt extract controls, but was a pale yellow. Czapek's agar supported no germination and the R.m. grew poorly. Potato-dextrose agar gave results nearly comparable to those obtained with the malt extract controls. There was no germination without the R.m., and with the R.m., the germination was 75-85 percent of that on the malt extract controls. The growth of the R.m. was not quite as great 36 as with the malt extract, and the colonies had a watery appearance. Malt extract agar did not support germination. At this point, the writer calls attention to the fact that Difco Malt Extract Agar is not, as a glance at the .Ap- pendix will show, merely Difco Malt Extract to which agar has been added. 37 Manipulation of the Incubation Conditions and Characterization of the Action Provided by the R.m. One Layer Versus Two Layers of Medium Bulmer (1961, and 1962) suggested that the function of the added layer of medium above the seeded layer might be to impose semi-anaerobic conditions on the incubating spores. The results in Table VIII show that the amount of germina— tion was essentially the same whether the spores were seeded into the bottom, top, or mixed into both layers. Furthermore the data suggest that the top layer of malt extract serves to provide for an increased amount of yeast growth and subsequently more germination. This is further supported by experiments with the basal medium (Table VIII). This information shows that, with l or 2 layers of basal medium, the R.m. growth was the same, and hence there was no major difference in the amount of germination. This evidence suggests that the action of the yeast is to provide a stimulator rather than remove an inhibitor from the malt extract . The reasoning is that the added malt extract would also add more inhibitor and thus cancel the effect of the increased amount of yeast growth. Effect of the Number of Layers of Medium Table VIII 38 Number of Germination per Million Diameter Layers #1601 #1608 #1611 of R.m. Malt "X" 1 Layer 234 . 204 318 1912 2 Layers (spores in bottom layer) 306 277 471 2512 2 Layers (spores mixed in both layers) 341 265 456 2512 2 Layers (spores in top layer) 312 261 478 2512 Basal Medium plus: Glucose 1 Layer 2.1 1.7 7.6 812 2 Layers 2.3 1.6 7.9 812 Maltose 1 Layer 37 33 - 812 2 Layers 38 31 — 8+2 Sol. Starch 1 Layer 13 47 22 812 2 Layers 13 42 21 812 Medium used: basal plus 1.0% carbohydrate and 1.0% malt extract 0.5% KNO3: 39 Effects of Petri Plate Orientation Bulmer (1960) reported that the amount of germination was reduced when the plates were incubated in an inverted position as compared to those incubated upright. The knowledge gained from Table IX explains this observation on the basis that a greater amount of yeast growth occurs on upright plates. Table IX Effect of Petri Dish Orientation Orientation of Germination/Million Diameter the Plates #1601 #1609 of R.m. Plates Inverted 226 186 20t2 mm Plates Upright 291 262 27:2 Medium used: 1.0% malt extract; 1 layer of medium Effects of the Incubating Atmosphere on Germination Experiments were designed to determine if the R.m. could stimulate germination if it was isolated from direct contact with the seeded medium. The first was to determine if a volatile stimulator was produced. To do this, tops of 50x55 mm Petri plates were placed as "wells" inside 30x100 mm plates, and sterilized. Sterile malt extract agar was poured into the wells and the yeast inoculated. Finally, seeded medium was added to the area outside the 40 wells. The plates were incubated in both inverted and upright positions. Controls with the wells seeded were used as well as 4-point plates. In none of the trials was germination observed outside the wells. Three sporophores were used. To further study the effects of the atmosphere above the incubating spores, various trapping substances were placed in center wells, and the area surrounding the wells were seeded and inoculated as usual. After sterilization, 4 ml of the following solutions were added to the wells: H O, conC. H SO , 2N KOH, K 2 2 4 MnO , and mineral 011. The 2 4 results showed that there was no effect due to the presence of the various trapping substances. The final attempt to study the possible effects of the incubating atmosphere was done by exchanging the air in the plates. The air was exchanged by opening the plates while in the upright and inverted positions. The plates were opened on the 3, 5, 8, 10, 13, 15, 18, 21, and 25th days after seeding and inoculating. The results failed to show differences between the plates that had been opened and the controls. 41 Effects of the Number of R.m. Colonies Since previous data suggested that the amount of ger- mination was a function of the R.m. growth, it was ques- tioned what the effect of different numbers of R.m. col- onies would be. The results (Table X) show that, although the total yeast growth is approximately the same, germina- tion increased with the number of colonies added. This data indicates that possibly the amount of diffusion is important, or that the postulated stimulator breaks down as it diffuses outward from the colony. The results were determined after 55 days of incubation. Table X Effect of the Number of R.m. Colonies Number of Colonies Total Area of Germination/106 R.m. Colonies 2 l (l-point) 1024 mm 59 2 (2-point) 1230 mm2 123 4 (4-point) 1092 mm2 259 Medium used: 1.0% malt extract; 1 layer or medium; sporophore #1601 Diffusibility of the Action Provided by the Yeast To determine the effect of distance over which the action of the yeast was effective in stimulating germination, 42 the following experiment was done. Large prescription bottles were seeded, the medium allowed to solidify (bottles were placed flat-side down), and the R.m. inoculated at the open end. The results (Figure I) bring to light two points; first, germination occurred in the greatest percentage near the yeast colony and fell-off sharply as the spores became more remote to the colony, and second, the action of the yeast took place over a distance of at least 15 cm. One observation which is not shown by the data is that the spores more remote from the yeast were sometimes the first to germinate; this might be accounted for by differences in growth rate rather than germination rate. In experiments where the amount of medium in the top layer was varied, it was found that more time was required for germination when the top layer was increased in depth. To further study the diffusion of the action provided by the R.m., the apparatus in Plate II was chosen. The basis for using this apparatus was that a stimulator should diffuse over the "bridges" and stimulate germination in the seeded medium. On the other hand, if the yeast acted by removing an inhibitor, all of the inhibitor might not diffuse over the bridges to be neutralized by the R.m. 43 mH va A.mEUV >QOHOU .E.m Eoum mocmumflm NH OH m w w N HO©H# QOHDMQHEHOO no acoHoo .E.m Eonm mucmumem mo pummwm H MmDOHm OH om Om burqeurmlas SBIOdS go JeqmnN 44 PLATE II Experimental apparatus for "agar bridge" tests. Inner plate (well) contains malt extract agar and R.m. colony. The outer plate has malt ex— tract agar which is seeded with basidiospores of Q, gigantea. The well is connected to the outer plate by paper bridged which are covered with agar. 45 The bridges were made of filter paper, cotton string, and glass strips. The plates were assembled and the bridges inserted prior to autoclaving. Next, the center wells were filled with malt extract agar, and the outer plates seeded. The final step was to add molten water-agar to the bridges with a pipette; several layers were added so that the agar was 1-2 mm thick. No germination was found to occur with the two sporophores tested. Transfer of the Postulated Stimulator via Agar Blocks The basis of this experiment was much the same as that of the classical auxin experiments in which the auxin was allowed to diffuse into agar blocks from a cut-off cole- optile tip. This block in turn was transferred to the stump of a decapitiated coleopile and the elongation re~ sponse observed. Thus, if a compound is produced by the R.m. which diffuses into the ambient medium, it should follow that this compound would be present in the medium. Proceeding on this assumption, one might suppose that by removing blocks of the agar near the yeast colony and transferring these blocks to previously seeded plates, the compound would diffuse from the blocks into the seeded plates; then the germination response would occur. 46 To determine if this proposal was true, a series of Petri plates was seeded. At the same time, a second series was seeded and inoculated with the R.m. The third series was seeded and inoculated. The following procedure was then carried out after 5, 10, 15, 20, 25, and 30 days of incu- bation. Blocks were cut from the seeded and inoculated plates; similar blocks were cut from plates that had been inoculated only. These blocks were then transferred to the plates which had been seeded. An illustration of the ex- perimental arrangement is shown in Plate III. The plates were held for 8-10 weeks after the blocks had been trans— ferred. The results showed that none of the basidiospores in the plates receiving the agar blocks germinated. The agar blocks from the series of plates which had been both seeded and inoculated did have a few colonies. These colonies were present only in those blocks which had been incubated for 20, 25, and 30 day periods. This latter observation sup- ports the results of Bulmer (1960). He found that growing mycelium of Q, gigantea did not enhance germination. 47 PLATE III An illustration of the attempt to transfer the postulated stimulator from an R.m. inoculated plate (left) by agar blocks to a plate seeded with g. gigantea basidiospores (right). 48 Effects of Separating the R-m. from the Spores with a Dialysis Membrane Studies were undertaken to ascertain if the stimula— tory action of the yeast on the spores would take place if a dialysis membrane was inserted between them. To accom- plish this, the experimental arrangement shown in Plate IV was used. Although germination did occur, it was observed that it was retarded approximately 2 weeks in comparison with 4-point control plates. To show that the yeast had not escaped, the bags were removed and streaked onto malt extract agar plates. Smears were also made of the seeded medium which had been in contact with the bags. In both cases the smears were negative; indicating that the yeast had not penetrated the bags. Sporophores #1601, #1605, and #1609 were used. To quantify any differences in the amount of germination due to the presence of the dialysis membrane, the experi- mental apparatus shown in Plate V was employed. The results in Table XI show that the membrane de- creased both the rate and the total number of spores ger- minating. Smears showed that no yeast had escaped through the membrane. 49 PLATE IV Colonies derived from germinated basidiospores of Q, gigantea after 9 weeks of incubation. The R.m- was retained within a dialysis membrane. 50 PLATE V A comparison of the amount of germination when a dialysis membrane was used to separate the basidiospores of g, gigantea from the yeast. The plate on the left has a membrane inserted between the yeast and the spores. The plate on the right is a control without the membrane. 51 Table XI Quantitative Results Using Dialysis Membrane to Separate the R.m. from the Incubating Spores Days after Seeding Germination per Million and Inoculating #1601 #1603 Membrane: present absent present absent 30 O 87 O 93 6O 78 252 84 279 90 132 273 143 291 Medium used: 1.0% malt extract; 1 layer of medium with chlormycetin added Germination in Liquid Malt Extract via Dialysis Membrane The procedure followed for these experiments was much the same as in the preceeding experiments. However, smaller dialysis tubing and 125 ml Erlenmyer flasks which contained 50 ml of malt extract broth were used (Plate VI). The results were obtained by two methods. One method was to examine the broth microscopically for the presence of germ tubes. Aliquots were also centrifuged to concentrate the spores. The other method was to pipette aliquots of the broth onto malt extract agar (plate-out), and wait for the appearance of visible colonies. Microscopic examinations for germinating spores were negative for the first nine weeks of incubation; after this, germ tubes began to emerge. The plated—out aliquots did not show colonies until the 11th week (suspensions had been 52 PLATE VI Colonies of mycelium derived from germinated basidiospores of g, gigantea in malt extract broth. The R.m. is retained within the di- alysis membrane. The picture was taken 17 weeks after the spores and yeast were inoculated. 53 plated-out on the 9th week). This points out that 10-14 days are required for the germlings to grow to visible colonies. It should be pointed out that the yeast within the membrane was pale pink until about 4 weeks after in- oculation before it obtained the normal color. Quantitative results showed that approximately 40 percent as many spores germinated in this manner as did the 4-point controls. Sporophores #1601 and #1608 were used, and Chloromycetin was added to the broth. Attempts To Find Active Fractions of the Malt Extract or the R.m. Filtrates of Malt Extract in which the R.m. Grew Although one would expect a great deal of difference in the metabolism of an organism growing under different conditions, it was thought that perhaps the postulated stimulator could be produced in broth medium, or possibly that an inhibitor was removed or converted. To test this hypothesis, 2 percent malt extract was placed in flasks, and the flasks inoculated with the R.m. These flasks were allowed to incubate for periods of 10, 20, and 30 days. Prior to seeding, the yeast cells were removed by either Seitz or Millipore filtration and the broth collected. 54 To 12.5 ml of molten 4 percent water agar, 12-13 m1 of this broth was added. The seeding was done in the usual manner. Chloromycetin was added to the water agar. This experiment was tried several times, and 6 different sporophores were used. No germination was ever found to occur. The effects of Spent Agar Medium Malt extract agar plates were poured and inoculated with the R.m. They were allowed to incubate for 10, 20, 30, 40, 50, and 60 days. Next, the yeast colonies along with the medium below them were cut out, re—melted, and seeded. The remaining agar in the plates was treated in the same manner. No germination occurred on plates which were not inoculated with the R.m. Plates with the R.m. contained a reduced amount of germination, but the diameter of the R.m. colonies was equally reduced. These findings indicate that the action of the yeast is more than a simple starvation of the incubation medium. Attempts to Extract an Active Fraction of the R.m. The purpose of these experiments was to determine if a cell—free preparation could be obtained which would re- place the growing R.m. The R.m. cells were acquired from 55 different growing conditions, and the extracts were pre- pared in different ways. The cell-free extracts were prepared by breaking the cells in chilled Waring blendors which contained glass beads and 60 ml of homogenizing medium. The extracts were sterilized by Seitz and Millipore filtration, and aliquots of l, 2, 3, and 4 ml were added to 25 ml of sterile molten malt extract agar. The individual extracts were prepared as follows: extract #1 was prepared from cells which had been grown on malt extract agar slants for 23 days (10 slants were used), extract #2 was prepared from yeast that was re- moved from 10 4-point plates (5 weeks old), extract #3 was identical to #2 except that the R.m. was 8 weeks old, extract #5 was the same as #2 except that the yeast was boiled 10 minutes prior to homogenizing, extract #6 was prepared by homogenizing the cells in 1 percent malt ex- tract instead of distilled water (cells were 5 weeks old), and extract #7 was made by blending the agar medium, spores, germlings, and yeast from 10 4—point plates which were 5 weeks old. The results were all negative. 56 Chemical Analysis of the Incubation Media Since it appeared obvious from previous experiments that the yeast exerted an effect on the basidiospores via the agar medium, it was thought that perhaps differences in the composition between media with and without the R.m. could be found. The hope was that a unique compound could be found and eluted which, when added to incubation medium, would produce the same effect as the growing yeast. To study some of the possible Changes brought about by the yeast, paper chromatographic and spectrophotometric methods were used. The procedure followed for the chroma- tography was to remove plugs (8 mm in diameter) of medium from plates with and without the R.m.; the medium was then allowed to diffuse from the plugs into the paper. The solvent used was n-butanol: acetic acid: water (4-1-5 v/v). Several reagents were then used to determine if compounds appeared or disappeared due to the presence of the R.m. The reagents used were: (1) ammonical silver nitrate (Williams gt_al,) and periodate-benzidine (Litwack) for the detection of carbohydrates, (2) a modified ninhydrin reagent (Litwack) for ninhydrin positive compounds, (3) a molybdic acid reagent (Block) for detecting phosphate 57 esters, and (4) potassium permanganate for aliphatic acids (Block). The results of analyzing the defined medium showed only one difference that was a result of the R.m. The difference was that, when sucrose was the carbon source, it was hydrolyzed to glucose and fructose. A timed study showed that the hydrolysis began on the third day of incu- bation and was complete by the twelfth. Attempts to analyze the malt extract medium were unsuccessful due to the large number of compounds present. Attempts to analyze media spectrophotometrically were accomplished by preparing extracts of media with and with— out the R.m. The extracts were concentrated by lyophiliz- ation. The results showed no apparent increase or decrease in optical density in either the UV or visible regions due to the R.m. Time-Rate Studies on Germination To determine what effects prolonged incubation with- out the yeast would have on the spores; and if the presence of the yeast was necessary for an extended period of time, the following experiments were done. 58 Effects of Addinqithe R.m. at Various Time Intervals The first experiment to clarify these points was done by seeding plates at time zero. The yeast was then inocu- lated on to aliquots of the plates at time zero and at 5 day intervals thereafter. The results are shown in Figure II. These findings point out that 23—25 days of incuba- tion were required after the R.m. was inoculated before colonies appeared; and that most of the colonies appeared between the 28th and 35th day after the inoculation of the R.m. It also appears that the maximum amount of ger- mination was not affected by prolonged incubation without the R.m. Furthermore, since the lines are essentially parallel, it would seem that there is no differential in the rate of germination due to the prolonged incubation without the yeast. Judging from these figures it appears that the spores will remain dormant in the medium until the yeast is added. Similar results were obtained with #1608. cofiumcHEHmw c0 .mwumam common on» on .E.m may mcflumasoocfl mo mafia u H Amhmmv mcflpmwm Hmuwm mama mo 0% P Hooa# muonmomomm uomnuxm paw: 8H .mnm>mq m .e.m an» mo :oflufleea mo HH mmDon mm - ”EDHUOS mEHB mfiu mo muommmm rom u_ooH roma fioom 10mm room salods UOTIITW Jed uorqeurmxes 60 Effects of Removing the R.m. at Various Time Intervals This experiment was carried out in the following manner: (1) all the plates were seeded and inoculated (l-pint) at time zero, and (2) on various succeeding days, aliquots of the plates were Opened and the yeast colonies removed. The results are given in Figure III. It is obvious that the yeast must be present for at least 6-7 days before the spores are affected. Further- more, there is some action due to the R.m. between the 25th and 30th day of incubation; this can be realized if one subtracts the amount of germination in plates from which the yeast was removed on the 25th day from the controls. The data also suggest that the action of the yeast in evoking germination occurs mostly between the 13th and 20th day of incubation. Since the rate of R.m. growth would bear on the above experiments, the rate of the R.m. growth was also determined (Figure IV). The results demonstrate that growth the first 5 days represents nearly half of the total growth, and the growth was nearly complete by the 15th day. 61 ummmw on» wax» .E.m map mafiumHsuocH cam monomm mcflpmmm kumm Amwmnvwfiea mm C pm>OEmm oi) L? Q coeumflsusH mama om prmm Bonm mEHB ms» um .E.m Sues mmumam ON m . _ mama om kumm mmumHm Houucou \ \\ .\ msmo me umumm \ 0M\\\\mm>OEwm mmmo me Hmumm mmumam Houucou .E.m QUHB mmuma m umwmq H . cofiumcflfiumw so mmumam on» Eoum Um>oamm wuwB mwflcoHou H OH m m ooa# whoamouomm uumuuxm yam: XH ”Esepwz .E.m may mo Hm>OEwm mo muummmm HHH WMDOHm ON ow Om om om UOIIITW Jed uorieurmlas 62 mm ow coHumasoocH Hmumm m>wa CH OEMB mm om ummmq H .uumuuxm uamz ea resouo .s.m mo mumm >H mgOHm wumHm me Ammecoaoovmucflom ¢ “Wt mumHm uwm choHOUvucHom H "ESHUOS NH ma wm Om 'm'u go Iaiemerq sxeiamrttrw u: seruotoa 63 Effects of Different Species of Yeast and Bacteria There is an obvious question that could be asked about the relationship between the yeast and the germination process: is the R.m. unique in stimulating the germina- tion of the basidiospores? Tables VIII and IX show that other species of Rhodotorula are effective in stimulating germination. Furthermore, the different species affect the germination oféspores from various sporophores dif- ferently. For example, 3, rubra, and E, aurantiaca were more stimulatory to the germination of spores from #1609 than was the 3, mucilaqinosa. In addition, it is clear that when different carbohydrates were substituted into the basal medium, the amount of germination with a given yeast varied. Saccharomyces cereviciae (colorless yeast) failed to stimulate germination of spores from 3 sporophores. One observation in these experiments was that the 3. mucilaqinosa, 3, rubra, and B, aurantiaca were all deeply pigmented, while the others had less color. Possibly, one significant fact is that these three species appeared to stimulate the greatest amount of germination. 64 Effects of Bacterial Co-Cultures An accidental deep yellow gram-negative bacterial contaminant also stimulated the spores to germinate, but as Table X indicates, the bacterium did not stimulate germination as well as the R.m. The counts were made after 5 weeks of incubation. However, when the plates were checked 13 weeks later it was found that the number of germinations had increased approximately six— fold. Bulmer (personal communication) reported that the bacterium which he found to stimulate the germina- tion of the g, gigantea basidiospores was also bright yellow. Again, there may be significance to the fact that the stimulating organisms were pigmented. Bacilus subtilis, Pseudomonas aeruginosa, and Segr ratia marcescens did not stimulate the germination of any spores from #1601, #1605, and #1608. The latter two pig- ment producing organisms were green and red, respectively. Of the many other fungi and bacteria which invaded the writer's experiments, none was found to stimulate germination. 65 Table XII Effects of Using Different Species of Rhodotorula Rhodotorula species Germination per Million #1601 #1603 #1609 B, mucilaqinosa 379 349 97 3, aurantiaca 246 198 148 .3. rubra 98 212 148 B, minuta 21 186 161 3, pallida 23 19 0(7)* 3, flava 31 24 O(23)* no yeast 0 O 0 Medium used: 2.0% malt extract; 2 layer of medium; *counts made 2 weeks later Table XIII Effects of Different Species of Rhodotorula and Different Carbohydrates Rhodotorula Malt species Sucrose Glucose Maltose S.S. "x" B. mucilaqinosa 18 6 34 26 269 ‘3. rubra 4 5 15 17 89 B. aurantiaca - 8 - 21 146 B, minuta ll 3 10 16 18 B. pallida ll 1 12 21 24 5, flava 10 3 13 25 24 no yeast 4 3 14 18 000 Medium used: basal plus 1.0% carbohydrates and 0.5% KNO3; 1.0% malt extract; 1 layer of medium; sporophore #1605 66 Table XIV Effects of a Yellow Bacterium A. Type of Plate Germination per Million Diameter #1601 #1608 of R.m. Colony (mm) l-Point Plate 3, mucilaqinosa 35 25 28:2 Yellow Bacterium 7 10 12i2 4-Point Plate 3. mucilaqinosa 277 234 l7+2 Yellow Bacterium 17 22 8+2 Medium used: 1.0% malt extract; 1 layer of medium DISCUSSION The purpose of this investigation was to further elab- orate on the germination of the basidiospores of Calvatia gigantea, especially as the process is affected by the yeast, Rhodotorula mucilaqinosa. It was also hoped that the information found here would be applicable to the germination of basidiospores from other species of Gas- teromycetes which Bulmer and Beneke (1960, 1961, 1962, l964a,b) have shown to be affected by yeast. One of the major facets of this study was to estab- lish the mode(s) of action of the R.m. The several modes of action considered were: (1) the R.m. produced a metab- olite which activated the germination machinery, (2) the R.m. served to remove or convert an inhibitor that was of spore origin or present in the medium, or (3) the R.m. altered the medium in a physico-chemical manner. The approaches employed in this study were: to de- termine if chemical and/or physical treatments would affect germination, to study the nutritional aspects of the germinating spores, and to elaborate on the effects of the environment on the incubating spores. Attempts were also made to isolate a stimulatory compound from 67 68 the yeast, and the media were examined for differences due to the presence of the R.m. Timemrate studies were made, and other species of yeast and bacteria were considered for their effects on germination. In the interpretation of the results, one must con- sider every variable not only as it might affect the basidiospores but also as it might affect the R.m. It should also be reiterated that germination was measured by the presence of macroscopically visible colonies. Hence, the variables studied can not be confined only to the process of germ tube emergence, a parameter often used to study fungus spore germination, but rather, one must consider the effect of the variables on the growth of the hyphae derived from the germinated spores. Further- more, some spores could have germinated but were masked by the mycelium arising from earlier germinating spores. When it was found that a reduced number of spores would germinate on a chemically defined medium without the R.m. co—culture, the possibility of the R.m. acting solely by removing an inhibitor of spore origin was ruled out; however, the amount of germination was higher if the yeast was present. The amount of R.m. growth 69 was also reduced on the defined medium and could be in- creased with the addition of thiamine, however, the thiamine had no effect on germination. It was found that in general ammonium salts and amino acids did not sup- port germination unless the R.m. was present. Thus there is a suggestion that the action of the R.m. is to remove, or to convert reduced nitrogen sources to a usable form. The addition of peptone to the defined medium in-/ hibited germination even though the yeast was present. Malt extract, when added to the basal medium, inhibited germination if the yeast was absent, but enhanced both germination and yeast growth on the R.m. inoculated plates. This latter evidence indicated that an inhibitor is present in the malt extract and that the R.m. removes or converts the inhibitory substance; it does not, however, give evidence for or against the possibility that the R.m. produced a stimulator. It is also possible that the malt extract contains a germination or growth factor which becomes operative after the inhibitor is removed. Other types of media failed to support germination while in some cases, supported good yeast growth. This finding suggested that the presence of a large amount of 7O yeast growth. Plates incubated in the upright position showed more germination than those in inverted plates because of the greater amount of yeast growth in the former. The experiments in which agar-covered bridges con- nected the growing R.m. and the seeded medium provided possible clues for resolving the stimulator-inhibitor dilemma. The information was indirect in nature since the yeast did not stimulate germination via the bridges. The first possible solution is that the R.m. failed to remove the inhibitor from the seeded medium because all the inhibitor could not diffuse over the bridges (most likely). While, if a stimulator had been produced, it should have passed over the bridges. It is also possible that the stimulator was inactivated while passing over the bridge, but this is unlikely for reasons to be discussed later. Similarly, there are several possible explanations why the action of the yeast was not transmitted via agar blocks from yeast inoculated plates to seeded plates. One is that the R.m. acted to remove an inhibitor from the medium, enough of the stimulator may not have been transferred, or the stimulator was unstable. Another possibility is that the blocks were not added at the 71 correct time, however, the time rate studies indicated that the action of the yeast was continuous. Since the R.m. was effective in stimulating germination over a distance of 15 cm, it is suggestive that if a stimulator is produced it is stable enough to diffuse that far. That a volatile stimulator was not produced by the R.m. was demonstrated by the lack of germination when the yeast was isolated from direct contact with the spores. Opening the plates periodically during incuba- tion had no effect on germination, nor did the presence of trapping substances placed in center wells. This indicated the air surrounding the seeded spores is of little importance. Experiments demonstrating that the R.m. was active in enhancing germination through a dialysis membrane did not resolve the mode of action of the yeast. The amount and rate of germination, however, was reduced by inserting the membrane. This suggests that the molecule(s) involved was large enough to be hindered as it passed through the membrane. The use of different species of yeast and bacteria revealed several facts; the B, mucilaqinosa was not unique 72 in its effect on the germination of g, qiqantea basidio- spores, and the organisms which enhanced germination were pigmented. Furthermore, there was a correlation between the degree of pigmentation and the amount of germination. There was, however, a longer time period required before germination occurred when the yellow bacterium and some of the yeasts were used. The interpretation of these facts present two views. First, all of the organisms affecting germination did so by producing a common metabo- lite, possibly a by-product of pigment formation. Second, they all removed the substance(s) responsible for inhibit- ing germination. It is the writer's opinion that there probably are many organisms in the microbial world which also act on basidiospores of the Gasteromycetes. To demonstrate unequivocally that the yeast acted via producing a stimulator, one would have to isolate a frac- tion of the yeast or of the medium which would act in the prescribed manner; in the experiments done, this was not achieved. Likewise, attempts to analyze the incubation media, which had and had not been inoculated with the R.m. failed to show chemical differences due to the yeast. SUMMARY The germination of the basidiospores of Calvatia gigantea was studied; emphasis was placed on the elab- oration of the stimulatory role of the yeast, Rhodo- torula mucilaqinosa, on the process. A basal salt medium, to which various carbon and nitrogen sources were added, was found to support a reduced amount of germination even in the absence of the yeast. Maltose, soluble starch, and sucrose were the best carbon sources, and KNO3 was the best nitro- gen source. Ammonium salts and amino acids did not support germination. Malt extract inhibited germina- tion if the R.m. was not present. Potato-dextrose agar supported germination nearly as well as malt extract agar. The addition of excess malt extract agar increased the amount of germination by increasing the amount of R.m. growth. .Inverted Petri plates had more germination than those incubated upright; the increase was due to an increased amount of R.m. growth. Germination occurred at a distance of 15 cm away from the R.m., but the amount was inversely proportional to the distance from the yeast colony. 73 10. ll. 12. 74 There was no evidence that a volatile stimulator was produced by the R.m. The air above the spores was apparently unimportant in the germination process. Various extracts of the yeast and filtrates of the spent medium did not stimulate germination. The spores remained dormant in the medium for 23-30 days after the R.m. was added. The action of the R.m. in stimulating germination appeared to be con- tinuous after inoculation onto the seeded plates. The action of the yeast took place when it was separated from the spores by a dialysis membrane. Chemical analysis failed to show differences between medium that had been inoculated with the R.m. and the same medium that had not been inoculated. Several species of Rhodotorula and an unidentified bacterium stimulated germination. Although there were quantitative differences in the amounts of germination of spores from various sporo- phores, the qualitative action of the yeast, and the other variables affected germination similarly. LITERATURE CITED Beneke, E. S. 1963. Calvatia, Calvacin, and Cancer. Mycologia. 55: 257. Block, R. J. 1952. Paper Chromatography. Academic Press Inc. New York, New York. Brown, R. 1946. Biological stimulation in germination. Nature. 157: 64. Bulmer, G. S. 1960. The germination of basidiospores of calvatia gigantea and related genera. Ph.D. thesis. Michigan State University. , and E. S. Beneke. 1961. Studies on Calvatia gigantea I. Germination of the basidiospores. Mycologia. 5}; 123. , and . 1962. Studies on Calvatia gigantea II. Factors affecting basidiospore germination. Mycologia. 54: 621. , and . 1964. Germination of basidio- spores of Lycoperdon species and Scleroderma lycopoides. Mycologia. 565 70. _J 1964. Spore germination of forty-two species of puffballs. Mycologia. 56: 630. Cochrane, W. L. 1958. Physiology of Fungi. John Wiley and Sons. New York, New York. Cook, W. L. 1962. Physiological studies of spore strains of Calvatia gigantea. M. S. thesis. Michigan State University. Cunningham, G. H. 1944. The Gasteromycetes of Australia and New Zealand. Printed Privately by John McIndoe. Dunedin, N.Z. Dowding, E. S., and G. S. Bulmer. 1964. Notes on the cytol- ogy and secuality of puffballs. Canad. Jour. Micro. 19; 783. 75 76 Duggar, B. M. 1901. Physiological studies with reference to the germination of certain fungous spores. Bull. Torrey Bot. Club. 42: 313. Ferguson, M. E. 1901.-Germination of Basidiomycetous spores. Science, N. S. 155 407. Foster, J. W. 1949. Chemical Activities of the Fungi. Academic Press, Inc. New York, New York. Fries, N. 1941. Uber die Sporenkeimung bei einigen Gaster- myceten und mykorrhizabilden Hymenomyceten. Archiv. Fur Mikrobiologie. 12; 266. . 1943. Untersuchungen fiber Sporen—keimung und Mycelentwicklung Bodenbewohnen der Hymenomyceten. Synbolae Botanicae Upsaliensis. 6: 1. Good, H. M., and W. Spanis. 1958. Some factors affecting the germination of spores of Fomes igniarius var. populinus (Neuman) Campbell, and the significance of these factors in infection. Canad. Jour. Bot. 36: 421. Gottlieb, D. 1950. The physiology of spore germination in fungi. Bot. Review. 16; 229. . 1964. Germination of fungus spores. Endevor. 23: 85. Hafiz, A., and D. J. Niederpruem. 1963. Studies on basidio- spore germination in Schizophyllum commune. Amer. Jour. Bot. 59: 614. Kaufmann, F. H. O. 1934. Studies on the germination of the spores of certain basidiomycetes. Bot. Gaz. 26: 282. Knaysi, G. 1945. Investigations on the existance and nature of reserve materials in the endospores of Bacilis mycoides by an indirect method. Jour. Bact. 42; 617. Kneebone, L. K. 1950. An investigation of basidiospore ger- mination in the Hymenomycetes, especially in the Agaricaceae. Ph.D. thesis. Penn. State Univ. 77 Lilly, V. G., and H. L. Barnett. 1951. Physiology of the Fungi. McGraw—Hill Co. New York, New York. Lindeberg, G. 1941. Uber die Wirkung von Biotin auf Marasmius androsaceus (L) Fr. Arch. Mikrobiologie. 12: 58. Litwack, G. 1963. Experimental Biochemistry. John Wiley and Sons, Inc. New York, New York. Lucas, E. H. et a1, 1957. Tumor inhibitors in Boletus edulis andother Holobasidiomycetes. Antibiotics and Chemotherapy. Z: 1. . 1958-59. Production of oncostatic principles in_vivo and ig_vitro by species of the genus Calvatia. Antibiotics Annual. Medical Encyclopedia Inc. New York, New York. 493. Mandels, G. R. 1955. Biotin and interrupted growth of Myro- thecium verrucaria. Amer. Jour. Bot. 42: 921. Niederpruem, D. J. 1964. Respiration of basidiospores of Schizophyllum commune. Jour. Bact. 88: 210. Ratts, T. §t_§1. 1964. Physiological studies on basidiospore germination in Schizophyllum commune. Bact. Proc 111. Sedlmayr, M. 1960. Physiological studies of Calvatia species. Ph.D. thesis. Michigan State University. , E. S. Beneke, and J. A. Stevens. 1961. Physio— logical studies on Calvatia species I. Vitamin re- quirements. Mycologia. 5;: 98. , , and . 1961. Physiologi- cal studies on Calvatia species II. Carbon utiliza- tion. Mycologia. 53: 558. Smith, A. H. 1951. Puffballs and their Allies in Michigan. University of Michigan Press. Ann Arbor, Mich. Stanek, M. 1959. The germination of basidiospores of culti- vated mushrooms Aqaricus hortenis (Cooke) Pilat II. Chem. Abst. 35(2): 1960. 21980. Sussman, 78 A. S. 1953. The effects of furfural upon the germination and respiration of ascospores of Neurospora tetrasperma. Amer. Jour. Bot. 40; 401. . 1954. The sensitization of ascospores to Swartz, chemical stimulators by heat treatment. Mycologia. 46; 143. D. 1928. Spore germination of Lycoperdon pyriforme. Papers of the MiCh. Acad. of Arts and Letters. 2: 299. Williams, R. S. 1951. Biochemical Institute Studies IV. University of Texas Publications. Austin, Texas. AP PENDI X Background Information on the Sporophores Used for This Investigation Code Number Date of Harvest Location of Harvest 1601, 1602 Southwest corner 1603, 1604 of the M. S. U. 1605, 1606 September, 1961 Golf Course 1608 June, 1963 E. Lansing, Mich. 1609 September, 1961 Ada, Michigan 1611 October, 1961 Grand Rapids, Mich. Background Information on the Rhodotorula Species Used for This Investigation* Genus and Species Code Number Rhodotorula aurantiaca NRRL Y-1581 3. $1.33 NRRL Yv-1585 R. qlutinis NRRL Y-2502 3. minuta NRRL Y-1589 R. mucilaqinosa NRRL Y—174 _R_. pallida NRRL Y—339 R. 3.112;; NRRL Y~1592 *Received from: ARS Culture Collection Northern Utilization Research and Development Peoria, Illinois 79 80 Ingredients of Media Difco Malt Extract Agar Maltose 1.27% Dextrin 0.027% Glycerol 0.023% Peptone 0.008% Agar 1.500% Difco Malt Extract A dehydrated infusion of barley which contains: Diastase Dextrin Dextrose Protein bodies and salts from barley NIT ‘ ' J,11,;£:155 HICHIGQN STRTE UNIV. LIBRARIES ll1IIIHIVIIHIll!“I”WNWIHIHIIIIIHIIINHIIIlllHHHI 31293107295689