AI ii! i 1 iliii'lli 1‘! | i PHYSIOLOGICAL $TUDEES 0F SPORE $TRAi§ \‘S Q? CALVATi iA GiGANTfifiA Thesis ior the Degree oi M 5 Am EGAN STATE m 33m, Warren Lee Cook 1962 TH ESiS lliiiiiiiiiiiiiiimimiiil , . 3129310729 6802 LIBRARY Michigan State University MSU LIBRARIES “ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES wii] be charged if book is returned after the date stamped beiow. PHYSIOLOGICAL STUDIES OF SPORE STRAINS OF CALVATIA GIGANTEA By Warren Lee Cook A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1,, 1 ‘3 LI 734 1+ A a'li'é' ACKNOWLEDGEMENTS The author wishes to express his thanks and sincere appreciation to Dr. E. S. Beneke for his stimulation, interest and guidance both as an undergraduate and graduate student. The stimulation that Dr. Beneke gave the author in undergraduate school facilitated the writers entering graduate school in the field of myc010gy. Appreciation is also due Drs.‘W. B. Drew, J. L. Lockwood and A. F. Yanders for their advice, suggestions and constructive criticisms during the course of this investigation. This investigation was supported by the National Institutes of Health for which the author is appreciative. A very sincere thanks is due the late Mr. Donald w. Journeay for his concern with the author's education by endowing him and his family with financial assistance. To mw'wife Elaine and son Gregory ABSTRACT .. The purpose of this experiment was to determine physiolOgical differences between three strains derived from basidiocarp tissue and three strains derived from spores of Calvatia gigantea.by the study of the utilization of carbon and production of calvacin. There was a differential utilization of glucose, cel16biose, dextrin and glycogen by the three spore strains and their three parent strains of Calvatia gigantea. One spore strain, E—9-6, utilized each of the four carbon sources much more readily than any of the other strains. Strain E-9-6 exhibited more than a 50% increase in mycelial dry weight in the presence of aluminum squares possibly due to either mechanical agitation or utilization of small quantities of aluminum as a nutrient. Calvacin, an inhibitor of Crocker mouse sarcoma 180, was produced by all the strains of Calvatia Q'gantea used in this investigation. There was a differential production of calvacin when the various carbon sources were used. Cultures grown in celldbiose generally produced.the most calvacin, as determined 'by the Crocker mouSe sarcoma 180 test. There was no correlation between the amount of growth and the amount of calvacin produced.when the selected carbon sources were used. iv TABLE OF CONTENTS INTRODUCTION...........o..............l LITERNTURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . .2 MATERIALSANDMEYI‘HODS......................h Source of Calvatia gigantea Cultures. . . . . . . . . . . . b Media . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . 10 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . 15 Carbon Utilization . . . . . . . . . . . . . . . . . . . . .15 Aluminum in Medium . . . . . . . . . . . . . . . . . . . . .25 Calvacin Production . . . . . . . . . . . . . . . . . . . . 27 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32 BIBLIOGRAPHY..........................3h MDH O O O O O O O O O O o O O O 0 O O 0 O 0 0 O o O O O O 0 36 LIST OF TABLES TABLE 1. 2. 3. h. Carbon Utilization by Basidiocarp 1096C and Spore Strain E-9-6 based on the dry weight of the mycelium . . . . . . . . . . . . . . Carbon Utilization by Basidiocarp 1369 and Spore Strain F-2-3 based on the dry weight ofthemycelium.............. Carbon Utilization by Basidiocarp 99S and Spore Strain A-S-l based on the dry weight OfthGWCBliMocoooooooooooo Calvacin Production by Various Strains of Calvatia gigantea . . . . . . . . . . . . . PAGE . l6 LIST OF FIGURES FIGURE PAGE 1. 2. 3. Utilization of Various Carbon Sources by Mycelia Derived From The Parent Basidiocarp 10966 and its BasidiOSpore E-9-6 . . . . . . . . . . . . . .17 Utilization of Various Carbon Sources by Mycelia Derived From The Parent Basidiocarp 1369 and its Basidiospore F-2-3 . . . . . . . . . . . . . . 20 Utilization of Various Carbon Sources by Mycelia Derived From The Parent Basidiocarp 995911dit3BaSidj-OSPOI‘9A'S‘1 o o o o o o o o o o o o o c 022 LIST OF PLATES PLATE PAGE I Strain A-S-l of Calvatia gigantea showing almmmcapoanaSkooooooo000.00.000.09 II Strain E-9-6 of Calvatia gigantea showing a 28 day h point inoculation of "Medium A" agar..................0.0...00.11 III Strain A-S-l of Calvatia i antea serial subculture grown on “Nedium roth . . . . . . . . . . . 13 IV Strain A-S-l of Calvatia gigantea 28 day culture of "Medium A“ on left and cellobiose mediumontheright..........o..oo...o.2i4 viii INTRODUCTION The merciless killing and incapacitation of our Species caused by cancer has led to world wide research on the etiology and methods for treatment of this dreaded disease. Dr. E. H. Lucas (1960) in l93h, rediscovered folklore in the Bohemian.Forest near the Bavarian'border, where the natives associated the eating of one kind of mushroom, Boletus ‘ggulig, with the prevention of cancer. Investigation has demonstrated that aqueous extracts of immature (white) basidiocarps of Calvatia gigantea (Pers.) Lloyd, inhibited.Crocker mouse sarcoma 180 (Lucas, gt. 31., 1958-1959). Further investigation has shown that this antitwmor substance, calvacin, could.be produced by shake flask cultures of the basidiocarp (Lucas, 23. 21., 1958-1959) and.from.spore strains of Calvatia gigantea (Bulmer, 1960). A broad-screen tumor survey showed that calvacin possessed significant antitumor activity against 13 of 2h various mouse, rat and hamster tumors (Roland, st. 31., 1960). These discoveries have led to further investigations to determine better*means for Obtaining calvacin. The purpose of this investigation was to elucidate some of the carbon requirements of both basidiocarp and Spore strains of Calvatia gigantea and to determine which carbon source would give maximum yields of calvacin. LITERATURE REVIEW Carbon compounds are essential for'metabolism in the fungi for two reasons. First, they supply the carbon required for synthesis, which comprises approximately 50% of the dry weight of a living cell. Second, the oxidation of carbon compounds provides a source of energy for the organism. Therefore, cognition of carbon nutrition is basic to understanding the physiology of any fungus (Cochrane, 1958). An extensive literature review on the carbon requirements for organisms in the sub-class Homdbasidiomycetidae was given by Stevens (1957) and Sedlmayr (1961b). Since 1960 four publications have appeared concerning the utilization of carbon in this sub-class. Swack and Miles (1960) indicated that the carbon source most utilized by Schizophyllum commune for the production of mycelium was D-xylose, and those for highest pigment production were D-fructose and D-glucose. Good growth of this organism was shown with: L-arabinose, D-ribose, D-glucose, celldbiose, D-fructose, D—galactose, D-glucose, maltose, D-mannitol, raffinose, D-sorbitol and sucrose. Meloche (1960) referred to a possible metabolic pathway for the utilization of a carbohydrate by Lactarius torminosus in the sub-class Homdbasidiomycetidae. He stated that the oxidation of glucose proceeded via the pentose phosphate cycle. This cycle possibly is the major path of glucose oxidation in this organism. Aschan-Aberg (1960) indicated that the order of utilization of carbon sources by Collybia velutipes during the process of fructification are: glucose > sucrose >maltose . Sedlmayr (1961b) showed that Calvatia species were able to utilize a variety of carbon sources. The carbons best utilized.by all strains were: dextrin, ceIIObiose, invert sugar, glucose, glycogen and starch. Only one strain utilized mannose and fructose better than the other strains studied. Stevens (1957) and Lucas, 23. a1. (1958-1959) gave the only reports concerning antitumor agents produced by organisms in sub-class Homobasidiomycetidae. They discovered antitumor agents from Boletus edulis 235. pinicola; Collybia radicata var. furfuracea; and Calvatia gigantea. .MATERIALS AND METHODS Source of Calvatiaggigantea cultures Cultures of mycelia from'basidiocarps were obtained from immature fructifications collected by Dr. Joseph A. Stevens and Dr. Glenn S. Bulmer. The strains derived from basidiospores were obtained from Dr. Glenn 8. Bulmer. The following three basidiocarps and three of their epore strains were used in this study: 1. Spore strain number'EP9-6'was Obtained from‘basidiocarp number 10960. This colony is the fastest growing of the three spore strains. The surface of this strain has a white periphery with a light tan center and the overall growth is velvety. The underside of this culture is light buff. The tumor-retarding preperty of this culture is moderately good (1')! for a 1h day culture and good (3’) for a 28 day culture (Bulmer, 1960). ‘1‘ (-) Indicates a parameterl'of 75% or more as compared to the control tumor. (3') Indicates a parameter between 50 and 75% as compared to the control tumor. (SI) Indicates a parameter between 25 and 50% as compared to the control tumor. (+) Indicates a parameter of 25% or less as compared to the control tumor. 1 - The parameter is based on the average measurement of two diametere (one the greatest diameter and the other perpendicular to it). 2. Basidiocarp number 10960 was Obtained from Ithaca, Michigan, OctOber l, 1958. This culture has a moderate rate of growth. The surface of a plate colony shows no pigment production and has a white cottony to a velvety appearance. The underside shows a light buff color on a 21 day old plate culture. The tumor-retarding property of cultures Obtained from this basidiocarp is poor (-) for a lh day culture and.moderately good (2') for a 28 day culture (Bulmer, 1960). 3. Spore strain number F-2-3 was Obtained from basidiocarp l369. This strain has a moderate rate of growth. No pigments are produced on the surface of the plate culture. The surface of the colony shows a cottony white growth. The underside of the culture is light buff in color. The tumor retarding property of this culture is poor (—) for a 1h day culture and good (1*) for a 28 day culture (Bulmer, 1960). h. Basidiocarp 1369 was obtained from near Lansing, Michigan on September 28, 1958. This is the fastest growing of the three colonies developed from basidiocarps. The center surface of this colony has a light tan color with a white periphery and a velvety to a cottony appearance. The underside of this colony is a tan color. The tumorw retarding property of this colony was poor (-) for a 28 day culture; no information is available for the 1h day culture (Bulmer, 1960). 5. Spore strain number.A-5-l was obtained from.basidiocarp 995. This strain is the slowest growing of the three spore strains, but it appears to grow faster than basidiocarp strain 995. The surface of a 21 day old plate culture does not show pigmentation, is pure white, and the reverse is a light buff color. The plate culture has a velvety to cottony appearance. The tumormretarding property of this spore strain is moderately good (1”) after lh days of growth and poor (-) after 28 days growth (Bulmer, 1960). 6. Basidiocarp number 995 was Obtained from an undesignated area of Michigan between the years 1956 and 1957. On agar’medium, the mycelium is the slowest growing of the three basidiocarp strains studied, and has the darkest brown pigment. In a shake culture containing I'Mledium A" this strain produces a characteristic diffusible dark brown pigment. 0n solid medium, the periphery of growth in the culture is a light tan color whereas the center of the colony is a dark tan color. The surface of the colony has a velvety texture. The underside of a 21 day old culture is a dark brown color. Tumor-retarding property of cultures obtained from this basidiocarp were reported as poor (-) at the end of both lb and 28 days of growth (Bulmer, 1960). These six cultures were chosen as three of the spore cultures were the best producers of a tumor retarding substance among those tested (Bulmer, l960). Two of these spore strains, E-9—6 and F-2-3, showed higher level of calvacin activity than any of the 251 mycelial cultures from'basidiocarps (Bulmer, 1960). These were compared with the three cultures Obtained from their parent'basidiocarps. 22:11:. Two types of media were used, a synthetic basal medium of Lindeberg (l9h6) supplemented with thiamine H01 (Sedlmayr, l96lb) and a modified Czapek's-Dox medium, designated by Stevens (1957) as "Medium A". Modified Lindeberg's.Medium: Glucose NHh-tartrate KHzi’Oh .MgSOho7H20 FeCl3 (sol. 1/500) ZnSOh (sol. 1/500) MhClz (sol. 0.1M) Ca012 (sol. 0.1M) Thiamine H01 DiStilled H20 "Medium A": Glucose Sucrose Bactopeptone Bacto-yeast MgSOh-7H20 KHZPQh K01 FeSOhOYHZO Distilled H20 20.0 grams 5.0 grams 1.0 gram 0.5 gram 0.5 ml 0.5 ml 0.5 ml 5.0 ml lOOEY/liter 995.0 ml 15.0 grams 15.0 grams 5.0 grams 5.0 grams 0.5 gram 1.0 gran 0.5 gram 0.01gran 1000.0 ml It was the author‘s impression that capping a culture flask with a substance other than the traditional cotton plugs would facilitate handling of the flasks. An additional reason for the undesirability of using cotton plugs, is the report that cotton introduces lint into the media. Some grades of cotton have been reported to release volatile substances into the culture flask which could affect mycelial growth and possibly calvacin production (Lilly and Barnett, l951). Aluminum foil was chosen as a cap for the culture flasks due to the ease of handling, low cost and readily accessibility of the foil (see Plate I). There is little information available in the literature concerning the effect of aluminum.on the growth of fungi. Cochrane (1958) stated that aluminum often exerts physiological effects and may improve growth; however, he further states that aluminum has not been reported to be essential for growth. It was reported that placing aluminum strips into a culture medium prevented growth of several strains of Aspergillus terreus and it was possible to overcome this toxicity by counteraction with magnesium ions (Lockwood.and.Reeves, l9h5). Due to these conflicting reports, it was decided to determine the affect of aluminum upon the growth of Calvatia gigantea. "Reynolds'Wrap" aluminum foil was used throughout the experiment. In a personal communication from Mrs. Elizabeth'w. Gauldin of the Reynolds Metals Company, it was stated that the only residual materials on the aluminum foil were rolling oils used as lubricants (which she felt should be destroyed upon autoclaving); and traces of silica, iron and minute amounts of magnesium. A total of 125 ml of Lindeberg's medium as modified.by'Sedlmayr (1961b), was placed in each of twentybfour 500 ml Florence flasks. Eight of these flasks were capped with cotton plugs; eight were capped with aluminum PLATE I Strain A-5-l of Calvatia gigantea showing aluminum cap on flask. lO foil and eight were capped with cotton plugs plus 18 one-half inch square strips of aluminum foil which was introduced into the medium. the above was autoclaved at l5 pounds of pressure, at 121°C for 15 minutes. A 10 ml inoculum of a lb day culture of spore strain E-9-6 was used as the test organism. The cultures were placed upon a shaker to enhance growth. By adding an excess of aluminum squares into the medium, it was considered that there should be a decided influence upon growth, either inhibitory or stimulatory. Four flasks in each group were harvested after lh days of growth and the remainder after 28 days. Dry weight of mycelium was used as the determination of growth. Procedure The culture flasks used.were acid washed 500 ml Florence flasks, as recommended by Stevens (personal communication). These were acid washed for 10 minutes in lN H01 solution followed by rinses with both tap and distilled.water. The various carbon sources used with Lindeberg's medium were autoclaved separately as a water solution, then added aseptically to the cooled basal medium. Cultures were Obtained from stock cultures that were maintained in nMedium A" covered with 5 ml of sterile Bayol Oil F (Penola Company). These cultures were inoculated into a petri dish containing 30 ml of I'MediumA" and serial subcultures every 28 days (see Plate II). When a flask culture was desired, nine point-inoculations from a 1h day culture were made in an area of approximately 1 inch square on 25 ml of "Medium A" agar. Approximately twentybone days later, __ .—————- .__..___. PLATE II Strain E-9-6 of Calvatia gigantea showing a 28 day four point- inoculation of "Medium A" agar. 12 a 1% inch square of the culture was removed and.blended with 52 ml of distilled water for 60 seconds in a sterile 360 ml semi-micro monel metal'waring blendor. Ten ml of the blended mycelium was inoculated into a 500 ml Florence flask containing 125 m1 of "Medium A", and placed on a reciprocal shaker. In order to keep good mycelial inoculum, subcultures from flask cultures were made every 1h days (see Plate III). At this time the mycelium was removed from a flask culture and blended with 50 ml of water for 60 seconds. To remove traces of "Medium A" from the mycelial fragments, the mycelium was washed three times by centrifuging with sterile distilled water for 20 minutes at 1500 R.P.M. After the third washing, the mycelial fragments were placed into a sterile flask and diluted with sterile water to approximately six times the volume of the fragments, giving 500 ml of the inoculum. A 10 ml inoculum suspension was combined with 125 ml of the experimental medium and incubated at an optimum temperature of 25°C (Stevens, 1957) and (Sedlmayr, 1961a) on a reciprocating shaker which had a stroke of one inch and gave 90 two-inch excursions per minute. This speed for the shaker was optimum for calvacin production (Stevens personal comunication). It was determined (Stevens, unpublished data) that the rate of growth increased with an increase in the Speed of the shaker; however, the amount of calvacin was reduced greatly. The pH of all media after autoclaving was 5.5. A pH of 5.5 was determined by Sedlmayr (1961a) to be optimumfor growth of Calvatia sp. At the conclusion of an experiment the pH was determined again. 13 PLATE III Strain A-5-1 of Calvatia gigantea serial subculture grown on "Medium A" broth. 1h Stevens (1957) had shown that maximum calvacin production was found after 28 days of mycelial growth on "Medium A" with some calvacin production after 1h days. The mycelial harvests were made at lb and 28 days. Due to shortage of shaker space and cost of media, there was a total of four replicates for every harvest. Three of the replicate flasks were used for determination of mycelial growth and one was used to determine calvacin production. The amount of growth was determined by mycelial weight after drying at 95°C for 2h hours. The replicates in all cases were nearly equal on the dry weight basis, indicating sufficient inoculum was used. The mycelium derived from a basidiocarp and the mycelium derived from its spore strain were cultured simultaneously, permitting comparative data on carbon utilization. The contents of the flask that was tested for calvacin production was blended for 60 seconds and the solid portion was separated from the liquid by suction flask filtration. One hundred ml of the liquid and 1 m1 of 1% merthiolate (1 gram.merthiolate, l.h grams borax per 100ml of water) was placed in a sterile rubber capped serum bottle. The solution was stored'by freezing. The merthiolate solution does (not affect the results of the tumor assay (Stevens, personal communication). The samples were shipped frozen to the Armour Research Foundation of Illinois Institute of Technology, Chicago, Illinois, for the Crocker mouse sarcoma 180 tests. 15 RESULTS AND DISCUSSION Carbon Utilization The utilization of sugar by living cells has long been observed as one of the major sources of energy for maintaining activity of a cell. The breakdown does not occur in one giant step from carbohydrate directly to carbon dioxide and water; rather, a series of stepwise reactions occurs involving a number of intermediate compounds (Pigman, 1957). The purpose of these experiments was to indicate various carbon sources utilized for growth as a means of indicating metabolic differences between spore strains of Calvatia gigantea and their parent basidiocarp strains. The sugars that were selected in this experiment were among the best utilized in Sedlmayr's (1961b) experiment. The basal Lindebergis medium contains 8 grams of carbon per liter. In this experiment an equivalent of 8 grams of carbon for each substituted carbohydrate was placed in the medium. Lindeberg's medium, plus thiamine hydrochloride as modified by Sedlmayr, minus the carbon source and.MMedium A" were used as controls. - Mycelium of basidiocarp strain 1096C did not grow as rapidly as its spore strain E-9-6 (Table 1 and Figure l). The main differences in utilization of carbon by these two strains is that strain E~9a6 was able to utilize glyc0gen as a carbon source and showed good growth after both lb and 28 days. Strain 1096C obtained all of its measurable mycelial growth in this medium after 1h days and after 28 days showed apparent autolysis. Glycogen was not a good carbon source for 16 0.0 m.amH.H*.H.m m.HmH.H a.m o.mm m.m ~.H: H.: 0.40m m.: o.oom* 0.: m.mool. m.: H.5mm a.m a.aoe e.: «.mam m.m a.aam m.s a.amm me we me we MNmo mm mNmo 4H human .oosfleaouoo a0: Edasoosfl mo pewwoz .moemoaaaon sweep seep mmoq * 3.0 m.NHN.H w.m H.eaa m.m H.om m.m 0.4m m.m a.mm m.m p.mm m.: ».o:H* :.m m.mo :.: :.mmH v.4 h.~HH 4.: o.HwH*_ m.: e.mma oomoa .ssflaoohe one we enwfioz.hho one no oommp_oumum cashew spoon ens oomoa aauooaeamwp.ap :oapwuaaaau cognac . 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SWEDITIDJ NI .LHDIEM KEG 23 Using cellobiose as a carbon source, strain 995 showed better mycelial growth at 28 days than lh days, whereas a decrease in weight occurred, possibly due to autolySis in strain AaS-l, with the same sugar at the end of 28 days. Strain 995 grew faster than strain A-S-l after lh days on "Medium A", but strain A-S-l showed greater mycelial growth than strain 995 after 28 days (see Plate IV). For the statistical analyses which were made for Tables 1, 2 and 3, see Appendix. The source of nitrOgen, ammonium tartrate (ChH ), is an 12N206 organic compound and could be construed as a carbon source. The inocula of the four strains (l369, F-2-3, 99S and A-S-l) were weighed before placing in the control media and at the end of the experimental times. Usually, no growth increase was measurable in the control flasks, and autolysis appeared between the time of the inoculating and harvesting of the mycelium in the culture flask. This lack of increase in mycelial dry weight in the controls would indicate that Calvatia gigantea was not able to utilize ammonium tartrate as a carbon source under these conditions. Sedlmayr (1960) had shown that of the four nitrOgen sources studied, aSparagine, NHuCl, KNO3 and ammonium tartrate that ammonium tartrate was the best source of nitrogen for growth of Calvatia Species. No additional tests were made to determine if the carbon requirements of these various strains were consistent over a period of time. There was no visible evidence of morphological change while these strains were on plate culture. Stevens (personal communication) stated that calvacin production and mycelial growth rates were nearly consistent if methods and the amount of inocula remained constant. He also found 2h ¥ 7—-wo, PLATE IV Strain A-Snl of galvatia‘gigantea 28 day culture of"Medium A" on left and cellobiose medium on the right. 25 that tissue from different portions of one basidiocarp would consistently produce similar quantities of calvacin when tested. These experiments have been repeated numerous times with similar results. Maximum growth prdbably was not achieved in all of the carbon experiments. A mycelial growth curve would be desirable for each carbon utilized to determine the period of maximum growth time, if it were possible to have more than four replicates in each of the experiments. This would indicate whether or not variations occurred in the maximum time required for each basidiocarp strain and spore strain to utilize the carbon source. At no time did any of the strains show mycelial growth in test media as great as that in "Medium A", indicating that growth factors must be present in "Medium A" that were not present in the modified Lindeberg's medium. The carbon utilization by all the spore strains was different from that by their respective parent basidiocarp strains, as shown by the variations in mycelial dry weight. This would indicate metabolic differences in the former when compared with the latter. It is possible that these differences are due to genetic recombination which occurred during formation of the basidiocarp, in spore germination, or in the somatic phase, but the evidence is merely indirect. Aluminum in Medium The results by average weight of four replicates of spore strain E-9-6 and the average pH are as follows: 26 1h days 28 davs mg pH mg pH Cotton-plugged flask 509.2 h.h 625.9 3.7 Aluminum-capped flask 505.8 h.3 612.h 3.7 Cotton plug plus aluminum squares 5h9.9 h.2 1,053.h h.0 No significant mycelial growth differences were noted in cotton- plugged flasks and aluminum-capped flasks. The weight difference between the 1h day cultures was 3.h mg, a slight weight increase for mycelium in cotton-plugged flasks. The difference between 28 day cultures was 13.5 mg with growth of mycelium in the cotton-plugged flasks being slightly greater by weight. These differences are considered to be insignificant, but the lesser growth of mycelium in the aluminum-capped flasks as compared to the cotton-plugged flasks might have been due to leaching of the cotton. The addition of approximately 18 square inches of aluminum foil to the medium caused a marked increase in mycelial growth, giving an increase as compared with the average weight of the other two groups of 37.h mg for 1h days and h3h.2 mg after 28 days. The mycelial pellets were noticeably smaller on the flask with the aluminum squares. This was possibly caused by mechanical agitation due to the movement of the movement of the squares while the culture flasks were on the shaker, or its utilization of aluminum. Upon examination of the aluminum strips after 28 days it objectively appeared that the squares were thinner, and that the edges had eroded. Further tests should be made to determine if the increase in growth was due to mechanical agitation of the aluminum squares (thereby, 27 having a greater surface area of mycelium to enhance growth); or if the increase in mycelial growth was due to utilization of the aluminum through an enzyme action, as a nutrient, or through partial decompOSition of the squares due to the hydrogen ion content of the culture. Calvacin Production Stevens (1957) had shown variations in tumor retarding activity of cultures of Collybia radicata, var. furfuracea when various different carbon sources were utilized. In this study a comparison was also made with Calvatia gigantea to determine if variation in calvacin production was evident when other carbon sources were substituted in the basal medium. The results of the Crocker mouse sarcoma 180 tumor tests using six mice in each test are indicated in Table h. The designation T/C in Table h is determined.by taking the average tumor weight of the experimental mice, injected with a 1:1 dilution of the calvacin solution, and dividing this figure by the average tumor weight of the control mice, which were injectedwdth normal saline solution. This routine method of determining tumor retardation by the Armour Research Foundation is different than the method used by the Sloan-Kettering Institute for Cancer Research. The designation used by Sloan-Kettering Institute has been reported by Clarke (1955) and Stevens (1957). This is the ratio between the average parameter change of the treated animals to the average parameter change of the control group and is designated as (+), (1+), (: -)’ and (-) as previously indicated in materials and methods. 28 pnuflmz posse Homecoo one mp voofl>flo pzmfloz posse Hmpcosfipoaxm n o\e coapmocsom commemom asoEp< h; come mpmop aoESP * aaa Noa ea 2% ea em sea mma Hm sea as ea mm mm as am as ea sea oma Ha as ooa ow om mma boa me me mm mm am as as am so Noa ma mm as as mm 2 mm 2. mm om mm emaasampmm 902 as so oaa on He as maa mm ma on as e\a mam o\Ha-m-a o\a meme 0\a m-~-a o\a oomoa *0\a osmsm caaasm aaaasm camapm eaaasm :amapm aaaapm mopnmwww mfipmbawo mo mafimppm msoflhm> hp soaposoosd aflom>dwo n : mqmda whee name what when mhmo ammo mhmb name when whee mm AH mm 2H mm 4H mm 4H mm zmuoowuo ZHmBNMQ mmOHmoqqmo mmOODAO 4 szmm: 29 The correlation and significance of these two methods of determining tumor retardation has not definitely been determined although it has been discussed extensively by specialists in this field. Table h shows that all strains could produce some calvacin with the different carbon sources as shown by the tumor tests in mice with the exception of strain 995 and strain A—5-1 with glycogen as the carbon source. Some of the strains produced more calvacin in Lindeberg's medium with different carbon sources than when grown in "Medium A". There were also small quantities of mycelium produced in relation to the amount of calvacin produced in Lindeberg's medium. Compounds in the filtrate that stimulate tumor growth appeared in some instances where the T/C is reported to be 100 or more. This has been shown to be due to the production of these substances in younger cultures prior to the development of calvacin (Lucas, gt. 31., 1958-1959). A tumor retardation curve would be desirable for each medium tested for calvacin production. It has been shown that calvacin, once produced 32 zitrg, can be lost with further mycelial growth or that more time may be needed for mycelial growth for maximum calvacin production (Stevens, 1957). Bulmer (1960) had these six strains tested for calvacin production in 1959 by the Sloan-Kettering Institute for Cancer Research. This method involved parameter changes in the mouse tumor. A11 strains tested continued to show some tumor-retardation either at 1h or 28 days in the current tests which were three years later than the 30 original tests. Strain E-9-6 originally was moderately good in 1h days, and good in 28 days, indicating an apparent loss of some tumor-retarding activity, while strain 1096C originally was poor to moderately good and during this test appears to have shown a marked increase in tumorbretarding activity. Strain F-2-3, originally poor to good, appears to have lost all or nearly all of its activity, while strain 1369 originally was poor and now appears to show some increase in tumor retarding activity. Strain A-S-l originally was moderately good to poor and now appears to have no or slight activity, while strain 995 showed very little activity at all times. The apparent lack of exact correlation of calvacin production with Bulmer's (1960) experiment is possibly due to two things. There was a different method used for culturing the fungi before taking extracts for the Crocker mouse sarcoma 180 tests. In Bulmer's (1960) experiment the stock cultures of the fungus were inoculated at 9-points on "Medium A" agar in a petri dish, ground, and then grown in "Medium A" broth. In these experiments, as described on page 12, the stock culture inoculum was maintained by serial subcultures on "Medium A" broth and then inoculated into the media with various carbohydrates. Better pellet formation occurred when the 9-point plate method was used. However, the 9-point method requires more time to develOp sufficient mycelial inoculum than when grown in liquid medium. In addition, individual variation in each flask may account for some of the small variation that occurs in the tumor testing results. A significant part of this experiment shows that when the various sugars were substituted in the basal medium as well as "Medium A" the 31 strains still produced some tumor retarding substance. Further investigation of other growth requirements of strains of Calvatia sp. and germinated spore strains should make it possible to develop a more exact medium for optimum production of calvacin. There is evidence in the results to indicate that there is no direct correlation between the amount of mycelial growth and the production of calvacin. 0f the sugars utilized in this experiment, more calvacin was produced by celldbiose than the other sugars, as seven out of twelve tests showed the mouse tumors were retarded to between 58 and 7h% of the controls. After 28 days the filtrate of 10960 grown in dextrin showed a marked amount of calvacin produced in comparison to the amount of mycelial growth. The variations in utilization of carbohydrates checked indicates that are some physiological differences between strains that would account for the differential calvacin production. In these experiments the differences in calvacin production between the basidiocarp strains and the spore strains would indicate that the metabolic requirements of these organisms vary considerably, and that it may be possible to select strains and develop media that would greatly increase calvacin production. 32 SUMMARY 1. Séx strains of Calvatia gigantea, three from.basidiocarps and three from Spore strains, were used to determine utilization of four carbon sources in relation to calvacin production. 2. The utilization of glyCOgen as a nutrient by the mycelium of basidiocarp strain 10960 was insignificant, whereas its spore strain E-9-6 readily utilized glyc0gen, indicating a physiological difference between these two strains. 3. The differences between basidiocarp strain 1369 and spore strain F-2-3 were marked. These two strains utilized glucose equally well up to 1h days, while at 28 days strain F-2-3 continued to increase in dry mycelial weight and strain 1369 showed no significant changes. Strain F-2-3 generally utilized all the sources of carbon better than Strain 1369 in Lindeberg's medium, but in "Medium A" strain 1369 grew better. h. Glucose was a good source of carbon for basidiocarp strain 995 but was a poor carbon source for Spore strain A-5-l. Both strains grew at about the same rate for 1h days when cellobiose was used as the carbon source. At 28 days, strain 995 showed additional growth, whereas strain A-Sal showed apparent autolysis. 5. In the absence of carbohydrates in the control medium, the six strains of Calvatia gigantea apparently were not able to utilize any of the carbon in the organic nitrogen source, ammonium tartrate. 6. All the strains were able to utilize the various carbon sources tested but in varying amounts. 33 7. There was no correlation between the amount of calvacin produced and the amount of mycelial growth of the various strains. 8. Calvacin was produced.when any of the four carbon sources were used. Celldbiose was generally the best carbon source for calvacin production. 9. The presence of aluminum squares in the media accelerates mycelial growth. 10. This study shows that two Spore strains, E-9—6 and F-2o3, utilized the four carbon sources better than the parent basidiocarp strains, while spore strain A—S-l did not utilize these carbon sources as well as the parent basidiocarp strain. 3h BIBLIOGRAPHY Aachen-Aberg, K. 1960. Genetical and hysiological studies on Collybia veiutipg . Svensk Bot. Tid. 5M2 :329-3u1 Bulmer, G. S. 1960. The germination of basidiospores of Calvatia gigantea and related genera. Ph.D. thesis. Michigan State University Clarke, D. A. 1955. Mouse sarcoma 180. Cancer Research Supplement NO. 3, 1’4'17 Cochrane, V. W. 1958. PhysiolOgy of fungi. John Wiley and Sons, Inc. New York Lilly, V. G. and H. L. Barnett. 1951. Physiology of the fungi. McGraw-Hill Book 00., Inc., N. Y., Toronto and London Lindeberg, G. 19h6. Thiamin and growth of litter decomposing hymenomycetes. Bot. Notiser. 19h6:89-93 Lockwood, L. B. and.M. D. Reeves. 19h5. Some factors affecting the production of itaconic acid by.Aspergillus terreus. Arch. Biochem. othS-h69 Lucas, E. H, R. U. Byerrum, Do A. Clarke, H. C. Reilly, J. A. Stevens and C. 0. Stock. 1958-1959. Production of oncostatic principles in vivo and in vitro by species of the genus Calvatia. Antibiotics Annual, MEEiE§I_Encyclopedia, Inc., New fork, N. Y. h93-h96 Lucas, E. H. 1960. Folklore and plant drugs. Papers of the Mich. Acad. of Sci. Arts and Letters. h53127-136 Meloche, H. P., Jr. 1960. Enzymatic utilization of glucose by a basidiomycete. Fed. Proc. 19(1 part 1):27 (abstract). Pigman, W. 1957. The carbohydrates - chemistry, biochemistry, physiology. Academic Press Inc., N. Y. Roland, J. P., Z. F. Chmielewicz, B. A. Weiner, A. M. Cross, 0. P. Boening, J. V. Luck, T. J. Bardos, H. C. Reilly, K. Sugiura, C. 0. Stock, E. H. Lucas, R. U. Byerrum and J. A. Stevens. 1960. Calvacin: a new antitumor agent. Science 13221897 Sedlmayr, M. 1960. Physiological studies on Calvatia species. Ph. D. thesis. Michigan State University Sedlmayr, M., E. S. Beneke, and J. A. Stevens. 1961a. Physiological studies on Calvatia species. I. Vitamin requirements. .Mycologia 53(1):98-108 35 Sedlmayr, M., E. S. Beneke, and J. A. Stevens. 1961b. Physiological studies on Calvatia species. 11. Carbon utilization. Mycologia 53--in press Stevens, J. A. 1957. Studies of environmental factors influencing in vitro growth of basidiomycetes and their elaboration of biologically active substances. Ph. D. thesis. Michigan State University Swack, N. S. and M. 0. Phillip. 1960. Conditions affecting growth and indigotin production by strain 130 of Schizophyllum commune. Mycologia 52(h):57h-583 36 APPENDIX T Test With N1 + N -2 Degrees of Freedom 2 T. X” - i1 Sp \r‘tl;Nl) + (12-532) 2 - 2 .2 . 2 SP2 8 2x112 _. Algal +2le -_(§+)___X O N1*N2"2 RESULTS * n Significant at 5% level. -*§ = Significant at 1% level. N.S. - Not significant at either level. Carbon utilization by Basidiocarp 10960 and Spore Strain.E-9-6 based on the dry weight of the mycelium analysed statistically. 1h day 28 dgy MEDIA T value Result T value 'Result GLUCOSE -S.21 *, ** ~69.1118 *, ** CELLOBIOSE -3.56 * «16.1h8 *, ** DEXTRIN w20.7h *, ** Not Analysed GLYCOGEN ~25.189 *, ** m23.589 *, ** CONTROL -5.58 *, ** w6.27 a,‘** MEDIUM A 3.26 * Not Analysed eat GLUCOSE CELLOBIOSE DEXTRIN GLXCOGEN CONTROL MEDIUMiA w GLUCOSE CELLOBIOSE DEXTRIN GLYCOGEN CONTROL MEDIUM A 37 Carbon utilization by Basidiocarp 1369 and Spore Strain F-2-3 based on the dry weight of the mycelium analysed statistically. ale @321 T value Result T value Result -5.83 *, ** -8.577 *, *s l7.h27 *,‘** —7.296 s, ** 2.99 * -2.2h N.S. —.603h N.S. 3.83 a. -.O99l N.S. -.081 N.S. 18.262 *9 ** 5,75 *9 as Carbon utilization by Basidiocarp 995 and Spore Strain A-5-1 based on the dry weight of the mycelium analysed statistically. use 2.9.93. T value Result T value Result 1.1766 N.S. 59.50 *3 ** -.09h N.S. 2.52 N.S. h.lh7 *' 3.368 * -l9.l79 *,‘** Not Analysed ~h.917 *. ass 4.16 {- 2.99h * c.0667 N.S. ROOM U SE ORLY Illllllllll1Ill!1|”11111111111111“ ”1111111111 293107296802