‘E‘Hfi GERMl-HATW 0F BASWlOSPORES o? cmvgm WAND RELATED GENERA Thesis for 9h. Dear» of Ph D. MICHKEM‘S STATE UNIVERSWY Glenn Stuart Bulmar W60 0-169 MHWH mm M M m3 1293 107 o'fl—fl This is to certify that the thesis entitled The Germination of Baaidioepores of Salvatia. gigantea and fieIated Genera presented by Glenn StuartiBulmer' has been accepted towards fulfillment of the requirements for m—degree mm (Dept. of Botany & Plant Pathology) 5%(1 /(’/r’-’c~_ rolessor E. S. Bgne a Date ember 21 1 60 LIBRA R Y Michigan State University MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. ul lil. 7%,} Ea.l’i"..i THE GERMINATION OF BASIDIOSPORES OF CALVATIA GIGANTEA AND RELATED GENERA BY Glenn Stuart Bulmer A THESIS Submitted to the School for Advanced Graduate Studies of Midhigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1960 g /s’// 2— 0/,/ 9/ (a I ii ACKNOWLEDGEMENT S The author wishes to express his sincere thanks to Dr. E. S. Beneke for an introduction to the fungi as an undergraduate student and the guidance, counsel and encouragement so freely given throughout the author's graduate studies. Without his support these investigations would not have been possible. Sincere appreciation is also due to Drs. W. B. Drew, J. L. Lockwood, G. L. Kilgour, R. L. Kiesling, G. P. Steinbauer, and J. A. Stevens for their many helpful suggestions throughout this investigation. The writer is indebted to P. G. Coleman for photo- graphic preparations; Sigrid Heine for technical assistance and the Parke Davis Company for their supplies of chloromycetin. This investigation was supported by the Rackham Research Foundation and the National Institutes of Health for which the author is indebted. Sincere thanks are due the Mycological Society of America for presenting the author with the 1958—59 Fellowship. To my wife and our three little Lycoperdons: Scott, Gary and Cindy. THE GERMINATION OF BASIDIOSPORES OF CALVATIA GIGANTEA AND RELATED GENERA BY Glenn Stuart Bulmer AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agricultura and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology Year 1960 Approved 933*42zéfigfl g::L¢ 75% of that of controls. A ? designation indicated an incomplete test owing to the death of two or more of the test mice. Mating Experiments Two cm3 blocks of Medium A.were placed 1 cm apart on a flamed microscope slide in a sterile Petri dish containing 5 m1 of sterile water. Small mycelial transfers from cultures of g, qiqantea were inoculated on the edges of these blocks. A flamed cover slip was then placed on top of these blocks, bridging them. When completed, this apparatus was incubated at 26°C. In 2-3 weeks the mycelium from each block had grown onto the underside of the cover slip. As the hyphae approached and overlapped each other, the cover slip was removed and permanently mounted on another microscope slide. Antifungal Activity Two techniques were employed in attempts to assay antifungal activity of g, qigantea mycelium. One method tested activity of g. qigantea mycelium 16 against cultures of Glomerella cingulata (Stoneman) Spaulding and Schrenk. A 50 m1 aqueous suspension containing 1.4 x 107 .Q. cingulata conidia was added to 200 ml of cooled (41°C) 2% agar enriched with 0.5% Bacto-Peptone. These plates were then divided into three groups: (a) cultures incubated at 26°C for 12 hours before they were inoculated with g, gigantea mycelium; (b) cultures incubated for 4 days at 26°C and then inoculated with g, gigantea mycelium: (c) cultures incubated for 4 days at 26°C, then killed by placing in a desiccator containing propylene oxide. Plates were removed after 6—8 hours, covered with one layer of sterile cheesecloth and allowed to air out overnight before they were inoculated with “Q. gigantea mycelium. The centers of all plates were inoculated with 1/4" cylinders cut from agar cultures of g. qiqantea. Mycelium of Q. gigantea was also tested for antifungal activity against Candida albicans (Robin) Berkhout, Colletotrichum phomoides Saccardo (Chester) and Trichophyton mentagrophytes (Robin) Blanchard in the following manner: Petri dishes containing Medium A were inoculated with three 1/4" cylinders cut from the edges of agar cultures of g. gigantea and incubated for three days at 26°C. At this time streaks of the test organisms were made on the medium radial to the g. gigantea cylinders. Two sources of Q. gigantes mycelium were tested: (a) mycelium originally derived from immature sporophores, 17 (b) mycelium derived from spores germinated on barley extract medium. In some instances, additional materials and methods are cited in results and observations. This was done to aid the reader in interpreting the experimental work. 18 RESULTS AND OBSERVATIONS Pgeliminary Investigations to Germinate BasidiOspores There appear to be no published reports of attempts to germinate the basidiospores of g, gigantea. With this in mind, it was decided to start these investigations by using various media at different temperatures, and by pre- treatment of spores with various methods. In the first series of experiments various media at incubation temperatures of 28°C and 35°C were used (Table l). Chloromycetin was not used in media in these experiments. Experiments were terminated after 30 days incubation. This period was selected because Fries (1941) noted that 30 days was the longest period required for colonies to appear from Lycoperdon s2, spores. Media were inoculated by lightly shaking a small portion of sporophore material above the plates thus dispersing spores onto the surface of the media. Contamination by fungi and particularly bacteria was a great problem in these experiments, but a sufficient number of clean plates were obtained to evaluate the media. None of the media supported germination of basidiospores from the sporophores used (Table 1). In further germination experiments spores of g, gigantea were subjected to various pretreatments. After each treatment the Spores were kept for 24 hours in sterile TABLE 1 19 Agar media used unsuccessfully in preliminary attempts to germinate basidiospores of Q. gigantea. incubated for 30 days at 28°C or 35°C. Plates were Media 1. Steamed Puffball Extrace 2. Blended Puffball Extract 3. Soil Extract 4. Sabouraud's inoculated at 4 places with R.M.a yeast 5. Malt Extract inouclated at 4 places with R.M. yeast 6. ,Soil extract inoculated at 4 places with R.M. yeast 7. Steamed Puffball extract inoculated at 4 places with R.M. yeast 8. Potato Dextrose 9. Litman's Oxgall 10. Difco Blood Agar base Sporophore Number 961, 961, 998, 983 985. 995, 995 995, 995, 995 948, 948, 989, 989, 985, 985, 984 983, 983, 954’ 984 989, 984 971, 970 971, 970 961, 986, 984 aR.M yeast (Rhototorula muscilaginosa var. sanguinea.) 20 distilled water and examined microscopically for germination. Treated spores were streaked on Medium A without chloro- mycetin contained in Petri dishes. These diShes were incubated for 20 days and examined periodically for germina- tion. many as treated 1. Owing to contaminants it was necessary to use as 20 plates of Medium A agar for each sample of pre- spores. Pretreatments of spores are recorded below. Spores from sporophore #995 were washed with 95% ethanol for periods of one, five and ten minutes. Spores from sporophore #989 were heated for 5 minutes at the following temperatures: 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C. Spores from sporophores #995 and #986 were dipped into the following concentrations of sulphuric acid for 5 minutes: 1, 10, 25, 50%. The walls of spores from sporophore #1373 were etched in a Nasal disintegrator. Spores from sporophore #984 were incubated for 20 hours in sterile solutions of Bile salts ranging from 0.25 to 15%. Spores from sporophore #939 were alternately frozen and thawed six times over a three day period. Spores from sporophores #972, #986 were heated at 50°C, 60°C, 70°C, and 80°C for intervals of 5, 10, 15, 20, and 25 minutes. 21 8. Spores from sporophore #1373 were placed in the following glucose concentrations for 24 hours: 0.5, 1.0, 5, 10%. 9. Forty gm of a Q, gigantea sporophore (immature) was blended for 5 minutes with 500 ml of distilled water. This preparation was filtered through a Seitz apparatus. Spores from sporophore #1373 were incubated for 3 days in this preparation. 10. Spores from sporophore #1380 were incubated at 28°C for 24 hours in a 1% solution of Pancreatin extract that had been filtered through a Seitz apparatus. None of these treatments was successful in germinating the spores of g. gigantea. Germination in Liquid Cultures Barley extract broth and spores of Q, gigantea #995 were used. All flasks were incubated on a rotary shaking machine. During the first two weeks of incubation, several of the flasks contained fungal growth which was later identified as a contaminant(s). The remaining flasks were left on the shaker for several more weeks. Finally after 9 weeks incubation, mycelium growing on the original piece of spore inoculum was observed in 3 of the 6 remaining flasks. Samples of this mycelium were transferred to Medium A agar. This culture could not be identified as any previously described fungus. Since there is apparently no fungus 22 generally classified as a laboratory contaminant that requires nine weeks from spore germination to colony development, this fungus was compared with a culture obtained from the immature sporophore. Both cultures were buff-brown in color. The centers were slightly raised and growth was relatively flat. They turned the medium slightly brown and three weeks were required for these cultures to reach one-inch in diameter. The hyphae were 2-3.5 microns in diameter and lacked reproductive structures except for occasional Chlamydospores. In subsequent experiments, using spores of Q. gigantea #995 in barley extract broth, germination was observed after 7-8 weeks incubation (e.g., Plate I). The number of germinating spores calculated after eight weeks incubation indicated that less than one spore out of 20 million had germinated. An occasional spore appeared to have an appressorium. However, these structures were never observed giving rise to hyphae. Advanced stages of spore germination were never seen, as observed microscopically, once the germ tube protruded to a length of approximately five microns, it appeared to be broken off by the action of the shaking machine. Capillitium was never seen giving rise to hypha, in fact, most of these threads did not contain complete cells. From the above evidence it was decided that spores from Q. gigantea had been germinated. Later, in support of this contention, mycelium derived in a similar manner un’,.fl‘.l I I’ll EL. 23 Plate I. A germinated basidiospore of g. gigantea #995 incubated on ' shaker for 8 weeks at 26°C in barley extract broth. 24 demonstrated antifungal and tumor-retarding properties. It was of interest to determine if the spores of Q, gigantea could be germinated in broth other than barley extract. Various broth media were, therefore, investigated (Table 2). Spores from sporophore #995 were incubated for 90 days at 26°C. If no growth occurred after this period of time, it was considered that the spores would not germinate under the experimental conditions. Spores from sporophore #995 were germinated in six of the ten media, the most successful of which were the barley and puffball extracts containing chloromycetin. Of the 18 flasks containing 9, gigantea mycelium 16 had chloromycetin incorporated into the broth. The media lacking the anti— biotic were mostly contaminated with bacteria. In later experiments, employing barley extract broth, spores from sporophores of g, gigantea #1373, 1096E, 1369, and 1389: L, pyriforme #1374: L. gemmatum #1383, 1403: and .E- echinatum Pers. #1384 were gernimated. No germination was observed using spores of g, gigantea #1370, 1376, 1380: _L_. gemmatum #1385: or _I_.. pyriforme #1386. These results indicated that spores from some 9. gigantea and Lycoperdon EB: sporophores could be germinated in barley extract broth. Spore Germination Results from Q, gigantea #995 grown in TABLE 2 Various Media 25 Chloromycetin a b Broth Incorporated FFF M.P. (l) Sabouraud's yes 2/5 1 plus one day autoclaved no 1/5 0 R.M. Yeast (2) Sabouraud‘s yes 2/5 0 plus 3 day autoclaved no 2/5 1 R.M. yeast (3) Sabouraud's plus yes 4/5 0 living R.M. yeast no 4/5 0 (4) Autoclaved yes 4/5 0 Soil Extract no 4/5 0 (5) Seitz Filtered yes 4/5 0 Soil Extract no 4/5 0 (6) Puffball Extract, yes 3/5 3 Autoclaved no 4/5 0 (7) Puffball Extract, yes 3/5 3 Seitz Filtered no 4/5 0 (8) Medium A yes 8/10 0 no 9/10 0 (9) Medium A yes 3/5 2 plus Pancreatin no 1/5 1 (10) Barley yes 9/10 8 Extract no 7/10 0 aRatio of flasks free from fungal contamination to original number of f1a9ks. b g, gigantea. Number of flasks containing mycelial pellets of 26 C. gigantea Spore Germination in Agar Medium Despite the fact that spores of g, gigantea had been germinated in broth, it was considered more desirable to germinate them in solid medium so that conditions necessary for germination could be more accurately defined and the chances of securing monosporous cultures might be increased. Fries' (1941) technique which was successful in germinating spores of Lycoperdon s2, was modified slightly for these experiments. Spores of an overwintered puffball (#1390A) were used. In this experiment 40 Petri dishes containing malt extract agar (without Chloromycetin) were seeded With spores, and 20 of them were inoculated at 4 equidistant spots with R.M. yeast. No R.M. yeast was inoculated into the remaining plates. Each dish contained 36 x 106 spores and was incubated at 28°C. Periodically, these plates were examined for evidences of spore germination. After 4 weeks, an average of 36 small white colonies (l colony per million spores inoculated) were seen growing in each Petri dish inoculated with the yeast. One hundred and eight colonies (3 colonies per million spores) were counted after 7 weeks' incubation. This number never increased, in spite of an additional 4 weeks incubation. Mycelium.was transferred from several of these cultures and placed on slants of Medium A agar. Two weeks later these cultures exhibited characteristics similar to the cultures which were derived from Q. gigantea 27 spore germination in barley extract broth (pp. 21. 22). It was decided that these were colonies of Q, gigantea. No colonies appeared in any of the plates that had not been inoculated with the yeast. Plate II shows a typical Petri dish containing R.M. yeast and §,'gigantea colonies. In subsequent experiments, using spores from a minimum of eight sporophores, it was observed that Q. gigantea colonies appeared only in malt extract agar that had been inoculated with the R.M. yeast. -Apparently this living yeast is necessary for germination to occur in solid media. Owing to the fact that these colonies appeared to originate $2 the medium it was not possible to determine if they were derived fromlg. gigantea spores. This difficulty was overcome by seeding 44 x 106 spores from #1390AF evenly on the surface of malt extract agar in Petri dishes. One- half of this seeded surface was covered with an additional layer of malt extract agar and inoculated with the R.M. yeast and incubated at 28°C. After 597 weeks the added section of medium from some of these dishes was removed and the germination of g. gigantea spores was observed micro- scopically (Plates III, IV, V). The remaining dishes contained 9, gigantea colonies after an additional 2 weeks' incubation. This was considered evidence that spores of Q. gigantea had been germinated in a solid medium. It was observed in this experiment that the medium which had spores uncovered supported an average of 8 colonies 28 Plate II. Colonies resulting from the germinated spores of g. gigantea in malt extract agar incubated at 260C for 9 weeks. The larger orange colonies are the R.M. yeast. Plate III. Germinated spores of g, o gigantea incubated at 26 C for 5 weeks in malt extract agar inoculated with the R.M. yeast. 29 it ‘. u "x Plate IV. Germinated spore of Q. gigantea incubated at 26°C for 6 weeks in malt extract agar inoculated with the R.M. yeast. 30 Plate V. Colony of g, gigantea derived from spore germination in malt extract agar inoculated with the R.M. yeast and incubated at 26°C for 7 weeks. Note numerous ungerminated spores and capillitium. 31 32 and the section that was covered had 65 colonies (Plate VI). The germination per million spores inoculated was 0.4 and 2.9 respectively (Plate VI). Throughout the ensuing months, the germination of spores from at least 24 different sporophores verified the observation that more spores of IQ. gigantea germinate when covered with a layer of medium. This rate was similar to that obtained when spores were seeded into the malt extract medium. Possibly spores from Q. gigantea require microaerobic conditions for germination. However, exceptions to this would be the spores that germinated on the agar surface and those in heavily aerated broths (i.e., barley extract). In concurrent experiments when spores from #1390AF were seeded into malt extract medium and inoculated with R.M. yeast the germination rate was 2.8 per million. Since 2.9 spores per million germinated when dispensed on the medium covered with additional agar medium, it was concluded that seeding spores at 42°C did not effect germination results. The Effect of Vitamins on Germination Fries (1941) attempted to replace the living yeast with various vitamins in his studies on Lycoperdon s2. spore germination. Although he failed, Cochrane (1958) mentioned that this co-culture with a yeast is suggestive of a vitamin effect. The folldwing vitamins were investigated. In every Plate VI. 33 Colonies resulting from germinated spores of g. gigantea #1390AF incubated at 26°C for 8 weeks. Spores on the right side of the Petri dish were covered with malt extract agar while spores on the left side were not covered. 34 case Petri dishes containing malt extract agar seeded with .9. gigantea spores and inoculated with R.M. yeast were a control. In these experiments spores from either #1400C or #1390AF were used. 1. Biotin: Concentrations of 5, 10, and 15 pg/l were attained in the following media: Medium A lacking yeast extract. Medium A lacking yeast extract and peptone, malt extract agar, and Magenmaltz agar (Fries. 1943). In preliminary experiments, without Chloromycetin, colonies which strongly resembled Q. gigantea appeared in some of the above. These were subsequently always considered to be 9. gigantea . colonies for reasons outlined previously. However, close examination revealed there were some bacteria present. In subsequent experiments, when Chloromycetin was used to inhibit bacteria, none appeared. 2. Thiamine: Concentrations of 75, 100 and 125‘pg/l‘were attained in the following media: malt extract agar, Magenmaltz agar, Medium A lacking yeast extract, and Medium A lacking yeast extract and peptone. In one instance 10 concentrations of thiamine (from 50 up to SOO‘ug/l) were attained in malt extract agar. Ithhese experiments no colonies of Q. gigantea appeared except in one of 10 plates containing malt extract. 350lug/l thiamine, and spores of Q. gigantea #1400C. This plate did not contain contaminating bacteria.' 3. Pyridoxine: Concentrations of 50, 100, and 200~pg/1 were attained in Medium A lacking peptone and yeast extract. 35 No colonies of Q. gigantea were observed. 4. Inositol: Concentrations of 50, 100 and 200.pg/1 were attained in Medium A lacking yeast extract and peptone. No colonies of Q, gigantea were observed. 5. Riboflavin: Concentrations of 10, 20, 40 pg/l were attained in malt extract agar. No colonies of Q. gigantea were observed. Attempts to replace the R.M. yeast with various vitamins were not successful. This should not be taken as evidence that a vitamin is not important in the spore germination of Q. gigantea. For example, the combination of two or more vitamins could be necessary. It is also possible that the yeast masks or neutralizes germination inhibitor(s). Spore germination always occurred in malt extract medium which was inoculated with R.M. yeast. In the biotin substitution experiments, a contaminating bacterium appeared to stimulate germination. To investigate this further, 30 plates of malt extract agar (minus Chloromycetin) were seeded with spores of Q. gigantea #1400C. Ten of these plates were inoculated with R.M. yeast in the usual manner. Another 10 plates were inoculated at 4 places with the unknown bacterium. The remaining plates were streaked at 2 places with this bacterium. After 7 weeks the control plates had an average of 7.5 colonies/million spores inoculated, the plates containing the 4 colonies of the bacterium had 4.6 colonies/million spores inoculated and the 36 streaked plates had 7.5 colonies/million spores inoculated. This demonstrated that this bacterium can quantitatively replace the function of the R.M. yeast. Effect of Chloromycetin Early attempts to germinate the spores of g, gigantea were continually hampered by bacterial contamination probably from the gleba. Swartz (1929) noticed this and reported on the microflora in the basidiocarp of various puffballs. It was decided that a bacteriostatic compound must be incorporated into the media. Chloromycetin was selected since it was readily available and maintained its integrity throughout autoclaving. After 6 weeks in an exploratory experiment, with spores from Q. gigantea #1390A, dishes without Chloromycetin contained an average of 31 colonies per million spores inoculated, yet the medium containing 100 ppm of bacterial inhibitor had only 18 colonies per million of g. gigantea. Six concentrations of Chloromycetin (1.0, 2.5, 5.0, 10.0, 15.0 and 30.0 ppm per liter) were tested using spores of #1397A. Plates were incubated 7 weeks at 26°C. The results are shown in Figure 1. The medium lacking Chloromycetin contained 16.5 colonies per dish. With 1, 2.5, and 5 ppm of Chloromycetin an average of 27, 20, and 18 respectively were observed in each dish. As the concentration increased further the number 37 H\Emm CH cflpmumfiouoHno m0 cofiumuucmocoo mm om ma OH m o _ P! b ——.p... _ 1 _ — _J O 1... cm «nmmae mwucmoflc aw mo coflumcflfiumw whomm :0 com sameMEouoHfio pommmm one H wnsmflm qsrq txqeg/seruotoa go JeqmnN ebexenv 38 of colonies per plate gradually fell off until, at 30 ppm of chloromycetin. only 12.5 colonies per dish were recorded. A concentration of 6 ppm of Chloromycetin was selected for use in future experiments based on two considerations: (1) at this level the average number of colonies per plate (or germ/million) was similar to the medium containing no Chloromycetin: (2) this concentration was sufficient to inhibit most bacteria. 1 It was interesting to note that the greatest number of colonies per dish was obtained at a concentration of 1 ppm chloromycetin. This increase might well be an oligodynamic effect. This term, as used by Foster (1949) states that ". . . strong poisons actually‘may be stimulatory if furnished in subinhibitory doses." Plating of portions of the medium from these plates onto Sabouraud's agar Showed no evidence of bacteria. Thus the increased number of colonies was not due to bacterial contamination in these dishes. Manometric studies were undertaken in an attempt to ascertain the extent of oxygen uptake depression by a concentration of 6 ppm of Chloromycetin onspores of g. 'gigantea. Figure 2 records the effect of various doses of Chloromycetin on the oxygen uptake by g, gigantea spores #1390AF. All readings were taken 2 1/2 hours after the start of the Warburg. Concentrations of Chloromycetin up to 2.5 ppm did not 39 H\Emm CH GHDCUMEOHOHno mo cofiumupcwucoo Om mm ON ma OH m «l _ P L— . - m --m E i _ ..’-1' sexods UOTIITH euo/exeqdn ZOIfi seconds mmucmmam no. mo monomm >3 meDQD cwmwxo :0 6mm cfluwomfioonSO Dommmm 035 N musmam 40 appreciably effect the oxygen uptake by spores of g, gigantea #1390AF. At a level of 3.7 ppm the uptake was slightly depressed. At 5.0 and 7.5 ppm this depression was more extensive but had leveled off. Concentrations above 7.5 ppm resulted in a more extensive reducing of the oxygen uptake. In all instances the negligible number of bacteria in each Warburg flask and the ccmplete lack of a lag phase demonstrated that the recorded oxygen uptake was not due to bacteria. These studies indicated that a dosage of 6 ppm Chloromycetin does reduce oxygen uptake but not nearly as much as a concentration of 15 ppm. This evidence was taken as support for not using Chloromycetin in excess of 7.5 ppm for spore germination studies. The increased reduction in oxygen uptake beyond this dosage might reflect some extensive metabolic changes. Effect of C._gigantea Mycelium It was previously found that all colonies of Q. gigantea appeared in the first 8 weeks following seeding. However, if the Q. gigantea mycelium was eventually stimulating further spore germinations this could be difficult to determine if the newly developed germinated colonies from germinated spores occurred adjacent to or under previous Q. gigantea colonies. In an attempt to ascertain if g. gigantea mycelium 41 could stimulate germination of Q. gigantea spores (#1390AF), 30 Petri dishes containing malt extract medium were prepared. Ten of these plates were inoculated with R.M. yeast and the remainder were inoculated with cultures of Q. gigantea which had been obtained from a former germination experiment using spores from sporophore #1400C. After 2 months incuba- tion at 26°C the plates with yeast contained 13 colonies/million spores. The plates containing the g, gigantea culture contained no colonies even after 4 months incubation. Thus it seems doubtful that mycelium of g, gigantea can stimulate spore germination in malt extract agar. Other Factors Possibly Affecting Germination Throughout the fall and early part of the winter of 1959 the spores from various sporophores of g, gigantea were tested for germination. The results of these germination experiments are recorded in Table 3 (28°C incubation). This data indicated that several factors probably affected spore germination: l. The concentration of spores per Petri dish. The number of spores per million that germinated from Q. gigantea sporophores #1391A, and #1389 appeared to vary With the spore concentration. 2. .The sphorophore. Spores from certain sporophores (#1096E, 1370, 1376 and 1400A) did not germinate in malt extract agar containing R.M. yeast. m.mm o.ona n m mm Nova 0 o w.a m woova o o H.H m mooea Q o o n.h a moova e.v 6.6H 6.m «\H aamma o.~m mo.mmm a.ma a\H = o.mm mo.m~m m.oa e\H . o.oma no.666H 6.6 a o.~m 6.6mm m.¢ «\H «Hams 6.NH o.ea m.m N\H as = m.mm o.ooa m.a N\H NH . H.vm o.oma h.H «a n.m o.m¢ m.ma m domma v.0h o.oma h.m ea ¢.m mo.om¢ o.oma H momma m.o m.v m.m oma : o o 0.0 oma = o o H.o oma mama m.H h.ma m.m h omma H.o m.o m.m om mnma m.m o.¢m o.va m mhma o o h.NH Ha ohma n.mm o.mga ¢.m mm mhma o o o.m~ m = o o m.o an o o m.m m osma o o m.a am 0 o m.hm m mmmoa S\Eumm Qmflp nmflp 00m .wnuE ca 2\Ehwm Swap zmflp Mom .mQuE CH muonm 000mm m\HOO 0H X 0:00 0mmuoum whomm m\HOU OH x 0200 commoym Iouomm 0 000mm 0 000mm Doom am UwumfldocH 00mm pm UmumnsucH Aoomm .>mv .meu EOOH um cmuoum m0H0£mouomm .< .mmudumumm80u usmummmap mosey um pomegm 0H03 £0H£3 mmuoamonomm mmucmmflm am Hmu0>0m mcflms mcoflumcflfinwm 000mm pmumfimuum mo muaflmmm m MAM m.N h o o n.m N mamma m.mm o.ooa N.H 0H o.MH o.ma o.H m : h.¢m 0.0m m.N 0H H.m 0.0H H.H h z o o m.Nm m = o o o.N v : 0.0m mo.wmm H.0H N mmomma E\EH00 Qmflp nmflp Mom .mnuE CH 2\Ehwm Swap Swap 00m .mfluE CH 000:& 000mm m\H00 OH x 0:00 mmmuoum whomm m\H00 OH x 0c00 mmmnoum Iouomm 0 000mm 0 whomm comm um embmnsucH comm 0m cwbmnsucH UONH um Uwuoum mononmouomm .m ooschucoonum mamas 44 3. Storage conditions. Spores from half a puffball stored at room temperature (#1390A) gave a germination rate of 2.7 per million. Yet the other half of this sporophore (#1390AF) stored for the same length of time at 12°C had a germination rate of 30.8 spores per million. As the storage time increased the number of spores from sporophore #1390AF which germinated decreased. Thus an experiment was designed to investigate the effects of spore concentrations and temperatures of storage and incubation on spore germination. Three sporophores of ‘Q. gigantea all collected from the same location and on the same day were selected for this purpose. The dried peridia and dark gleba of these sporophores indicated that all of them were mature. One sporophore (#14008) was stored at 26°C, a second (#1400F) was stored at 12°C and the third puffball (#1400C) was kept at -l8°C. These 3 sporophores were stored at these temperatures for 2 months before the germination experiment. Six spore concentrations ranging approximately from 1.0 to 20.0 X 106 spores per ml were prepared from each of the three sporophores and seeded in malt extract agar which was inoculated with R.M. yeast. Plates were incubated at 20, 24, 28, and 32°C. Effect of Incubation Temperature The germination results with spores of sporophores 45 #1400C. 1400F, and 14003 at 20, 24, 28 and 32°C incubation are recorded in Table 4. Very few spores from the latter two sporophores germinated. The effect of incubation tempera- tures on spore germination of Q. gigantea #1400C indicates that the incubation temperature of 24°C resulted in the greatest germination per million. Fewer spores germinated at 20 and 28°C incubation. No germination was recorded at 32°C. To more precisely define the incubation temperature for spore germination (#1400C) an additional experiment was set up in which plates were incubated at 24, 26, and 28°C. In this experiment a spore concentration of 4.7 X 106 per dish was seeded into malt extract agar and inoculated with R.M. yeast. After 8 weeks incubation at 24, 26, and 28°C respectively, germination rates were 3.6, 3.8, and 1.4 per million. These results tended to support those of the previous experiment and suggest that 24-26°C is the most suitable for germination of spores from Q. gigantea #1400C (Fig. 3). Effect of Spore Concentration The effect 6 spore concentrations from puffballs stored at 3 different temperatures had on germination is recorded in Table 4. Only 1 spore from sporophore #14008 germinated out of the ten replicate plates seeded with 2.5 X 106 spores 46 coaumcflemucou ou mGHBO Umuumomacll 0 Q oumHm 009 mwflcoaoo wmmuw>0|lm\om COHHHHE mom COHDMCHfiummIIz\0 0 0 0 0 0 0 0 0 0.0a 0 0 0 0 0 0 0 0 N.0 A000N .>0 0 0 0 0 0 0 0 0 0.0 .mfiwu .Em 0 0 0 0 0 0 00.0 0H.0 0.N um Umuogmv 0 0 0 0 0 0 0 0 0.N 0 0 0 0 0 0 0 0 0.H m000a 0 0 No.0 05.0 HH.0 00.0 H0.0 00.0 0.0m 0 0 No.0 60.0 HH.0 00.N 00.0 00.0 0.0a 0 0 0N.0 mh.0 0 0 0 0 h.m 00NH 0 0 0 0 0 0 0 0 m.m um Umuoumv 0 0 0 0 0 0 0 0 0.N 0 0 0 0 0 0 0 0 m.H @000H 0 0 00.0 00.H 0H.0 00.N 0N.0 00.N 0.0N 0 0 0N.0 0N.m mh.H 05.0H Hm.0 00.0 N.HH 0 0 0m.0 0N.N m>.H 00.MH 00.0 00.m 0.5 oomHI 0 0 mm.H 00.0 0m.m 00.0H mn.0 00.m 0.0 um 00HODmV 0 0 0 0 0 0 0 0 0.N 0 0 0 0 00.H 00.0 0H.0 00.0 H.N 0000a OH x 2\0 m\0 S\0 m\0 S\0 Q\0 QS\0 mm\0 .ocow whomm wuoflmouomm 00Nm 000N 000N 000N mmnsumuwmfima QOHDMQSUCH .AmSDQOE N 000 000000 Haflv .m0HOSQOH0mm mwucmmflm .M 00030 E000 mmhomm mo coflumcflfiuwm so 00: mmusumnmmfimu COHUMQSUCH UQM .wmusumuwmfimu mmmwouw .COHumHucwocoo 000mm uomwww 05B 0 mqmfie 47 vamumflucmo mmwnmmov mwusumuwmfiwe COHDmQSUQH Nm Om mm 0N 0N NN ON _ _ _ _ _ _ iii! \7 _ \f _ O UOITIIW/UOTQEUINIBO 00Hx .UQOU whomm M$\\\\.\.\NV fl L 0.0 0000a mmwgmmflm am we Goflumcflfiumw 0Homm :0 00m wusumuwmfimfi COHDmQSUCH uommmm 039 m musmfim 48 and incubated at 20°C. In the case of spores from the sporophore #1400F germination occurred only when the spore concentration per dish was 3.7 X 106 spores or greater. The greatest number of Spores germinated from sporophore #1400C which was stored at -l8°C. Figure 4 shows the effect on germination of spore concentration. At 20, 24, or 28°C incubation the greatest number of spores from sporophore #1400C germinated when approximately 5 million spores were present in each Petri dish. As the spore concentration increased above this amount the germination rate decreased until it was negligible at a concentration of 26.0 X 106 spores per dish. This data suggests that increased spore concentrations inhibit spore germination. However, since germination per million was determined by the number of visible colonies, it is possible that at higher spOre concentra- tions small, invisible colonies might be produced (although microscopic examination of such plates did not reveal any minute colonies). The fact that below a spore concentration of 4.5 X 106 spores per dish the number of spores that germinated decreased was studied further 7 months later. At that time, 40 plates of malt extract agar were seeded with spores (2.0 X103) from sporophore #1400C. Ten additional dishes, each containing 5.2 X 106 spores, were prepared. All plates were inoculated with R.M. yeast and incubated at 26°C. The results showed 49 AQmHD Hup0m H000 00Hx 00Hu000000000 00000 mN ON 0H 0H m o im.o Io; Tm.H D s no.m -m.m oomm u I oovm n D ro.m ooom n O 0H500H0QE0B GOHDMQDUCH gl0.0 0000H# 000000H0 aw EOHM m0nomm mo coHumcHEH00 so 00mmcoHumnucmocoo 0H0mm u00mmm 0:8 0 0H50Hh uorttrw/uorqeurmles 50 that 14.4 spores per million germinated in the control dishes containing 5.2 X 106 spores. In the dishes each containing 2.0 X 103 spores, only 1 colony was observed. Since a total of 160 X 103 spores had been tested in these plates. the germination rate was approximately 6 spores per million. This data supported the previous experiment showing at least for spores from Q. gigantea #1400C. there was a decrease in germination as the spore concentration decreased below 4.6 X 10° per dish. In subsequent germination experiments using spores from 15 sporophores stored from 7 to 33 months at three temperatures (Table 3, 26°C) the germination rate also was affected by spore concentration. With spores from sporophore #1400C, after 9 months storage at —18°C a greater germination rate was observed at a spore concentration of 5.2 X 106 than at 3.5 X 10°. However, the greatest number of spores to germinate from other sporophores (germinated at similar storage temperatures and time intervals) was when the concentration was between 1 and 3 million spores per Petri dish. Furthermore, the "ideal" concentration varies from one sporophore to another. This number may even change as the germination rate increases within a specific sporophore. For the most part, concentrations of 1-5 million spores per dish results in the best germination rate. 51 Effect of Storage Temperature The effect of the three storage temperatures on the germination of 3 puffballs is compiled in Table 4. V Only one spore germinated from sporophore #1400B which had been stored at room temperature for 2 months. The greatest number of spores that germinated from sporophore #1400F which had been stored at 12°C for 2 months was 0.2 per million inoculated. For the most part a germination rate of < 0.1 spores per million was recorded from this sporophore. Of the spores stored at -18°C for 2 months the greatest germination per million was 3.39 spores. By far, more spores stored at -18°C germinated than when stored at 12°C or room temperature. Seven to 10 months later it was decided to retest spore germination from some of the previously tested sporophores and also to attempt spore germination of other sporophores that had been stored at various temperatures for several months. Data from these experiments is recorded in Table 3 (26°C). Spore germination ability was maintained for spores stored at 12°C and -18°C. One sporophore had been divided into two pieces when collected. One half (#1390A) was stored at room temperature and from 12.6 to 94.1 spores germinated per million. The other half of this sporophore (#1390AF) was stored at 12°C, and from 34.7 to 83.3 spores germinated per million. However, some spores 52 stored at room temperature germinated well. In most cases it appears that storage at either 12°C or -l8°C results in the greatest number of spores germinated. Effect of Storage Intervals The spores from some sporophores were germinated after varying lenghts of storage (Table 3). When spores from sporophore #1390AF were stored an additional 6—7 months the number of spores that germinated increased 3 to 8 fold. Spores from #1391B did not germinate after 2 months storage yet 280.0 spores per million germinated after 7 months' storage. Additional storage periods also increased the number of spores that germinate from sporophores #1390A and #1391A. These data indicate that for many of the sporophores tested, the spores require an after-ripening period. The extent of storage time required for the greatest spore germination to occur has not as yet been ascertained; however, 8-14 months appears to be optimal for spores from some of the sporophores although this varies from one sporophore to another. All the data relating to spore concentration and storage conditions indicates that individual sporophores, or groups of sporophores, may vary in their specific require- ments for germination. It was interesting to note that Snell (1922) stated in reference to wood destroying 53 .Basidiomycetes, "it is possible that spores collected from different fruit bodies of the same species of different degrees of maturity, from different climatic conditions and under different conditions of casting might give varying per cent values (in germination)." Other Spore Storage Methods Two attempts were made to determine if spores of Q, gigantea could be stored other than in the intact sporophore. (a) Plates of malt extract agar seeded with spores from sporophore #1400C (concentration of 4.8 X 106 per dish) were divided into 4 groups: (1) inoculated with R.M. yeast 24 hours later; (2) stored at -18°C for 17 days, removed, and inoculated with R.M. yeast; (3) stored at -18°C for 24 days, removed and inoculated with R.M. yeast: (4) stored at -18°C for 38 days, removed and inoculated with R.M. yeast. The only spores to germinate were those which had not been stored at -18°C in malt extract agar. (b) Aliquots of a spore suspension (#1400C at spore concentration of 3.5 per ml) containing 20 ppm Chloromycetin were stored in sterile polyethylene bottles at 26, 10, or -18°C. At intervals of 5 and 7 months, samples were removed from all bottles and tested for germination in malt extract agar plus R.M. yeast. Only in one instance did germination occur. After 5 months storage at -l8°C only 1 spore out of 5 million germinated: moreover, bacterial contamination was 54 extensive in all plates. Both of the above techniques failed as a method of storing #1400C spores. Effect of Inverted Petri Dishes In some experiments a few Petri dishes were inverted. When the number of colonies in these plates was eventually determined the germination was slightly less than in the upright plates. In a more controlled approach to this problem, 20 Petri dishes were incubated inverted and 20 upright. All these plates were seeded with spores from sporophore #1400C (stored at -l8OC for 9 months) at a concentration of 3.5 X 106 spores per dish. After 8 weeks incubation the upright plates had 20.0 colonies per million of g. gigantea as opposed to 15.0 per million in the inverted Petri dishes. The reason for this depression of germination is not understood. Manometric Studies Throughout these germination investigations the author attempted to find some quick method by which germina- tion potential of the spores in a given sporophore could be determined. Since dehydrogenase activity could not be detected with a 1% solution of 2, 3, 5-triphenyltetrazolium Chloride, several attempts were made to correlate oxygen uptake by spores with germination rate. 55 In a series of three separate experiments an RQ of 0.5 was obtained for spores from g. gigantea #1400C. This figure was similar when calculated from the total number of spores per flask or when converted to one billion spores. An R0 of this magnitude indicates that the spores might be metabolizing lipid while in the dormant state. In several studies the endogenous oxygen uptake by various spores fluctuated between 30 and 65 micro liters per one billion spores after 2 1/2 hours. There appeared to be no correlation between this and the germination ability of spores from a specific sporophore. Attempts to increase the oxygen uptake by additions of 10 concentrations (0.001 to 2.0%) of glucose or succinate gave erratic data. Studies were undertaken comparing oxygen uptake by spores with spores autoclaved 20 minutes at 121°C and 15 pounds pressure. Autoclaved spores recorded an oxygen consumption of 25—35 micro liters per 1 billion spores; however, spores treated in 40% formalin for one hour did not take up oxygen. In all these studies 2 ppm of Chloromycetin was used, and since no lag phase was observed it was decided that the erratic data was not a result of bacterial contamination. Sexuality of C. gigantea The mycelium of g. gigantea which produces the tumor- 56 retarding agent(s) was originally derived from the tertiary mycelium in the gleba of immature sporophores (Stevens, 1957). When spore germination in liquid media was achieved it became important to know whether or not the mycelium arising from spore germinations was of the primary or secondary type. Should this mycelium be primary then matings would be necessary to produce secondary mycelium. If, however, the secondary mycelium were present then perhaps strahs were already on hand that could produce the oncostatic principle(s). These questions became one of basic sexuality, e.g.. is g, gigantea a "homothallic" or a "heterothallic" fungus.2 Whitehouse (1949b) indicated that of the 230 species of Hymenomycetes and Gasteromycetes investigated only 10% were homothallic. He also reported that none of the species from the Lycoperdales had been investigated in this respect. Bessey (1950), in reference to the Gasteromyceteae spores. said, "These are almost always at first uninuCleate but in. most of the few cases studied they early become binucleate by the division of the original nucleus. Whether such binucleate spores give rise to the dicaryon phase of mycelium is not 'proved, but seems probable." Bessey believed that except for karyogamy in the young basidium sexual reproduction is practically unknown in these fungi. 2A homothallic fungus is capable of completing its life cycle from a single spore. Karyogamy is alternated with meiosis without the necessity of plasmogamy between mycelia derived from the germination of an other(s) spore. "Hetero— thallism," unlike homothallism, requires plasogamy between mycelia derived from the spore germinations of two spores of opposite sexes. 57 Fries believed that the Lycoperdon EE- which he had investigated from 1940—1943 were homothallic although he did not publish his opinions owing to the 1aCk of positive proof (personal communication, 1959). A series of experiments was conducted in attempts to clarify this question of sexuality. As soon as spore germination of g, gigantea was accomplished in broth on shakers an additional 40 flasks were inoculated with spore material from sporophore #995 and placed on the reciprocal shaker for 8 weeks. Isolates were taken from over 200 of the mycelial pellets and inoculated into slants of Medium A. Cultures gave slight variations of color and growth characteristics. Some were dark brownand others were pale tan. Between these 2 extremes there existed 2 or 3 color intermediates. Some of the cultures grew in a more heaped up fashion in the center. Similar color variations were observed later in cultures derived from spore germinations in malt extract‘ agar. Plate VII shows the variations. Over a three year period Stevens (personal communication) removed mycelial isolates from approximately 1000 immature lg. gigantea sporophores. These exhibited at least 8 distinct growth patterns including color and morpholigical variations. From each fruit body he removed mycelium from 5 different areas of the gleba. The resulting cultures from a single sporophore were almost always uniform in color-- only rarely did variations occur. 58 Plate VII. Cultures grown for 3 weeks on slants of medium A. Note color variations. These cultures were derived by germinating the spores of g. gigantea #1390A in malt extract agar inoculated with the R.M. yeast. .4423 Eli?“ 59 Comparing the uniformity of the colonies derived from immature sporophores with the variations observed in cultures from spore germinations suggests that colonies derived from germinated spores could be different. Attempts were made to mate cultures derived from spore germination of sporophore #995 in barley extract broth. Ninety-five combinations of the variously colored cultures were used. In only one instance was plasmogamy observed between two isolates. In this case no different appearing mycelium was observed as a result of this union. This was considered to be plasmogamy at the somatic level--not as a sign of heterothallism. In the other cases, where plasmogamy was not observed. the myCelia seldom grew together extensively. (a) Fruiting Attempts Several attempts to fruit Q. gigantea were made with cultures obtained by germinating the spores in barley broth. Three media (all containing 50 ppm Chloromycetin) were tested in the investigation: medium A agar, malt extract agar and medium A broth. Seventy-five and 100 ml amounts of these solid media were poured into 250 and 500 ml Erlenmyer flasks respectively. In several instances mycelium from only one culture was inoculated into each flaSk. On other occasions as many as eight transfers from spores that had been germinated from different sporophores were placed into one flaak. In all cases, after 8 months incubation at 26°C 60 no signs of fruiting could be observed. These cultures had heaped up somewhat at the original point of transfer. The cultures from spore germinations of different sporophores did not completely grow together. As each culture approached the other it appeared to slow down in growth, possibly as a result of nutritional competition, or inhibitory metabolic products. Mycelium from spore germinations in barley extract broth of four different g, gigantea sporophores was inoculated into a 6 liter flask containing 5 inches of sterilized soil which had been collected from the exact location where aIQ. gigantea sporophore had grown the previous year. Delivery tubes were inserted into this large flask in such a manner that Medium A broth could be siphoned into it or drawn off for disposal. This semi-constant-flow apparatus was maintained at room temperature (av. 26°C). The cultures of g, gigantea grew vigorously in the soil. Very little mycelium remained on top of the soil but large amounts of mycelium grew down to the bottom of the flask. At 2 week intervals approximately 100 ml of Medium A broth was flushed into the flaSk to main- tain a 1/2 inch level in the bottom of the flask. Each month all the broth was drawn off and fresh broth was added. In these conditions pure cultures of g, gigantea were grown for 8 months. No signs of fruiting were seen. No fruiting experiments were made using Q. gigantea mycelium derived from the spore germinations in malt extract 61 agar. However, on occasion, mycelium from single cultures were transferred into plates containing malt extract agar. After 2, and 4 months incubation (26°C) a great many Chlamydospores were observed in most cultures. In some instances these formations were club shaped (appearing similar to basidia found in the gleba of g, gigantea) but for the most part they were rounded to oval (Plate VIII). Occasionally swellings at the hyphal tips were seen. At first they . were taken for young microconidia (or oidia?). However, on further examination septations could never be seen between this swelling and the hypha proper. These were thought to be young terminal Chlamydospores (Plate VIII).' (b) Nuclear Condition Preliminary attempts to stain the nuclei of g, gigantea mycelium resulting from spore germination were unsatisfactory. Some of the stains tried were aceto-carmine, aceto-orcein. giemsa and haemotoxylin. Basidiospores and mycelium (from spore germination in malt extract agar plus R.M. yeast) treated with the Fuelgen method gave excellent nuclear staining (confirmed by G. B. Wilson, Mdchigan State University). The spores of g, gigantea appear to be binucleate (Plate IXa). Each nucleus was less than 1 micron in diameter. The mycelia appear to be binucleate (Plate IXb) or possibly multinucleate. Owing to the fineness of the mycelium. its interwoven nature and the distance between septa it was w‘ .1 ' Plate VIII. Chlamydospores in mycelium which was derived from germinated spores of g. gigantea. 63 PLATE IXa. Dikaryotic basidiospores of Q. gigantea. PLATE IXb. Nuclei in hyphae of g. gigantea the mycelium of which was derived from spore germination. 64 difficult to find complete cells. However, two nuclei were associated quite close together and it appeared that the cells were binucleate. This data suggests that the secondary mycelium is derived from germinating spores. Antifungal Properties of C. gigantea The results of evaluating strains of g, gigantea for antifungal activity by inoculating Glomerella cultures are shown in Table 5. When grown on killed cultures of g. cingulata, no evidence of antifungal activity was observed with any of the Calvatia strains tested. In the case of killed cultures, antifungal activity was determined by measuring the lytic zone. Antifungal activity was demonstrated by Calvatia strains when inoculated onto the surface of Glomerella which had been growing for 12 hours (Plate X). In Table 5 strains isolated from immature sporophores of Calvatia, #995 to #1369, showed greater antifungal activity against Glomerella mycelium than strains that developed from germination of Q. gigantea #995 spores (numbers A1-2 to A15-8). When strains of Q..gigantea were placed on plates of Glomerella grown for 4 days, only 3 of these had produced zones of inhibited growth, of which Ad5—8 was the most effective. When these zones were tested with pH indicator paper it was evident that this phenomenon is not a function of hydrogen ion concentration. When attempts were made to reproduce these results 4 .85 :H 0:0N >HH>H000 H00:SMHH:0 mo msH0mu 0000H0:H 00::0Hmm 0 0H 0 0 mlde 6 m o mus: 0 0 0 Nim: m n 0 NIH¢ 0 0H 0 0hMH 0 0H 0 HFMH 0 MN 0 m0MH 0 m 0 m0MH 0 0H 0 M000H 0 ON 0 000 mam: m when m mam: m 0000H500:H 00u0H500:H 0000950:H 0:0 m>m0 0 How 0:0 muse: NH 0:0 m>m0 0 H0000 MMMMWMMWm.m :3000 mHH0H0E0H0 How :30Hm 0HH0H0E0H0 00HHHx 0HH0H0EOH0 . 0000H50:H0 0HH0H0E0H0 mo EDHH00>E um:Hmmm 000:0mHm .w mo >DH>Huom H00:smHu:< m mAmQ .000N pm 0066Q50:H 600656060QE06 6:060MHH0 um mHm>60u:H 0EHu msoH6m> 600 006060 0603 £0H£3 60H00mm 66660050 0 806m 0060mm mo :OHum:HE600 h mflm00 06500600509 EOOMIIBmU .:0H00:HE00:00 00 0:H30 0006000H0 0000H0 Q .00065000:H 00:500 0:0H00 000E 0000H0 000 :H £0306060>0 0>Hm:00x00 6 66 6 66 66.6 66 6 6.6 66 6.6 66 66.6 66 6\6 6.6 66 6.6 66 66.6 66 6 6.6 66 6.6 6 66.6 666 6 33% 6.66 66 6.6 66 6.6 66: 6 6.6 66 6.6 66 6.6 66: 6 6 66 6 66 6.66 66' 6 6 66 6 66 6.6 66 6 6.66 66 6.6 66 6.6 66 6 6 66 6 66 6.66 66 6 6.666 66 6.6 66 6.6 66 6 6.6 66 6.6 66 6.6 66 6 6 66 6 66 6.66 oo66 6 2\E600 0000350:H S\E600 0000950:H OH x 0:00 .0809 0006000 .0308 :H 6000 0000 0 06000 0006000 60:HE 00mH>om 0050H0COUIIH qu200 per million. These spores had been stored at 10°C for 8 months. The greatest number of spores from‘L. marginatum germinated (>250 per million) after -18°C storage for 8 months. The greatest number of §-.Ei22£ spores to germinate (125 per million) were those stored at 26°C for 8 months. The data from germinating spores of these 3 species suggests: (1) either 10°C or -18°C maintains germination ability better than room temperature storage: (2) an after- ripening period increases germination ability. Between 3 and 5 spores per million from S. lycoperdoides germinated at zero storage time, (room temperature) 0.2 per million germinated after 2 weeks storage and no spores germinated after 3 months storage. It appears that loss of germination ability is very rapid at room temperature for spores of this species. Germination comparisons were made between seeding spores stored for 2 months at room temperature on the surface 77 of medium and seeding them into the medium. L. miggg spores germinated at a rate of 0.70 spores per million when seeded on the surface of the medium and 0.65 if seeded into the medium; L. curtisii spores germinated at a rate of 1.0 spores when seeded on top of the medium and 0.50 when seeded into the medium. This could imply that spores from L. curtisii require more oxygen to germinate. This does not seem to be the case with spores from L. EEBQE- A modification of Fries' technique was used in an attempt to germinate spores of L. curtisii and L. marginatum. The modification consisted of using R.M. yeast as a substitute for "Hefe X." Fries (1941) inoculated "Hefe X" onto malt extract agar and incubated it for 7 days, then removed the yeast and autoclaved the medium. By seeding Lycoperdon spores on the medium Fries was able to germinate Lycoperdon gp. spores without the living yeast. By using Fries' method spore germination for both fungi was observed. For L. curtisii a germination of 0.7 per million was recorded. For L, marginatum species 0.5 spores germinated per million. Since these sporophores had been stored for 5 months at room temperature the expected germination rate was low. The metabolic products of the yeast in the autoclaved medium apparently did not aid in germinating these spores. 78 DISCUSSION It is not difficult to postulate why the author's early attempts in these investigations to germinate g. gigantea spores met with failure. In most cases the experimental spores had been stored at room temperature, quite often for long periods of time. More than likely, these spores were not viable. It was expected that if certain treatments were successful then spore germination could be detected in a matter of days or at the most, less than 30 days. Such an assumption was incorrect. Also, it was not expected at the time, that should germination occur it would be as low as few spores per million. Once spores had been germinated in barley extract broth, it became apparent that some of the early notions were faulty. The barley method left a great deal to be desired so far as understanding the conditions necessary for germina- tion. However, it did suggest that spore germination of g. gigantea (a) may require periods in excess of 30 days to produce visible colonies, (b) might occur at a very low rate, (c) was conceivably possible only with spores from certain sporophores. The diversity of broths in which the spores germinated was and still is a puzzling matter. Their only apparent similarity was that all were decoctions of natural products. Once the spores from an overwintered sporophore were 79 germinated in malt extract agar inoculated with a living yeast it was suspected that spores from Q. gigantea may require one or all of the following conditions: (1) storage at a temperature below the freezing point; (2) overwintering; (3) after ripening: (4) vernalization. Further experiments indicated that room temperature storage resulted in low or negative germination results. Storage at —18°C appeared to be the best. However, there were exceptions. During the ensuing months the effect of storage temperature on germina- tion was studied with spores from many sporophores. These investigations revealed that the storage question was not clear cut. Some spores germinate well when stored at 12°C or room temperature yet spores from other sporophores never germinated when stored at room temperature. The varying results from spores of different sporophores is probably attributable to genetic or environmental factors. The maturity of a sporphore when collected may effect its subsequent germination rate. However, for the most part, storage at 12°C or -18°C is most suitable, i.e., no deleterious effects were ever noted at these temperatures. Spores of Q. gigantea appear to require an after- ripening period which seems to vary for individual sporophores. In some cases only a few weeks are necessary while for others this becomes a matter of several months. Approximately 12 months may be the peak of this period for the majority of i’rl - II n. 1 ...~_ ~ _- .36.. "'5 H Mae-‘2']. 80 sporophores. Whether or not more extended periods of after— ripening than have been examined continues to increase the number of spores that will germinate has not been ascertained. Only one sporophore was tested for germination after an extended period. This one had been stored at room temperature for at least 10 years and only one spore germinated out of 2 million under observation. The variability of some germination results should be noted. Although attempts were made to control many experi— mental conditions there still exists the possibility that some factors have not yet been observed. The concentration of spores placed in a Petri dish affect the number of colonies Which appear as a result of spore germination. The concentration is not an arbitrary number. First experiments indicated that approximately 4.6 X 106 spores per dish resulted in maximum germination results. However, further experiments indicated that this figure decreases as the number of spores which will germinate increases. An incubation temperature between 24 and 26°C results in the greatest germination percentage. When spores from.g. gigantea were germinated in malt extract agar, a living yeast was necessary as a co- culture. The effect of the yeast raises many intriguing questions. Since various vitamins did not replace the yeast it appears doubtful that the yeast has the sole effect of 81 supplying a vitamin required for germination. However, a vitamin could be necessary as a stimulative agent with the yeast performing a second function——such as neutralizing or masking inhibitor(s). Until the spores can be germinated in synthetic media the conditions necessary for germination also relate to factors affecting the metabolism of the yeast. The sexuality of Q. gigantea still remains unknown. The major criterion used by investigators to demonstrate heterothallism in certain Basidiomycetes is the observation of clamp connections in the secondary mycelium and lack of such structures in mycelium derived from germinating spores. When these characters are not seen in secondary mycelium. as is the case with g. gigantea, it becomes a more difficult problem to determine sexuality. The author attempted matings and fruiting experiments which were unsuccessful. The fact that mycelium from germinated spores is similar to known secondary mycelium and that it appears to be dikaryotic does suggest homothallism. Successful fruiting experiments along with further cytological evidence is needed to either support or refute this suggestion. The germination of g. gigantea spores as a method of obtaining strains with a greater capacity for producing the oncostatic principle(s) appears to hold great promise. Future investigations will reveal if the malt-yeast technique can successfully be applied to germinating.the spores of the many other species of puffballs. Thus far illl.’ 82 all attempts to germinate spores from related genera have been successful. This includes one Scleroderma gp., one Bovista sp,. and two Lycoperdon gp, Such preliminary studies indicate that this technique may bring about germination of species from several related genera. Several of the conditions necessary to germinate the above-mentioned puffball spores seem to be similar to those required by spores of Q. gigantea. In all cases malt extract medium plus R.M. yeast was required for germination. How— ever, in the case of spores from L. curtisii and L, marginatum it was demonstrated that this yeast need not be living. It is not known, although it is a real possibility. that the germination of spores from related genera are affected by the number of spores in a Petri dish and an after-ripening period. Germination of any puffball spores should be studied with these possible effeCts in mind. Storage conditions have not been well defined owing to the lack of present knowledge regarding the effect of spore concentration on germination results. In light of available data the spores from the different species studied appear to require different storage temperatures for maximum germination ability. The spores from g. lycoperdoides would not germinate after 3-4 months storage at room temperature although only strains obtained from South America were used in these investigations. 83 S UMMARY l. The basidiospores of Calvatia gigantea have been germinated: (a) in 5 different broth media: (b) in malt extract medium inoculated with Rhodotorula muscilaginosa var. sanguinea. 2. Spores of Lycoperdon gemmatum were germinated in barley extract broth. 3. Spores obtained from the following species were germinated in malt extract agar inoculated with R. muscilaginosa var. sanguinea: L, marginatum, L. curtisii. Bovista minor and Scleroderma lycoperdoides. 4. Some of the factors affecting spore germination of Q. gigantea which have been investigated include: length and temperature of storage; the concentration of spore inoculum;.replacement of the yeast by vitamins, a bacterium, or Q. gigantea mycelium: incubation temperatures; and use of Chloromycetin as a bacteriostatic agent. 5. Basidiospores and the mycelium derived from spore germination of g, gigantea appear to be dikaryotic. Attempts to fructify this organism were not successful. 6. Some of the cultures derived from spore germinations of g, gigantea have shown an increase over the parent mycelium in the capacity to produce in liquid culture materials which retard Sarcoma 180 tumors implanted in mice. /_ _ 1 84 LITERATURE CITED Bessey, C. E. 1884. An enormous puff-ball. Am. Nat. 18:530. Bessey. E. A. 1950. Morphology and Taxonomy of Fungi. Blakiston Co., Philadelphia, Penn. Brefeld. O. 1877. Basidiomyceten I. Untersuchungen aus dem Gesammtgebiete der Mykologie. ;: 1877. . 1908. Die Kultur der Pilze. Untersuchungen aus dem Gesammtgebiete der Mykologie. L4: 1908. Clarke. D. A. 1955. Mouse sarcoma 180. Cancer Research. Supplement No. 3, l4. Cochrane,K. W., and E. H. Lucas. 1958—59. Chemoprophylaxis of Poliomyelitis in mice through the administration of plant extracts. Antibiotics Annual, Medical Encyclopedia Inc., New York. Cochran, V. W. 1958. Physiology of Fungi. John Wiley & Sons, New York. Cool. Catharina. 1912. 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Studies in Botany. .2: l. 88 APPENDIX 89 Information on Collected Sporophores Calvatia gigantea #939. 970, 984, 995, #1096E #1368 #1369 #1370 #1371 #1373 #1376 #1378 #1379 #1380 #1389 #1390 #1391 #1397 948. 971, 985, 998 954, 972, 986, 961, 983, 989. Collection Date 1956-57 10/1/58 9/15/58 9/28/58 9/15/58 9/30/58 9/15/57 10/8/58 10/17/58 10/17/58 11/1/58 Approx. 1949 4/16/59 9/1/59 10/59 Information From various areas in Michigan Lansing, Michigan South of Lansing, Michigan Lansing, Michigan Livingston County. Michigan East Lansing, Michigan Lansing, Michigan South of Lansing, Michigan Baker Wood Lot. Michigan State University Baker Wood Lot, Michigan State University Utica, Michigan Michigan State University Northern Michigan. A - Stored at Rm. Temp. AF — Stored at 12°C Michigan State University wood lot. A — stored room temp. B - stored 10°C C - stored deep freeze Okemos, Michigan A - stored rm. temp. B - stored 10°C 90 Information on Collected Sporophores-—Continued Collection Calvatia gigantea Date #1400A. 1400B. 1400C. 10/28/59 1400F, 1400G Lycoperdon pyriforme #1374 10/1/58 #1386 10/58 Lycoperdon gemmatum #1383 10/58 #1385 10/58 #1403 9/57 Lycoperdon echinatum #1384 10/58 Scleroderma lycoperdoides 4/60 Lycoperdon marginatum 9/59 (Lycoperdon curtisii Bovista minor Information 10 miles south of Lansing. Michigan Michigan State University wood lot. Michigan State University campus. Michigan State University campus Detroit. Michigan Michigan State University wood lot Michigan State University wood lot. Brazil. South America Michigan National Guard grounds, Lansing, MiChigan .c ' a. ' «m ‘0 u. ‘ ‘ _ l . ‘ ' -... L_.' "I111111111011111“