PHYSEC‘LOGICAL ACTIVFEEES IN SPQRODINIA GRANDIS LINK Thesis §or Hm Degree of DE. D. MECHIGAN STATE UNIVERSFPY Frederick E. Van Nostran 1956 THE‘IS This is to certify that the thesis entitled PhysioloQiCul activities in §Bprodinia Grandis presented by Frederick E. Van Nostran has been accepted towards fulfillment of the requirements for Ph_D_ degree in Kycologj in the gotany and Plant Pathology Department Major professor E- 3. :eneke Due November :1, 1956 PHYSIOLOGICAL ACTIVITIES IN SPORODINIA GRANDIS LINK By Frederick E. Van Nostran AN ABSTRACT 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 DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology Year 1956 Approved by “Ow—wig. ZZ— § (8 W161, Frederick E. Van Nostran ABSTRACT 1 Physiological studies of Sporodinia grandis Link showed that two factors of the medium affected the production of zygo- spores in this fungus. An (Nhu)2SOu concentration of the medium greater than 3.5 percent was found to be necessary for zygo- spore production. In a medium containing h.0 percent (Nflh)230h zygospores were not formed when the initial pH of the medium was less than 5.7. Buffering the medium with CaCO3 enhanced sporangiospore formation to the apparent exclusion of zygospores. The forma- tion of melanins in the sporangiospores was greatly increased by buffering the medium with CaCOB. The intensity of melanin formation in the sporangiospores was reduced when 2.5 percent glucose was replaced with 0.5 percent in the buffered‘medium. Ascorbic acid and thiamine enhanced growth and zygosporfiv formation under the conditions of the experiment. Malonic acid greatly increased growth and zygospore formation. Thiourea neither enhanced nor reduced growth in this organism, but zygospore formation was inhibited. The color of the.mycelium was yellow when zygospore production was inhibited by thiourea. Neither polyphenol oxidase nor cytochrome oxidase was present in the fungal tissue. The oxidation-reduction potential of the mycelial extract was highly poised at the time gametangia are formed on the mycelium. The oxidation-reduction potential of the medium was well poised at the time zygospores were maturing on the mycelium. ”00.. Frederick E. Van Nostran 2 The pH of the mycelium of Sporodinia grandis under condi- tions unfavorable for zygospore formation was determined to be 6.2 after three and one-half days of incubation. PHYSIOLOGICAL ACTIVITIES IN SPORODINIA GRANDIS LINK By Frederick E. Van Nostran 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 DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1956 ACKNOWLEDGMENTS -The author wishes to express his sincere thanks to Dr. Everett S. Beneke under whose helpful criticism and guidance this investigation was undertaken. The writer is also in- debted to Dr. Robert S. Bandurski for his valuable suggestions and use of his laboratory. VITA Frederick E. Van Nostran candidate for the degree of Doctor of Philosophy 1 Final examination: November 16, 1956 ' Dissertation: Physiological studies in Sporodinia grandis Link Outline of Studies: Major subject: Mycology Minor subjects: Bacteriology, biochemistry Biographical Items: Born, November 12, 1926, Canton, Ohio Undergraduate Studies, Marshall College, 19h6-51 Graduate Studies, Marshall College, 1951-52; Michigan State University, 1952-56 Member of Mycological Society of America, Botanical Society of America, Society of Sigma Xi TABLE OF CONTENTS CHAPTER I. INTRODUCTION AND LITERATURE REVIEW.................. II. METHODS AND MATERIALS............................... III. EFFECTS OF AUTOCLAVING ON THE pH OF THE MEDIUM...... IV. EFFECTS OF pH ON GROWTH AND ZYGOSPORE PRODUCTION.... A. Medium'Unbuffered I. Effects of pH on growth ................ 2. Effects of pH on zygospore production... B. Medium Buffered 1. Effects of buffering on growth.......... 2. Effects of buffering on zygospore and Sporangial production................... V. EFFECTS OF NUTRIENT CONCENTRATION ON GROWTH AND ZYGOSPORE PRODUCTION................................ A. Varying the Concentration of (NHh)2SOh........ 1. Effects on growth....................... 2. Effects on zygospore production......... B. Varying the Glucose Concentration............. 1. Effects on growth....................... 2. EffeCtB on zygospore pPOduCtionooooeooeo Link with Varying Concentrations of and NHuNOB oeooooooeoeoooooooooeeoooooooocoo 0 C. A Growth Comparison for Sporedinia randis lN 7280 VI. EFFECTS OF VARYING CONCENTRATIONS OF TWO VITAMINS ON GROWTH AND ZYGOSPORE PRODUCTION.................. A. Effects of Varying Concentrations of Thiamine on Growth and Zygospore Production............ PAGE 12 l7 17 2O 26 32 32 33 33 35 39 39 is h? TABLE OF CONTENTS CHAPTER I. INTRODUCTION AND LITERATURE REVIEW.................. II. METHODS AND MATERIALS............................... III. EFFECTS OF AUTOCLAVING ON THE pH OF THE MEDIUM...... IV. EFFECTS OF pH ON GROWTH AND ZYGOSPORE PRODUCTION.... A. Medium.Unbuffered 1. Effects of pH on growth ................ 2. Effects of pH on zygospore production... B. Medium Buffered 1. Effects of buffering on growth.......... 2. Effects of buffering on zygospore and Sporangial production................... V. EFFECTS OF NUTRIENT CONCENTRATION ON GROWTH AND ZYGOSPORE PRODUCTIONOOOOOOOOOOC0.00000IOOOOOOOOOOOOO A. Varying the Concentration of (NHh)ZSO ........ 1. Effects on growthOOOOOOOOOOOOOOOOOOOO... 2. Effects on zygospore production......... B. Varying the Glucose Concentration............. 1. Effects on growth....................... 2. Effects on zygospore production......... C. A Growth Comparison for Sporodinia randis Link with Varying Concentrations of lN hizso and NI'I“N03 .0...0..COO...00.000000000000000. . VI. EFFECTS OF VARYING CONCENTRATIONS OF TWO VITAMINS ON GROWTH AND ZYGOSPORE PRODUCTION.................. A. Effects of Varying Concentrations of Thiamine on Growth and Zygospore Production............ PAGE 12 l7 17 20 26 32 32 33 33 35 39 39 N6 N7 VI VIII. . IX. ( TABLE or CONTENTS (Cont.) CHAPTER 1. Effects on growth........................ 2. Effects on zygospore production.......... B. Effects of Varying Concentrations of Ascorbic Acid on Growth and Zygospore Production........ VII. EFFECTS OF VARYING CONCENTRATIONS OF TWO ENZYME INHIBITORS ON GROWTH AND ZYGOSPORE PRODUCTION........ A. Effects of Varying Concentrations of Malonic Acid on Growth and Zygospore Production........ 1. Effects on growthooooooIoI-oooccone-0000. 2. Effects on zygospore production.......... B. Effects of Varying Concentrations of Thiourea on Growth and Zygospore Production............. 1. Effects on growthOCOOCII0.00.0.0'00000... 2. Effects on zygospore production.......... C. Combined Effects of Malonic Acid and Thiourea on Growth and Zygospore Production............. VIII. THE pH OF THE MYCELIUM OF SPORODINIA GRANDIS LINK.... IX. OXIDATION-REDUCTION POTENTIAL STUDIES................ A. Methods and Materials.IIOOOOIIOOOOOIOIOOCOOO0.0 B. Resu1t8000....0...‘...I.II.OOOOOIOOOIOOOIIOI... 1. Changes in the pH of the medium during metabolism of Sporodinia grandis Link.... 2. Oxidation-reduction Potential Studies.... (a) Potentials determined at pH 6.2.... (b) Potentials determined at pH 7.0.... x. DISCUSSION AND CONCLUSIOIJS.-...-II............'.'.... XI. SWARYOCIOOOOOOOIOOOOCIOOOIOCIIOIIOOIOOOCCOOOOOOI... XII. LITERATURE CITED.-00.00.00no...I..uoocuouool0¢000n0n| PAGE 8 its 50 52 53 53 S3 56 56 56 S9 62 67 73 76 76 77 77 82 90 100 103 LIST OF TABLES TABLE 1. Evaluation of replications of mycelial weight for Sporodinia grandis Link grown at various pHs...... 2. Effects of autoclaving on the pH of the medium with 0.1 percent (NHh)280u as the nitrogen source. 3. Effects of autoclaving on the pH of the medium with h.0 percent (NHN)ZSON as the nitrogen source...... b. Effects of pH on growth in Sporodinia grandis Link.. 5. Effects of pH on 2 gospore production in Sporodinia grandis Link on K.0 percent (NEH)ZSOH"‘°"‘°""' 6. Effects of buffering the medium on growth and zygospore production in Sporodinia grandis Link... 7. Influence of buffering on growth and zygospore pro- duction in Sporodinia grandis Link when 0.5 percent glucose is incorporated in the medium............. 8. Effects of varying the (NH ) SO concentration on growth and zygospore prohugti n in Sporodinia Brandi-8 LinkOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.... 9. Influence of glucose concentration on zygospore production and growth in Sporodinia grandis Link.. 10. Growth of §porodinia grandis Link with (N ) $0 of NHuNO3 as the nitrogen source........ ,?,,Q,,,, 11. Effects of varying concentrations of thiamine on growth and zygospore production in Sporodinia Brandi-8 Lin-k0...OOOOOOIOOOOOOOOOOO0.00.00.00.00... 12. Effects of varying concentrations of ascorbic acid on growth and zygospore production in Spprodinia Brandi-3 LinkOOOOOOOOOOOOOOOOOOOO00000.0.0.0000...- 13. Effects of varying concentrations of malonic acid on growth and zygospore production in Sporodinia Brandi-8 LinkOOOOO0.00000000IOOOOOOOO0.0000....O... 15 18 22 25 30 31+ 38 h2 M9 51 Sh LIST OF TABLES (Cont.) TABLE 1h. Effects of varying concentrations of thiourea on growth and zygospore production in §porodinia grandia LinkCCOOOOO00.00...OOOOOOOOOOOOOOOOOIOOOOOOC 57 15. Combined effects of thiourea and malonic acid on growth and zygospore production in Sporodinia Brandi-8 LinROOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. 60 16. Determination of pH of mycelium.in Sporodinia Brandi-8 Link..........0.0000CCOCOCOOOOCCOOOCOOOO0.. 61+ UT 10. 11. FIGURE 1. 2. 3. 9. 10. 11. LIST OF FIGURES Growth of §porodinia grandis Link on (NHM)ZSO at various pH levels.........................g........ Influence of glucose concentration on growth in Sporodinia grandis................................. A growth comparison for Sporodinia grandis Link on varying concentrations of (NHLJBSOLL and NHhN03.'.'. Effects of malonic acid on growth of Spprodinia grandis LinkOOCOOOOOOOOOOOCOOCOOOOOOOOOOOOOOOOOOOOOOOOOOCOO Changes in the pH of the medium during metabolism of Sporodinia grandis Link............................ Oxidation-reduction potentials of the medium determined at pH 6.2.0....OOOOOOOOOOOOOCOOOOO...OOOOOOOCCIOOOO Oxidation-reduction potentials of mycelial extracts determined at pH 6.2.000.00.0.000000000000000000000 Oxidation-reduction potentials of the medium determined at pH 7.000000000000000.coo...oooooococooooooooooot Oxidation—reduction potentials of mycelial extracts determined at pH 7.0.0.0000000000000000000000000IOC A comparison of oxidation-reduction potentials of the medium and mycelial extracts when the initial pH Of the medium is 50700000000000.00000000000000.0000 A comparison of oxidation—reduction potentials of the medium and mycelial extracts when the initial pH Of the medium is 6.00.00.00.00...00.000000...00.... 19 37 Ala 78 80 81 83 85 87 88 1e; flit CHAPTER I INTRODUCTION AND LITERATURE REVIEW Most fungi undergo a series of morphological changes during their life histories. From the time of spore germination to the initiation and maturation of the fruiting structures a con- tinuous chain of reactions takes place. The type of fungus spore formed is dependent upon and governed by external and intracellular stimuli. Light, temperature, pH, aeration, etc., are external factors or stimuli which influence growth and spore produc- tion in fungi. The effects of these factors have been exten- sively investigated. The question of how external stimuli influence the mycelial threads to undergo morphological changes remains to be answered. Due to its most comprehen- sive scope and time consuming areas for investigation into the nature of morphogenesis little work of this type has been undertaken in the fungi. A correlation of external and internal factors on growth and sporulation presents a most interesting investigative ap- proach to morphogenesis and its implications in §porodinia grandis Link. Sporodinia grandis Link is a phycomycetous fungus which lends itself well to both areas of investigation. The con- ditions important for the formation of zygospores by this W the (Elk homothallic species have long been a controversial subject. Internal changes of the mycelium.have never been studied to date. Klebs (18) showed that zygospore formation in §porodinia grandis depended upon the presence of light and a high rela- tive humidity. Others such as Falk (10) indicated that the composition of the medium is of importance in the type of spore formed in this fungus. l§pprodinia grandis produces sporangiospores and zygospores. Klebs (19) after additional work along similar lines continued to claim the most impor- tant factors to be light and humidity. Baker (2) in a more comprehensive work with Sporodinia found that: F a) changes in relative humidity (the concentration of the medium and temperature remaining constant) do not notice- ably alter the type of spore formed, except in special border cases, i.e., extremes of relative humidity or concentration, b) both zygospores and sporangia are formed over the whole range of relative humidity from O to 100 percent, c) nitrogen in some complex form, carbohydrates, and mineral salts are essential for good zygospore formation, d) zygospore formation is possible provided that both the glucose and asparagine constituents reach certain concen- trations, e) at low concentrations of one of the constituents (glucose or asparagine) increase in concentration of the other constituents increases zygospore production, f) the minimum concentration of asparagine necessary depends upon the glucose concentration, g) the use of sucrose or fructose instead of glucose as a carbohydrate source has very little effect on the quantity or type of spore formed or the concentration required for spore formation. Robinson (29) grew liquid cultures of Sporodinia grandis with the flasks corked above the cotton plugs and measured the respiration rate throughout the life cycle of the fugus. Robinson found that respiration of §porodinia grandis was highest after one day of growth. Between the first and second day of incubation the respiration rate rapidly decreased. After the second day of incubation, respiration declined gradually. Respiration in this case was determined by measure- ment of 002 evolved. Further reference will be made to this work. As for the cause of zygospore and sporangiospore forma- tion in the fungus Robinson says: "In the determination of the transition from purely vegetative growth to the develop- ment of either type of reproductive structure in Sporodinia, the primary factors appear to be the internal metabolic changes associated with a diminution of growth. The changes envisaged are, up to a point, common to both types of reproductive bodies. From.this stage the course of subsequent metabolic processes and the consequent morphological development are determined by the nature of the medium and the degree of humidity of the air around it.” Barnett and Lilly (5) in.more recent work have investigated the factors influencing zygospore formation by Choanephora cucurbitarum. Since this work closely parallels the investi- gations included within this paper it will be considered fully under the heading of Discussion and Conclusions. CHAPTER II METHODS AND MATERIALS Test Organism. Sporodinia grandis is a saprophyte in the order Mucorales of the class Phycomycetes widely found on decaying organics, especially on species of Russula and Lactarius. According to Dr. W. C. Hesseltine1 (personal communication), from whom the culture was procured, the correct name of this organism is Syzygites megalocarpus. This binomial is the accepted name according to British workers such as G. C. Ainsworth and G. R. Bisby. Nowhere in the literature reviewed for this paper was the name Syzygites megalocarpus encountered. Therefore, for convenience, the organism will be referred to as‘§pgrodinia grandis2 throughout the text of the paper. Maintenance of Subcultures Stock cultures were maintained on potato dextrose agar for periods of six to seven months. After this time the 1In charge of culture collection unit, Fermentation Sec- tion, Northern Utilization Research Branch, U.S.D.A. 2Sporodinia grandis Link NRRL 2h06 is the designation for the strain used in this study. cultures were transferred to fresh PDA slants in order to main- tain the viability and vitality of the original strain. More frequent transfers were avoided to reduce variation in the stock cultures. The Medium The basal medium.used throughout the project was a modi- fied Lilly and Barnett (23) glucose asparagine medium with (NHh)é80h replacing asparagine as the nitrogen source. The modified medium is as follows: KHZPOu 1.0 gm, MgSOu.7H20 0.5 gm. microelement solution* 1.0 ml. biotin 100 microgms. thiamine 5.0 microgms. glucose ' 25.0 gms. (NHh)280u as desired Throughout the period of research variations of the basal medium.were used. In each case these variations in the nature and concentration of the constituents are clearly outlined in the text. *In units per liter of medium: Na28h07, 8.8 x 10"5 gms.; (NHu)6MO7)2h 9 60’... x 10-5 8318.; FBCL306H203 9.6 X 10-“- 81330; ZnSOhJHZO, 8.8 x 10"3 gms.; CuSO , u.9 x 10‘"LL 31:13.; male. 7.2 x 10'5 gms. In...» . , *rr- m1 .4 as i Preparation of the Medium A concentrated (usually double strength) aliquot of the medium was adjusted to pH 6.5 with the aid of a Beckman glass electrode pH meter and KOH or HCl as required. After the pH was adjusted the solution was made up to 96 m1s.with distilled 1 water in a graduated cylinder. Twenty-four mls,of this medium were dispensed into each replicate 250 ml. Erlenmeyer flask.2 The flasks were plugged loosely with nonabsorbent cotton and ( autoclaved at 2h0°E.for ten minutes. Autoclaving for 10 min- utes at 2h0°E.sterilized the medium with a minimum amount of constituent breakdown. The media were removed from the auto- clave immediately to minimize further exposure to heat. The Inoculum Transfers from the permanent stock cultures of the organism were inoculated onto corn meal yeast extract agar and allowed to incubate in a constant temperature room at ca. 25°C. for seven days. Numerous sporangiospores have developed from the mycelium during this period. Since this organism.is highly phototropic, the sporangia and sporangiophores usually tend to aggregate around the periphery of the petri plate thus facilitating their removal. The sporangiospores along with the mycelium.were removed from the petri plate with the aid of 2Addition of one m1. of inoculum to the 21; mls. of medium gave a final volume of 25 mls. of medium. a sterile hooked inoculating needle and placed in a flask with sterile distilled water. The flask was gently shaken periodically to wet the mycelium.and suspend the sporangio- spores in the water free from the mycelial mat. Spore counts were made with the aid of a Spencer haemocytometer from the resulting suspension and dilution or concentration adjustments made accordingly. The method of inoculum preparation proved to be an ex- ceedingly reliable one for obtaining sporangiospore suspen- sions. The occurrence of mycelial fragments in the spore inoculum was indeed rare, when checked microscopically. Size of Inoculum Preliminary studies concerning the size of the inoculum indicated that a small spore concentration (approximately 200 per ml.of medium) resulted in poor agreement of replications. However when the spore load was raised to 8000 spores per ml. of medium statistical agreement of replications was excellent as shown in Table 1. In no instance did the replications of any treatment dif- fer by more than 0.h of a milligram.and even though two repli- cations (where 9 3 25.9 and 26.7) differed only by 0.8 of a milligram.these replications were significantly different at a 5 percent error margin.3 3Significant differences calculated from.a normal curve distribution. EVALUATION OF SPORODINIA GRANDIS LINK GROWN AT VARIOUS pHS ~ TABLE 1 REPLICATIONS OF MICELIAL WEIGHT FOR pH at flgms. offiyggelium * ** Inoculation Rep 1 Rep 2 Rep 3 6; Ag 11.6 26.0 25.8 25.9 25.9 .008 5.2 26.6 26.9 26.5 26.7 .017 5.7 29.2 29.2 29.3 29.2 .001 6.2 35.8 35.6 35.1; 35.6 .016 * 69‘. refers to average of the replications “'5 - refers to the standard deviation calculated from é: \ /_i_75_3'_( £191 at 5 percent error (d I .05) Y\ fl where x mycelium, and n equals the replications in milligrams of equals the number of replications. Incubation Conditions After inoculation of each flask the pH of one replication wa 5 determined. The remaining three replications for each tr.eatment were placed in a refrigerator-incubator at 26°C. and a31':_-1,<:wed to incubate for 6 days. The incubator remained in general use so that the cultures were exposed to intermittent 11. sht- Harvesting the Mycelium At the end of the growth period, the flasks were removed from the incubator, the pH of the medium determined and the mycelium harvested in the following manner: the mycelial pad was spread on a nylon cloth suspended over a large beaker with the aid of a large mesh metal screen and washed three times with approximately fifty milliliters of distilled water. After- each washing the pad was squeezed by hand in the nylon CIOth. The damp pad was then placed on an ordinary absorbent hand towel in numbered squares. The towels were supported by and attached to sheets of cardboard. The cardboard with the mycelial pads were placed in a hot air oven and allowed to dry f” 2(4- hours at 60°C. The loose dry pads were lifted from the paper with the aid of forceps and weighed to the nearest tenth of a milliSram °“ 3 R01 ler Smith Precision Spring Balance. _.._...___..__.--.-.—-—-—-l-E-I- ——"‘ ‘- 11 Qualification for this harvesting method is evident in the j’gznificance of the replications (Table I). 9 Glassware Cleaning Procedure Glassware was washed in a commercial detergent, rinsed thoroughly with tap water and, after draining off tap water, r111 sed in distilled water and dried at room temperature. Additional methods and techniques employed in this project were not of a general nature and therefore will be included under separate headings. Chemicals employed were all of reagent grade. CHAPTER III EFFECTS OF AUTOCLAVING ON THE pH 01“ THE MEDIUM Numerous examples of the effects of autoclaving on media are lmown. Maillard (214.) reported that a brown color developed when reducing sugars such as glucose, fructose, etc. were auto- claved with amino acids. Hill and Patton (16) have shown that growth Of Streptococcus W is reduced when tryptophane is autoclaved with sugars. Stanier (30) found glucose which had been heated at a temperature as low'as 50°C. produced less growth of various species of Cytophaga, a mycobacterium. The pH of media are also affected by autoclaving during sterilization. Therefore, since autoclaving was to be used for sterilization of media in this project, an attempt was made to find an appropriate pH for sterilization of the two princi- pal types of media. A pH for good growth was desired in addi- tion to one which did not affect a large amount of decomposi- tion of the medium constituents during sterilization. Method and Materials Two media containing different amounts of (NHh)280u were tested at various pH values to determine the extent of con- stituent breakdown when autoclaved at 2h0°F. for 10 minutes. The media were set up in the routine manner as described 1 the preceding chapter with 1.0 percent (N 30 and (4-0 We a 991' cent (NHH)28014. as the nitrogen sources. The pHs were ad- jug ted to various levels before autoclaving as shown in Tables 2 and 3. After the flasks were autoclaved and cooled to room beIn;>erature, the pH was determined and the average pH for each tr-e atment was recorded. Results Medium Containing 0.1 Percent MRI-92801; The results in Table 2 indicate that the higher the ini- tial pH of the medium the greater the loss in pH units when the medium was autoclaved. Up to an initial pH of 6.6 there was a gradual increase in loss of pH units. Beyond an initial pH of 6.6, i.e., pH 7.2, an abrupt increase in loss of pH units occurs. When the initial pH of the medium was 6.6 a 0.1;. pH unit 1088 occurred, whereas at an initial pH of 7.2 there was a loss of 0.7 pH units . Medium Containing ”-0 P°r°°nt (Nflh)2soh. The results in Table 3 indicate that the higher the ini- tial PH of the medium the greater the loss in 9‘1 units when the me"iii-1.1m is autoclaved. TABEE 2 EFFECTS OF AUTOCLAVING ON THE pH OF THE MEDIUM WITH 0.1 PERCENT (NHu)280 AS THE NITROGEN SOURCE ’47 1+ ggifiéfiufifi .. zigzszwm 3.283.121. __, 2.5 2.5 0.0 ‘3.1 3.2 -o.1 .3.5 3.6 -o.1 14.2 u.o -o.2 ll.6 h-B -0.3 5;.0 h.6 -o.u .£;.5 5.2 -o.3 6 . 1 5 .7 -0 .14 6 . 6 6. 2 -O.u 7 - 2 6.5 -o.7 TABLE 3 EFFECTS OF AUTOCLAVING ON THE pH OF THE MEDIUM WITH 14.0 PERCENT (NHu)280h AS THE NITROGEN SOURCE ____—-—_.-::_.——"*r Initial pH pH after Autoclaving Loss in of Medium pH Units 7 14.0 3.9 -0.1 11..S h.l -0.Il 5.0 14.5 -0.5 5.3 5.2 -o.1 5.5 5.1; -0.1 6.0 5.6 -0.h 6.2 5.7 -0.5 6.5 5.9 -0.6 6.7 6.0 -0.7 7 -0 6.0 -l.0 7.2 6.2 -l.0 In this case a stable increase is evident from an initial 91'; of 6.7 to 6.0 whereas below pH 6.0 the loss in pH units v19- 9 somewhat erratic. At and above the neutral pH level, i.e., 7 - O and 7.2, a loss of one pH unit for each treatment was e,._,;1_cient. The results show that not only does the breakdown of car- bonydrates incorporated in media contribute to the hydrogen ion concentration but also in these cases to the breakdown of (NH)... ) 2801+. The rather erratic changes of pH in both instances at and around pH 5.5 may be due to increased escape of ammonia from the medium since (NHH)2801+ is almost completely dissoci- ated at pH 5.5 Alkaline hydrolysis of glucose may be a factor in reducing the pH when the media were autoclaved at pH 7.0 and above, con- comitant with breakdown of the (Nah)280h° CHAPTER IV EFFECTS OF pH ON GROWTH AND ZYGOSPORE PRODUCTION A. Medium Unbuffered Eff e cts of pH on Growth The growth of Sporodinia grandis at various pH levels with two concentrations of (HEIRZSOH was determined and re- corded in Table 14.. The lower concentration (0.1 percent) is commonly used for nitrogen sources in growth studies. The higher concentration (14.0 percent) is ho times as great and therefore has a high osmotic pressure which is not tolerated by most fungi; hence the two extremes. The various pHs and concentrations were adjusted in a routine manner and similar procedures were followed throughout the remainder of the experiment. In Figure 1, growth with 11-0 percent (NHu)2SOu is repre- 1212501; is These data for Figure l are pre- sented by curve (A); growth with 0.1 percent (N represented by curve (B). sented in tabular form in Table 14.. The amount of growth of the organism with 14,.0 percent (NHLL128014 (see Figure 1, curve A) increased gradually to pH 5'“ at which point there was a marked increase in growth to pH 6-0- From pH 6.0 to 6.2 there appeared to be a tendency f or the rate of growth to increase only slightly. Growth, TABLE h 18 EFFECTS OF pH ON GROWTH IN SPORODINIA GRANDIS LINK 0.1 Percent (NEH)2SOM h.0 Percent (Nfih)230u pH of Medium Mgms.of pH of Medium Mgms. of When Inoculated Mycelium When Inoculated Mycelium 2.5 0.0 3.9 2h.8 3.2 16.1 h.1 28.5 3.6 22.2 h.5 28.7 h.0 2h.9 ' 5.2 32.8 h.3 25.6 5.h 36.0 h.6 25.9 5.6 38.7 5.2 26.7 5.7 kl.6 5.7 29.2 5.9 h2.6 6.2 35.6 6.0 #7.? 6.5 u2.1 6.2 h9.0 as indicated by curve B, increased more or less gradually to pH 6.5 with all indications of some further rise at increased pH values. The difficulty of obtaining a pH above this point when the medium is autoclaved is evident in Tables 2 and 3. While fungus growth has a tendency to be equal at the lower pH levels, at higher pH values the fungus is able to grow more rapidly in the h.0 percent (NHh)ZSOu'medium than in the 0.1 percent (NHh)280h medium. In general, greater growth of the organism occurs at higher pH values in the u.0 percent (NHu)230u medium than in.the 1.0 percent (NHh)280)4 medium. Also, an apparent growth peak of the organism is reached in the h.0 percent medium be- tween a pH of 6.0 and 6.2. This apparent growth peak does not occur when the fungus is grown in 1.0 percent (NHLL)ZSOL;. Effects of pH on Zygospore Production Eight flasks for each treatment were autoclaved at a predetermined pH. After autoclaving, the pH of five replicate flasks was determined and an average of these taken as the true pH. The three remaining flasks were inoculated and incu- bated for six days as in general procedure. After the six day incubation period the three replicates were removed from the incubator and the medium of each flask was very gently poured into one-half of a petri plate. The InMalia]. mat, which floats partially submerged in the medium, -.--—.' The was carried intact on to the petri plate with the medium. medium was carefully pipetted from the plate leaving the mat resting on the plate. The mat was carefully spread over the petri plate so that all zygospores on the superficial hyphae were apparently exposed for counting. The plate was placed in a bacteriological colony counter and the number of zygospores for each replicate was determined. Results. The number of zygospores produced (as shown in Table 5) was greatest when the initial pH of the medium was 6.2. The number of zygospores produced at pH 6.0 re- mained approximately the same. With a decrease of 0.1 pH unit, however, the average number decreased almost one-half, from 350 at a pH of 6.0 to 138 zygospores produced at pH 5.9. A, decline of 0.2 of a pH unit from 5.9 to 5.7 caused an addi- tional reduction of almost one-half the number of zygospores. Below pH 5.7 zygospores were not produced under the conditions of the experiment. The inhibitory effect of media which are too acid or alka- line upon the growth of some fungi or bacteria is well known. While many studies have been made to determine the pH at which growth and reproduction cease, few quantitative studies or this nature have been undertaken under conditions which are less severe than those which inhibit growth and reproduc- tion entirely. Lilly and Barnett (22) have noted that Aspergillus rugulosus Produces many perithecia and few conidia at an initial pH value TABLE 5 22 EFFECTS OF pH ON ZYGOSPORE PRODUCTION IN SPORODINIA GRANDIS LINK ON h.O PERCENT (NHh)ZSOu Initial pH Number of Zygospores Average Number of of Medium. per Replicate Zygospores per Treatment“ 6.3 229 367 366 h01 6.0 k32 280 350 3&3 5.9 105 209 138 102 5.7 79 an 71 92 5.6 0 0 .5.2 0 0 ‘TBased on the average of three replications of 6 to 8 whereas no perithecia form at pH 3 to 14.. These con- ditions are extremes of course and give little indication of mean production. In an earlier paper (21) lilly and Barnett also observed that when Sordaria fimicola was grown at an initial pH of 3.6 to 3.8 normal growth and reproduction were inhibited. The addition of thiamine or its pyrimidine moiety overcame this inhibition at a low pH. Perithecia formed only after the pH of the medium had risen above 6.0 but this pH alone was not sufficient to ensure sexual reproduction. Robbins and Schmitt (28) found that failure of Phycomyces blakesleeanus to form progametes at 26°C. on a basal medium of mineral salts, asparagine, dextrose and thiamine was caused by the development of too umch acidity in the agar. The addition of potato extract, neutralized protein hydrolysates or neu- tralized glutamic acids favored gametic production because these supplements buffered the medium or increased the rate of growth so that the hyphae met to form progametes before the critical hydrogen ion concentration was reached. Whereas the previous literature citations point out that PH has a general effect on the sexual reproduction of some fungi, the initial pH of the medium has a direct and critical bearing on both the number and presence or absence of zygo- Spores in Sporodinia grandig. This effect can be noted in Table 5 - B. Medium Buffered That buffering the medium has an effect on growth and spore production is well known. Some fungi grow well in buf- fered media, some do not. The pH of the medium may be controlled within limits by The effects on Sporodinia the addition of calcium carbonate. grandis of incorporating calcium carbonate into the medium can be seen from the results of this experiment. Two media were selected: the first with 1.0 percent (NH’4)ZSO7-1- as the nitrogen source (see Chapter V, A-2), the other with 14.0 percent (NHH)ZSOL; as the nitrogen source. The media were adjusted to their respective (NHMZSOL‘. concentra- tions and 0.5 grams of calcium carbonate added to each test flask (see Table 6). All flasks were then autoclaved and inoculated. Effects of Buffering on Growth In all cases the mycelial weight produced in the buffered media was at least three times that amount produced in the un- burfered media (Table 6). Growth on the buffered medium was greater when 0.1 percent (NHNZSOH medium as compared with growth in the presence of 11..0 percent was incorporated in the (NH ) 80 as a nitrogen source. ’4- 2 u 25 +++ H.md o.N H.o a 0.: a + m.o:a «.0 0.0 + 0.: o . m.H: a.m ~.e - o.H m 1 Emma :.m To + o...” < noaomnowhN 8:30th «3:95 doeumHSoodH veaemgm domNAdmzv . n escapamse no cocononm Mo mfiwz no»: we pd mm ooso udooaom x23 manéa asvm amnesz mwozimz dommadmz. z accouom pcoepaona mompom zmoomaHz see me mozemz mo edemaemzv maHz ezHe mHez250 1 x 10"“ 6.0 3.1 1.5.5 202 3 x 104* 5.9 3.0 1.6.1 85 7 x 10‘14- 5.9 3.0 145.); 0 1 x 10-3 6.0 3.0 Lane 10 \ “Based on the average of three replications The control colony counter following the procedure on page 20. flasks were counted last, thus alleviating the task of counting the total number of spores in those flasks. A sufficient number was counted, however, to indicate significant differences in the control and test flasks. The average number of zygospores in the control flasks was greater than 250 per replicate (>250 in Table 114.) whereas the average of the test flasks was never more than 202 zygospores. The addition of 1 x 104'" M thiourea reduced the average grave—em?” number of zygospores from 250 to 202. A three-fold increase to 3 x 10-1; M resulted in a further reduction to 85 zygospores for the replicate average. At 7 x 10"!4 M thiourea zygospores were not evident in any replication but at l x 10'"3 M thiourea an average of ten zygospores was formed. Dietinct differences in color, of the mycelial pads were also obserVed. Diverging from the topic of zygospore forma- tion, it one compares a mycelial pad grown under conditions feVorable for vegetative growth only (non-zygospore forming) with a mycelium on which the production of zygospores has been completely inhibited with the aid of thiourea a definite dif- ference is noticed. In 1line flasks treated with thiourea the mycelial mat ap- Pears yellow whereas the non-zygospore forming mat appears white- treat:he texture of the mate is also different. The thiourea d that is smooth to the touch and feels like wet cotton w hen gent . 1y rubbed between the fingers. In comparison, the non-zygospor~e forming mat is more loosely woven and more coarse to the touch, All mycelial pads growing in the treated medium were examined under a binocular scope at a magnification of 30x for any evidence of hyaline zygospores, i.e., zygospores with no melaznin formation. No hyaline zygospores were observed. The ZYgospores appearing on the thiourea treated mycelial f” pads as compared with the untreated ones showed a distinct d1ff°r°n°° in aggregation on the pad. When zygospores occurred on th" treated pads they were aggregated in very small areas Ju‘h.‘ ".-u 1 . with six or seven zygospores in close proximity. 6' Combined Effects of Malonic Acid and Thiourea on Growth and Zygospore Production Since the effects of malonic acid and thiourea were dif- ferent in both formation of zygospores and mycelial growth they were incorporated into the same medium to observe their combined effects. Thiourea at a concentration of 2 x 10"3 M and ma‘lonic acid at a 2 x 10"3 M concentration were combined in the Same medium. The results of this combination on SPOWth and zygospore formation can be seen in Table 15- Tx‘eatments of media containing malonic acid (0). and a combination of both enzyme inhibitors (D) show that there is an in‘331‘ease in mycelial weight above the controls (A) arid the medium with thiourea (B). A comparison of treatments of media COI'lta1 1d (D) 1Iing thiourea (B) and both thiourea and malonic ac ‘ ‘ ‘ TABLE 15 COMBINED EFFECTS OF THIOUREA AND MALONIC ACID on GROWTH AND zycospoas PRODUCTION IN SPORODINIA GRANDIS LINK“ 60 ”2:22“ “W” “2:22“ Insist... M533- “$3?“ Growth yce um a ng A - - 6.0 2.8 h8.9 ++ B + _ 6.1 3.0 no.6 1‘. C ~ + 5.9 2.9 56-1 H“ D + + 6.1 2.9 58.6 _+_ \ * Result 8 in triplicate - ' New. -—-—-.-.‘.7. . ._ 61 : indicates that the presence of thiourea at this concentration inhibits the formation of zygospores. The rating 1 for treat- ments B and D signifies the appearance of 15 to 25 zygospores in one replication of both treatments. Zygospore formation was not entirely inhibited, but restricted to the same degree in both E and D treatments. 62 CHAPTER VIII TIE pH 0F MYCELIUM OF SPORODINIA GRANDIS LINK The charge on the amphoteric colloids of cells is regu- lated by the pH of the solution in which they are immersed. Banning ( 8) showed that when the mycelium of Aspergillus £11593 was immersed in solutions with a pH greater than 8.0, the pH of the mycelium ranged from 7.0 - 7.5. When the mycelium was immersed in solutions of 6.5 to 7.0, the mycelial pH was 5.0. 501111310118 of pH 2.5 to 3.0 induced a mycelial pH of AA. The PH of the mycelium was determined by differential uptake of various dyes. Banning also found in Aspargillus niger with the aid of the same method that nitrate absorption was a function of the pH of the medium. At pH 3.0 anionic dyes and nitrate were as- similated and stored. Above this pH, however, certain dyes were taken up and stored whereas anions, including nitrates, diffused from the mycelium. The above examples indicate that the pH of the mycelium changes as the pH of the medium Changes and that these changes in the IHedium influence the adsorption and possibly the meta- bollsm or certain substrates. The oxidation-reduction potential of any system, regard- legs or its simplicity or complexity, is regulated by the hydr- ' 089!) ion concentration. The internal metabolic processes ,.._..___.__,, . at the time of zygospore formation in §porodinia grandis would therefore be influenced by the pH of the mycelium. A modification of the Robbins method (27) was used to determine the pH of the mycelium. A series of petri plates was set up, each plate con- tainins a buffer of known pH as shown in Table 16. the test cut from pads and fuchsin. paper to immerse (1 1+8 hour 3 . Pieces of K mycelium approximately one square centimeter were actively growing, three and one-half day old mycelial stained with one percent eosin or 0.05 percent basic After staining the pads were squeezed in filter remove the excess dye. The prepared pads were then in their respective buffer solutions continuously for The solutions were replaced with fresh aliquots 0f the buffers after u, 8, 12 and 36 hours- The mycelium was grown under controlled conditions with the exceUtion that the flasks were incubated at ca. 25°C. and t he basal medium 1.0 percent (Nflh)280u as the nitrogen source. The A. mycelial I'esults recorded in Table 16 are outlined here. Eight hours after staining and immersion of the pad: 1. eosin - minimum color in. the mycelium at pH 6.0. 2. basic fuchsin - minimum color in the mycelium at [DH 5080 After twenty-four hours: 1. eosin - no color at pH 6.0 and pH 6.3. Some color remaining at pH 6.1. TABLE 16 DETERKINATION 0? pH OF MYCELIUM IN SPORODINIA GRANDIS LINK 61; pH of _ 3 hours 214. hours 36 hours 14.8 hours fitter Rosin 332:: in Be sin $33.51;. Eos in 3321112111 30 sin 3: 2:: in 3 3.1L +++ +4.4. +++ +++ +++ + +++ + 307 m +++ +++ «H4» +++ + +++ + “'5 *‘H' +++ +++ +++ +++ +4» +++ +4» Q 5'8 ”'* ++ ++ ++ + ++ +4» +4- 60° *"' +4.4 0 +++ 0 H o + 6'1 ”’* +++ + +++ 0 +4 0 + 6'3 ”’"’ Hut 0 Hut 0 «Hut 0 +++ 6 '5 ““" +4.; - ++ 4- - +++ - +++ 7'2 +++ +++ 4- 4+0 0 +++ 0 ++ —— \ 0 : :2 Egirer used J : alightozojigrthe mycelium 4-» g mOder'ate color intense color 65 2- basic fuchsin - minimum color at pH 5.8. C. After thirty-six hours: 3.. eosin - the color intensity of the mycelium was very low at pH 5.8; color was absent in the range of pH 6.0 and higher pH values. 2. basic fuchsin - the color intensity of the mycelium.was at a maximum in the buffers of pH pH 6.3 and above, remaining at the same inten- sity at pH values above 6.3. D- thter forty-eight hours: ‘1. eosin - minimum color intensity of the mycelium was at pH 5.8. No color was present in the range of pH 6.0 and above. 2. basic fuchsin - most intense color of the mycelium was at pH 6.3, with a notable decrease in color intensity below this pH. The pH of the mycelium of Sporodinia grandis was determined aft er thlI‘ee and one-half days of incubation. After three and one- halr days both gametangia and zygospores are forming on the same mycelium when the organism is grown under conditions stimulating zygospore formation. The initial pH of the medium in thia‘ experiment was sufficiently high for initiation of zygospores (pH 6.0). However, the concentration of (NHu)230u as reduced to one percent, a concentration at which zygospores are 11 0“ produced in Sporodinia. Therefore the pH of the 66 mycelium, if influenced by the external pH of the medium, should be e essentially the same as the pH of a mycelium when the fungus is grown in a medium with sufficient (NHLL)ZSOI+ for zygosp 0 re production. ~.'.l‘ ‘ xix—nu- -. Th7.~' . : CHAPTER IX OXIDATION-REDUCTION POTENTIAL STUDIES Oxidation and reduction are inseparable within any given system in which one or the other occurs. Without one the other cannot occur in normal biological systems. A review of selected references on oxidation will be considered first. There are three principal types of oxidation. The first may be, as the term implies, the addition of oxygen to some oxidizable substance as in the following: (1) A heKose is oxidized to C0 and 320. a common biological 2 ”30131011. The reverse of this reaction, the formation of a hem“ from 002 and H20, is therefore a reductive process. A secBond type of oxidation may involve the removal of h d y rogen from an oxidizable subsgance such as hydroquinone H 0 ll (2 ) D ,_,. + H2, 0 H T H r113 type of reaction is imgortant in some types of oxi- dases, The hydr'oquinone is said to be oxidized to the quinoid Struct lire at the right, with the concomitant removal of A third form is that of electron removal from a substance as 1 n the case below: ‘ : ziii‘a Fe. ‘41- --‘.r_- n «H (3) Fe ——-———-’Fe+++ + e The ferrous ion has lost one electron and has been con- verted to the ferric ion. This reaction is important biologic- ally as some iron containing enzymes function in this manner, i.e., the iron in the enzyme is oxidized or reduced depending ___ upon the valence of the iron in the enzyme. F Considering the three types of oxidation, the reverse ; of any one of these reactions constitutes a reduction. In E some systems especially (2) and (3) these reactions are equili- ; brated and constitute oxidation-reduction systems, so important to the function of biological systems. These systems are said to be reversible. Bancroft (2) may have been the first to investigate the Oxidation-reduction potentials of reversible systems, a system in Which both oxidized and reduced forms occur simultaneously. 0 'H H 0 .v 0 H n O The same amount of electrical energy is required for oxi- dation of the reduced form as is required for the reduction of the oxidized form. Other factors, such as pH, govern the p”CPO-1“";ion of each form. However, this reciprocal trade of energy is mostly a theope tical treatment. Oxidation-reduction potentials are 1157 designated by the symbol Eh . Eh is the potential Ih¥ 69 in volts that an inert electrode assumes when referred to the hydrogen electrode. By definition: .32 Ox Eh =- E0 + nF In E.“ ’ is the oxidation-reduction potential referred to where Eh R the hydrogen electrode; E0 is the observed potential; T is the absolute is the gas constant of 8.315 joules; temperature; n is the number of electrons involved in the transfer; F is equal to 96,500 coulombs; 103 x and EOxJ and filed] are the concentrations of the oxidized and reduced forms respectively. In general, one can conclude from the equation that the ln equals 2.303 greater the proportion of the oxidized form the higher the Potential and conversely the greater the proportion of the I"educed form the lower the potential. For a more complete ac><‘=<3u.nt of this phenomenon see Hewitt (15). Most early work on oxidation-reduction potentials was centered around bacterial cultures in connection with poten- tials of the medium. Different phenomena of growth occur under various oxidation-reduction potentials of the medium. Potter (26) showed that the growth of Escherichia coli in a Sterile medium'lowered the electrode potential (oxidation- reduction potential) of the medium. The yeast Saccharomxces W also produced this effect. However, Chromobacterium v M. Pseudomonas W and Sarcina lutea were ¥ His study demonstrated that unable to grow in this medium. the reducing condition of the medium was influenced by the concentration of the nutrients, size of inoculum and tempera- ture. Yudkin (31) measured the oxidation-reduction potentials of media which Bacterium 39;; and Bacterium alkaligenes were r Metabolizing. The measurements were determined both electro- { metically and colorimetrically. Yudkin found that the oxidation- : reduction potential of the medium was reduced during the meta- F bolism of the media by these two organisms. Bacterium alka- ligenes, a strict aerobe, reduced the oxidation reduction Potential of the medium from 40.1450 volts to 0.0. The medium used was tryptic digest of caseinogen. Allyn and Baldwin (1) indicated that the oxidation- Peduction potential exerts a decisive influence on the ability 01' certain aerobic bacteria (rhizobia) to initiate growth. VaPious forms .of Rhizobium trifolii were unable to utilize a “1 trate-mannitol medium which exhibited an initial potential The species could utilize the The °f approximately +0.1i50 volts. same medium when yeast water was substituted for nitrate. initial oxidation-reduction potential of this medium was approx- 1mate 1;, +0.1400 volts. Upon addition of the optimum concen- tration of 0.0075 percent thioglycollic acid to the nitrate- mannitol medium the potential dropped to aPPPOX1mately “050 VOltg- As consequence the bacterium was able to reduce the nit . rates and grew well. The conclusion is that "greater 71 .» reduction intensities seem to be required to reduce nitrate preparatory to the synthesis of protoplasm." Gillespie (11) has shown that Bacillus mycoides grew in a medium.of -.050 volts potential gradually increases this potential to approximately +0.350 volts. Bacillus coli increased the potential of the same medium from -0.200 bolts to +0.600 Ft vOlts. Another soil aerobe reduced the medium, i.e., raised the potential from.-0.200 to -0.150 volts. ‘In this case the oxidation-reduction potential was increased. '_.1'“_3"‘TTT h... ‘ . k' Theoretical interpretations of oxidation and reduction in plant respiration are based on the step by step transfer of electrons by subsequent oxidations and reductions of sub- strates until the highest potential is reached in the respira- tory process: that of combining hydrogen with oxygen to form ."ater. Theoretically, a potential of +0.815 at 25°C must be refiched before this reaction is able to take place. At this Paint it is well to examine some oxidation-reduction poten- tialg of some isolated biological systems, all calculated at PH 7.0 (from Bonner, 7): wuced Form Oxidation Products If] T°C 320 $02 + 2H + as +0.815 25° cateChol benzoquinone + 23* + 29 4.0.792 25o r Simona cytochrome ferric cytochrome oxidase unknown -- xidase + a 1'0 8 rrous cytochrome C ferric cytochrome c + e 4.0.290 250 u rggcinate fumarate + 211+ + 23 -o.026 250 11ch flavin enzyme oxidized flavin enzyme + 2, -0.063 38° 72 The systems are arranged in order of decreasing potential, that is, each system is capable of oxidizing any system below it in the table. _The reduction of oxygen to H 0 possesses the highest oxidation-reduction potential and is cipable of oxi- dizing all other systems. It is emphasized that both E0 , the oxidation-reduction Potential of a system at half reduction, and Eh , the observed oxidation-reduction potential, measure the intensity of the oxidizing or reducing tendency of the system and not the amount (If oxidation or reduction of which the system.is capable. The Eh is dependent upon the ratio of oxidized and reduced forms of the substances in question and not on their absolute quantities. Thus a 90 percent oxidized system will have the same electrode potential whether the total concentration is 0.01 percent or ten percent, but the poising effect will be ten t1Int-Es greater in the latter. Zygospore production in Sporodinia grandis is apparently IPegulated by the pH of the medium.1 The influence of pH on the oxidation reduction potential and biochemical reactions 13 We 11 known. Therefore the purpose of this experiment was to determine the differences in oxidation-reduction potential, it any, that exist in the medium and mycelial extracts of this fungus , \ lsee Table 5, page 22. A. Methods and Materials Preparation of Culture 5 Two five hundred ml.portions of the h. 0 percent (NH ) 2804 nmdium were adjusted to pH 6.5 and pH 6.0 and autoclaveduat 2h0°F. for 15 minutes at 15 pounds. After autoclaving the pH 0f the media was 6.0 and 5.7, respectively. Twenty-four milliliter aliquots of these media were introduced aseptically into each 250 m1. Erlenmeyer flask which had previously been sterilized in the following manner: after addition of one drop of distilled water into each empty flask, the flasks were plugged with cotton and autocalved at ZliO°F for 15 minutes at 15 pounds. After autoclaving the flasks were allowed to dry overnight before introduction of the medium. After introduction of the medium the flasks were then inOculated and incubated in the usual manner for 8, 29, 5h, 78, 103 and 177 hours. At the end of the incubation period three flasks were I‘SmOVSd from the incubator, the mycelial pads were removed from the flasks and the media from the flasks were mixed. An ali- quot 01‘ this mixture was withdrawn for the pH determination and the remaining portion used for the oxidation-reduction potehtial determinations. The mycelial pads were vacuum washed, collectively, with 250 mls. of pyrex distilled water on filter paper with the aid of a B"-Jltzhner funnel and exhaust flask. After allowing a few minutes for air to circulate through the resulting composite pad, the entire pad (weighed to the nearest milligram) was ground in a glass homogenizer in 300 times its weight of 0.1 M KZHFOLL in the cold (5°C.). The homogenate was then centrifuged at 12,000 r.p.m. for 10 minutes at 5°C. Immediately after centrifuging the supernatant extract was decanted and used in f“ the oxidation-reduction potential determinations. Temperature of De terminations All pH and oxidation—reduction potentials were determined in an incubator room at approximately 25°C. The temperature or all solutions was 25°C. The temperature of the solutions was determined by inserting a mercury thermometer directly into the solution. Additions of buffer solutions stored in the constant temperature room contributed to raising or lower- ing temperatures of the solutions. A water bath at room tem- Perature (25°C.) was also used for this purpose if the tem— per-a ture were not exactly at 25°C. pH AdJ'ustment of Test Solutions Buffers of pH 6.2 and 7.0 were prepared from .067 M KHZPOH. and -067 M Naz HPOH solutions in the prOportions required. Ar _ tel" these buffers were added to aliquots of the media and m ycelial extracts the pH of the buffered solutions was veri- fie d by means of a pH meter. _¥ Determination of Oxidation-Reduction Potentials A Beckman pH meter (Model G) was used for the determina- tions after a bright platinum electrode had been substituted for the hydrogen electrode. The calomel electrode served as a reference electrode, its potential determined to be +0.2h6 millivolts at 25°C. from the following equation: E31t.KCl 0.2a; - 0.00076 (T-25) EBBtJiCl is the potential of the calomel and T is the tem- Perature of 25°C. The platinum electrode was cleaned before each run with xylene, chromate-stou cleaning solution and then with dilute H01. Each of these treatments was followed by a washing in diSti lled water. Drift in Readings In the determination of oxidation-reduction potentials of val-"1011s biological systems one encounters drift in readings of the meter. Readings are taken with this phenomenon in mind. Readings in this particular study were taken as follows: readings of the same solution were taken until at least three consec"ltrlwe readings were the same. This reading or' ”drift reading " was established as the true reading. When no drift °°¢ur bed the stable potential reading was indicated. 76 . Preparation of Test Solutions Mycelial extracts. Immediately following the homogeni- zation of the mycelium two mls. of the extract were buffered at pH 6.2 and two mls. buffered at pH 7.0 and the temperature adjusted to 25°C. The total volume of the buffered mycelial F extracts was ten mls. “ Nitrogen gas was then bubbled through the solution from forty-five to sixty seconds after which the oxidation-reduction potential was determined . Medium. Two five ml. aliquots of the medium were buffered 8°Parately at pH 6.2 and 7.0. The remaining procedure is iden- tical with that described in Myc'elial extracts. The total VOIume of the buffered media was 25 mls. B. Results Changes in the pH of the Medium During Metabolism of Sporo- 21131.3 grandis PJr'evious reference has been made to the effects of Varying the pH on growth and sexual reproduction of Sporo- M‘ni‘a 83‘. andis Link.1 The appearance of gametangia and forma- tion or Zygospores through time warrants clarification GEL‘niletangia appeared approximately 5h hours after inocula- tion or the fungal spores. After 78 hours zygospores were Wident macroscopically as black dots on the superficial hyphae l rfable 5, page 22. ¥ of the fungal mat. A melanin type pigment appeared in the superficial hyphae as well as in the zygospores. The pigment intensity increased as the colony matured. Figure 5 graphically compares the pH changes in the medium through time when the initial pH values for the medium were 5.7 and 6.0. In both cases there was a sharp decrease in pH up to 78 hours of incubation after which a slow decrease in pH occurs up to 177 hours. The curves for the medium at a pH of 5.7 and 6.0 remain essentially parallel throughout the incubation Period. For convenience of presentation, throughout the results or this experiment presented here, reference will be made only to average potentials rather than to maximum or minimum p“afltial readings. Figures 6 to 11 inclusive present these maximum and minimum potential readings in graph form. ondation-Reduction Potential Studies ‘Potentials determined at pH 6.2. l. potentials of the medium F”Laure 6 presents a comparison of the oxidation-reduction peter“Eials of media during metabolism of Sporodinia grandis When the potentials were determined at pH 6.2. The initial pH of the medium was set at 6.0. Eight hours late r. aI‘ter the average of the three replicates was adjusted 92, the average potential of the medium was +0-1450 volts. # coaumnsoaa madam OH H8, 0 O mntpem JO Hd IIIIAI ._ 79 The potential had lowered to +0.1430 volts after 29 hours. Be- tween 514 and 78 hours a definite increase in the average reading occurred which was climaxed at 78 hours by a very stable reading of 40.505 volts. The potential after 78 hours declined to an average of +0.39S volts at 177 hours of incu- bation. The initial pH of the medium was set at 5.7. later, after the average of the three replicates was adjusted Eight hours to pH 6.2, the average potential of the medium was +0.h00 volts. After 29 hours the potential had risen to +0.1435 volts and then lowered to +O.’+30 volts at 5h hours. The potential readings reached a maximum after 78 hours of incubation with a poten- tial of +0.1460 volts. There was no stable reading at this P011113 as under the previous conditions when the initial pH of the medium was 6.0. However, there is an indication of ap- PrORChing stability as evidenced by the rise of the minimum readirlg between Sh and 78 hours. The maximum reading has re- Mined approximately at the same level. 2. potentials of the mycelial extracts Iyi—Enre 7 presents a comparison of the oxidation-reduction p°t°nt1als of mycelial extracts of Sporodinia grandis when the pOte’ntifills were determined at pH 6.2. The initial pH of the medium was set at 6.0. Seventy- ei ght h~<-‘M.41rs later, after the average of the three replicates was a’d-‘Iusted to pH 6.2, the average potential of the mycelial Simht.m. ~.'. .4 ..— _ _ ____ .4. (9410A) 1814 ............ ..... + + UOIqonpeJ-UOIQBPIXO .............. ...... 80 Hours of incubation ...... ..... + + + (sqIOA) {exquaaod uotqonpes-uorqsptxo ....... ...... ..... ...... Hours of incubation A . 1.3"" Ti ‘ The potential remained essentially extract was +0.505 volts. at the same intensity level up to 177 hours. The initial pH of the medium was set at 5.7. Seventy- eight hours later, after the average of the three replicates was adjusted to pH 6.2, the average potential of the mycelial extract was ‘04-‘90 volts. The potential trend was similar to the previous treatment but with an overall lower maximum poten- tial. Potentials determined at pH 7.0. 1. potentials of the medium Figure 8 presents a comparison of oxidation-reduction POtentials of media during metabolism by Sporodinia grandis When the potentials were determined at pH 7.0. The initial pH of the medium was set at 6.0. Eight hours 13“”: after the average of the three replicates was adjusted to PH 7.0, the average potential of the medium was +0.39O “1‘58- At 29 hours a slight lowering of the potential to .0335 Volts occurred. A rapid increase to 40.14145 V01t8 after 78 I‘m-11‘s , with excellent poising, climaxed the readings. The reading-s decreased to 40.385 after 103 hours and slowly rose to an &Verage of +0.1450 after 177 hours incubation. The high: well poised reading after 78 hours incubation was evident at the time the maximum number of zygospores appeared. The initial pH of the medium was set at 5.7. Eight hours ’ £1Ii‘ter the average of the three replicates was adjusted kt- ........ ........... ........... + + (8410A) {exqueqod notionpea-uotqeprxo .......... ..i ..‘. Hours of incubation to pH 7.0, the average potential was +0.39O volts (Figure 8). After 29 and Sh hours the potential was essentially the same (#0.395 volts). After 5h hours the potential rose from +0.39S to +0.h08 volts after 78 hours. No poising of the potential occurred after 78 hours of incubation. After 103 hours the potential was reduced to +0.385 after which it rose to +0.h08 F- volts at 177 hours. i 2. potentials of mycelial extracts Figure 9 presents a comparison of the oxidation-reduction fl— potentials of mycelial extracts of Sporodinia grandis when the potentials were determined at pH 7.0. The initial pH of the medium was set at pH 6.0. Fifty-four hours later, after the average of the three replicates was ad- justed to pH 7.0, a very stable reading of +O.h25 volts was ob- served. After 78 hours the reading had risen to a +0.h60 volt average. The reading was not well poised. The average poten- tial after 103 hours incubation declined to +0.h08 volts; how- ever, this decline was due mostly to a lowering of the minimum potential rather than an overall decline in readings. After 103 hours a slow rise in the readings occurred culminating at a potential of +0.h50 volts after 172 hours. The initial pH of the medium was set at pH 5.7. Fifty- four hours later, after the average of the three replicates was adjusted to 7.0 the average potential was +0.385 volts. :18 00mpared with the previous treatment, i.e., when the 'i‘i H . 1" S + + + (BQIOA) {exqualod uotqonpea-uotqaptxo I“, ..1‘ W + Hours of incubation initial pH of the medium was 6.0, this reading was not well poised. A maximum of +0.hOS volts and a minimum.of +0.36S volts gave the average reading of +0.385 volts. After 78 hours incubation the average potentialrose to +O.hhS volts, decreased to +O.h25 volts at 103 hours, and slowly rose to +O.hh8 volts at the end of 177 hours. The maximum potential for each treatment after 78 hours (Figure 9) was the same. The maximum number of zygospores appeared on the mycelium after 78 hours of incubation. Figure 10 compares the potentials of mycelial extracts with those of the metabolized medium through time when the initial pH of the medium was S.7. Figure 11 compares the potentials of mycelial extracts with those of the metabolized medium through time when the initial pH of the medium was 6.0. Well poised potential readings did not occur in either the medium or mycelial extracts when the initial pH of the nwdium was 5.7 (Figure 105. In Figure 11, when the initial pH of the medium was 6.0, stable readings occurred in both nmdium and mycelial extracts. A well poised potential occurred in the mycelial extract after Sh hours of incubation and at the end of 78 hours. Gametangia were formed after Sh hours; nmny zygospores had been formed at the end of 78 hours. As previously shown (on page'72) the higher the concentration of a submtance exhibiting a potential difference, the more stable the reading . 87 4. (euros) {staueqod uotqonpeJ-uotleptxo Hours of incubation 88 Hours of incubation 11’) Hr .n M. I I", Hi- 1 we, §.‘ In summary, when the potentials of the media were deter- mined at pH 6.2 a stable potential occurred in the medium when the initial pH of the medium was 6.0. When the potentials of the mycelial extracts were determined at pH 6.2, there was little difference in the readings. When the potentials of the media were determined at pH 7.0, a well poised potential occurred in the medium.when the initial pH of the medium was 6.0. No stable reading was ob- served in the medium when the initial pH of the medium was 5.7. When the potentials of the mycelial extracts were deter- mined at pH 7.0 a well poised reading occurred in the mycelial extract when the initial pH of the medium was 6.0. There was no stable reading in the mycelial extracts when the initial pH of the medium was 5.7. When the potentials of the mycelial extracts and medium were compared after the potentials were determined at pH 7.0, very stable readings were observed in the mycelial extracts only at the time many gametangia were formed on the mycelium. In the medium very stable readings were observed only after many zygospores were formed. 90 CHAPTER X DISCUSSION AND CONCLUSIONS The initial pH of the medium has a decided effect on growth and zygospore formation in §porodinia grandis. Growth of the fungus is better in a medium with h.O percent (Nfih)280h than with 1.0 percent (NHLL)280h at pH values from 3.0 to 6.3. Zygospores are produced in a medium with h.0 percent (NHh)2SOh at favorable pH values whereas zygospores are not formed with 1.0 percent (NHu)ZSOu in the medium. With h.0 percent (N SO,4 in the medium, pH has a most H132 critical effect on zygospore production. As the initial pH of the medium declines, the number of zygospores produced on each mycelial pad decreases. From a pH of 6.2 down to a pH of 5.7 zygospores are produced. At pH values lower than 5.7 zygospores are not produced on the mycelium. Barnett and Lilly (5) have demonstrated that pH is also a factor in zygospore production by Choanephora cucurbitarum. A special type of apparatus was used in which a fresh nutrient solution of known pH continuously flowed over a mycelium.pre- viously established on non-nutritive agar. In this way the fungus was in constant contact with fresh nutrient solution. Using;this method cultures were grown in media (malt extract 1 sun., yeast extract 0.5 gm.) at pH levels of 3.5 to 8.S. These authors found that: k~ ‘« Lb ',....—.-—- 91 l. Zygospores were produced quickly within a pH range of h.S to 8.5, developing mwre rapidly in the less acid media. 2. A few immature zygospores were present at pH h.0 after two days but none developed at pH 3.5. Barnett and Lilly conclude, after growing Choanephora cucurbitarum in various synthetic media including 0.1 percent (NHh)ZSOh medium that the failure of this organism to form zygospores in still liquid culture containing certain nitrogen sources was due to an unfavorable pH. Lilly and Barnett have previously pointed out (3, h) that the asexual stage of Choanephora cucurbitarum commonly found in nature is influenced by a number of environmental factors including temperature, light, humidity and amount of carbon dioxide over the medium. It was found in this project that the addition of calcium carbonate to the medium greatly in- fluenced the type of Spore produced by Sporodinia grandis, i.e., whether sporangiospores or zygospores were produced. Growth is also greatly influenced when pH decline of the medium is restricted by addition of calcium carbonate. Growth of mycelium is approximately three times that develOped in the unbuffered medium containing h.0 percent (NHu)280h . The buf- fered medium enhances the formation of sporangiospores to the apparent exclusion of zygospores. Buffering a medium.with a 1.0 percent (NHu)280u concen- trwrtion as a nitrogen source does not stimulate the production 01' zygospores. Buffering of both media 1.0 and h.O percent m-‘W .4_ i i ..1. . W319 280“. accelerates the appearance of sporangiospores and induces a.nmch.more intense black pigmentation of sporangia and sporangiospores. Effects of Carbohydrate Concentration in the Buffered Media Utilization efficiency of carbohydrate for cell formation 5 is increased when the medium is buffered. More mycelium is i produced in the buffered medium.when the concentration of glu- cose is reduced from 2.5 to 0.5 percent. This reduction in carbohydrate concentration has little influence on the number of zygospores produced, as measured by visual observation. The number of sporangiospores in the buffered medium, as meas- ured by visual observation, is dependent upon an adequate supply of glucose in the medium. Further, an adequate amount of glucose must be present for maximum pigment intensity in the sporangia and sporangiospores when this medium is buffered. Effects of Nutrient Concentration on Growth and Zygospore Production Baker (2) has shown that the concentration of the nitrogen source is a critical factor in the formation of zygospores by Sporodinia grandig. Baker, however, did not determine zygo- spore formation with (NHu)ZSOh in the medium. The general conclusions of Baker are substantiated in this work, i.e., the (Nfiu)2SOh concentration of the medium is a critical factor in initiation of zygospores. A high concentration in the medium ( 3.5 percent) is required for zygospore formation in this fungus. In contrast, Choanephora gucurbitarum, another phyco- mycete in the Mucorineae, is able to produce zygospores in a medium containing only 0.1 percent (NHu)230h (5). Whether the formation of zygospores is influenced by the concentration of (NHh)280h as an osmotic factor is unknown. It does appear, however, under the conditions tested that the concentration of glucose has no effect on the initiation of zygospores, but does T'EI‘II‘m—IT‘F have an effect on their abundance. Growth, as well as zygo- spore production, is reduced in a glucose concentration of h.0 percent in comparison to the results in lower concentrations of the sugar. Growth of Sporodinia on Two Inorganic Nitrogen Sources Growth of Sporodinia on (NHL)ZSOH and NH'hNO3 at equal nitrogen equivalents in the medium appeared to be approximately the same at various concentrations of each nitrogen source. The consequent hypothesis that Sporodinia was able to reduce nitrates was unproved since neither TPN-nitrate reductase enzyme studies nor growth studies showed that nitrates were being reduced. However, tangential information suggested that TPN-nitrate reductase was adaptive in Neurospora crassa as reported by its discoverers, Nason and Evans (25). m, w. Effects of Two Vitamins on Growth and Zygospore Production An increase in the thiamine concentration up to 6.3 x 10"6 M in the medium increased growth in §2orodinia. The production of zygospores increased with increase in the vitamin concentration up to 3.3 x 10"6 M thiamine, but at the maximum.thiamine con- F centration of 6.3 x'lO'6 M the number of zygospores showed a ) decrease. The reason for this continuous increase in growth —'lflfi‘-§v ”run: up to a maximum thiamine concentration and a reduction of zygo- spore formation at maximum thiamine concentration has not been explained. Ascorbic acid likewise causes an increase in growth of the organism up to the maximwm concentration incorporated in the medium (1.1 x 10"3 M). Maximum zygospore formation is, however, at the lower concentration of 5.7 x 10'.5 M. Growth is enhanced at all concentrations of both vita- mins. Sexual reproduction of the organism is increased only at specified concentrations. Effects of Two Enzyme Inhibitors on Growth and Zygospore Formation The dry weight of the mycelium increased with increase in concentration of malonic acid up to 2 x 107'3 M, the maximum concentration used in the experiment. A comparison of malonic acid and growth of the organism.auggests that this fungus utilizes this chemical as a nutrient, possibly in the citric I.‘ I. a VNM ,7 vim acid cycle. Zygospore production in Sporodinia was increased with an increase in the concentration of malonic acid. The incorporation of thiourea in the medium.produced an entirely different effect from malonic acid. Growth of the fungus was neither enhanced nor reduced. The mycelial weights (remained the same in the media that contained various concen- trations of thiourea. The number of zygospores produced was reduced at some concentrations of thiourea and zygospore for- mation was inhibited at others, with all indications that in- creased amounts of thiourea in the medium.would totally inhibit zygospore formation. Chodat, Fernand and Duparc (9) have shown that thiourea blocks the production of melanins from phenolic compounds in freshly cut potato. Potato contains a polyphenol oxidase. Since melanin-like substances occur first in the zygo- spores of Sporodinia manometric tests for polyphenol oxidase were conducted on mycelial homogenates at three points in the life cycle of the fungus; at Sh hours (gametangial production), 78 hours (zygospore production) and 103 hours. No polyphenol oxidase was present when tyrosine, cresol and catechol were used as substrates for the enzyme, although polyphenol oxidase was present in the control (potato juice). Manometric tests were also conducted at 5h, 78 and 103 hours after incubation for the presence of a cytochrome oxi- dase. No cytochrome oxidase was present in the fungus mycelium When p-phenylene diamine was used as a substrate, although cytochrome oxidase was demonstrated in the control (corn embryos). Tests for non-metal terminal oxidases were not conducted, but it is assumed that the terminal oxidase is some type of non-metal oxidase. It has been pointed out previously that mycelia treated with thiourea under conditions conducive to zygospore formation F‘ appear yellow in color and smooth in texture whereas those mycelia grown under conditions not conducive to zygospore for- 1 nation are white and more coarse. Barnett and Lilly (5) noted 5 that during routine culturing of Choanephora cucurbitarum in k liquid media that the mycelium in mixed (+) and (-) cultures become "bright yellow" whereas the mycelium of each sex when grown separately was only slightly yellow. The yellow pigments were extracted from the fungal mycelium and were determined to consist chiefly of beta-carotene. Microscopic study revealed a concentration of reddish-orange granules of carotene in the suspensors previous to zygospore formation. It is suggested here that an analogous situation exists in Sporodinia and that thiourea inhibits zygospore formation with a consequent yellowing of the mycelium as a result of increased carotene concentration in the mycelium. The addi- tion of diphenyl-amine, a known carotene inhibitor, to the culture medium resulted in inhibition of carotene production and a delay of zygospore formation in Choanephora cucurbitargm (5). Thiourea apparently does not inhibit carotene production I’LV c‘ IL, in.the mycelium of Sporodinia ggandig but does inhibit zygo- spore formation. Bodine and Fitderald (6) showed that thiourea forms a complex with copper in cupric acetate. Huelin and Stephans (17) indicated that thiourea reduced the copper catalized oxidation of ascorbic acid in fruit and vegetable suspensions. The addition of ascorbic acid to the medium increases zygospore production in §porodinia. The pH of the Mycelium Acid dyes ionize to give the dye portion (chromophore) of the molecule, or anion, a negative electrical charge. Eosin is an acid dye. Basic dyes ionize to give the chromophore of the molecule, or cation, a positive electrical charge. Basic fuchsin, as the name implies, is a basic dye. The chromophore of eosin will be attracted to positively charged portions (cations) of the cell colloids. When there is a greater number of cations in the cell colloids there is greater color intensity of the mycelium when stained with this dye. Conversely, the chromophore of basic fuchsin will be at- tracted to negatively charged portions (anions) of the cell colloids. If fewer anions are present, the color intensity of the mycelium is less. In basic fuchsin after h8 hours the pH of the mycelium was found by color change to be less than pH 6.3. A slight ~s~ou__._4.__.__-# ‘ AJ F ~ 2 IQ.“ it Lu :n.. . I ' 7:: . V 3'. r 1— LA- 5.4 "b amount of color was still retained in the mycelium stained with eosin at pH 6.1 after 2h hours. In eosin, the pH of the mycelium was determined by color change to be pH 6.0 or greater. Because of the very sharp decline of colorxintensity in basic fuchsin between pH 6.3 and 6.1 the pH of the mycelium was determined to be 6.2. f Oxidation-Reduction Potentials A The pH at which oxidation-reduction potentials of mycelial f-—m_ extracts is determined has a definite effect on potential readings. When the potentials of the mycelial extracts are determined at pH 7.0 distinct differences in poising occur as well as variance in the readings. When the potentials are de- termined at pH 6.2 there is little variation in potential readings beyond the time of zygospore formation. The reactions in the mycelium which prompt gametangial and zygospore forma- tion are in all probability dependent upon the pH of the myce- lium. The most interesting comparison is that of the mycelial extract potentials and the potential of the medium when the initial pH of the medium was 6.0. At the time of gametangial formation a very stable reading is observed. As previously pointed out, well poised readings are indicative of "high" concentrations of substances at that potential. Barnett and Lilly (S) have shown that large amounts of beta carotene are 99 present in the suspensors and mycelium Just prior to zygospore formation in Qhoanephora cucurbitarum. This stable reading may well be due to an abundance of carotene at the time of gametangial formation in Sporodinia. No information could be found on oxidation-reduction potentials of the carotenes. The potential of the medium at the time of zygospore for- mation is very stable, indicating an oxidation-reduction system of relatively high concentration appearing in the medium, pos- sibly secreted by the mycelium. This potential of +O.hhS volts is in the range of potentials for some phenolic compounds. How- ever, no phenolic compounds appeared to be present in the medium as indicated by the ferric chloride test after 78 hours of in- cubation. The results of the nitroprusside test at 78 hours showed that no compounds containing S-3 or S-H groups were present in the medium, or if present not in sufficient quantity to be demonstrable. The system in the medium at the time of zygospore formation is apparently reabsorbed or a new equili- brium has taken place. This is indicated by the decline in potential after zygospore formation. There is no noticeable change in the pH of the medium at the time of gametangial formation, at the beginning of zygospore formation or after zygospores are formed. The po- tentials of both media and mycelial extracts fluctuate inde- pendently of the pH of the medium.when the potentials are determined at a constant pH. 100 f SUMMARY 1. Breakdown of the medium constituents increases with increase in the pH of the medium as a result of autoclaving. At pH 7.0 and above constituent breakdown was more pronounced (a “0‘? than at lower pH values. Breakdown was determined on the basis of reduction in pH after autoclaving of the medium. 2. Growth is greater with increase in pH in unbuffered ; media. The number of zygospores produced by Sporodinia grandis Link is dependent upon the initial pH of the medium when un- buffered. At lower pH values of the medium (less than pH 5.7) zyEOspore formation is inhibited. 3. Buffering the medium.with calcium carbonate enhances sporangiospore formation when the fungus is grown in media favorable to zygospore formation. Very few zygospores are formed in the buffered media. Growth of Sporodinia grandis Link in media buffered with calcium carbonate was always greater than growth in unbuffered media. A. The amount of glucose in the medium.influences the number of zygospores produced in unbuffered media. In buf- fared media the glucose concentration affects the amount of melanin-like pigment in the sporangiospores. When low concen- trations of glucose are incorporated in the medium.the amount of pigment in the sporangiospores was less than when higher concentrations are present in the medium. 5. Growth of the organism increased with increase in con- centration of (Nah)280k in the medium up to h.0 percent (Nfih)280h° Zygospore production was initiated when more than 3.0 percent (Nfih)280h was incorporated in the medium. Zygospores are not formed when less than 3.0 percent (NHh)230h is incorporated in the medium. 6. Sporodinia grandis grows equally well in (NHh)280u and NHANO3 when equivalent amounts of nitrogen are incorporated in the medium as (NHh)250h nitrogen and NHuNO3 nitrogen. These data were interpreted as indicating that Sporodinia grandis Link was able to utilize nitrates. 7. Increasing the amount of thiamine in the medium in- creased growth of this organism. Zygospore formation was in- creased by incorporating thiamine in the medium, but at higher concentrations the number of zygospores was reduced. 8. Increasing the amount of ascorbic acid in the medium increased the amount of mycelium produced in this fungus. Erratic results were obtained in the case of zygospore produc- tion. 9. An increase in the amount of malonic acid in the medium resulted in an increase of mycelial growth of Sporodinia grandis Link. Zygospore formation was also increased with in- crease in concentration of malonic acid in the medium. 10. The incorporation of thiourea in the medium had no effect on growth of this organism. The number of zygospores formed in the medium, however, was reduced at some concentrations vF—suuusn— m , A _.— x. 102 I and inhibited at higher concentrations of thiourea. When zygospore formation is inhibited by the presence of thiourea in the medium the mycelium appears yellow. Under experimental conditions unfavorable for zygospore formation, the mycelium appears white. These results are similar to those in which beta-carotene has been indicated to be an important factor in sexual reproduction of Choanephora cucurbitarum. 11. The pH of the mycelium of Sporodinia grandis under M conditions unfavorable for zygospore formation was determined to be 6.2 after three and one-half days of incubation. 12. Oxidation-reduction potential studies show that the medium is in a highly oxidized state at the time zygospores a are formed. After zygospores are formed the potential de- creases. L It is shown that the mycelium contains an unknown sub- stance in quantity when gametangia are formed on the super- ficial hyphae as evidenced by the highly poised potential reading observed at this time. It is suggested that this substance is the previously mentioned carotenoid pigment | % shown to be a factor in sexual reproduction of Choanephora cucurbitarum. 1. 2. 10. 11. 103 LITERATURE CITED Allyn, W. P. and I. L. Baldwin. 1932. Oxidation—reduction potentials in relation to growth of an aerobic form of bacteria. J. Bact. 21:369. Baker, R. E. D. 1931. Observations on the conditions of spore formation in Sporodinia grandis. New Phytologist 39KB)=303-316. Barnett, H. L. and V. G. Lilly. 1950. vInfluence of nutri- tional and environmental factors upon asexual reproduction 8f Choanephora cucurbitarum in culture. Phytopath. g9: 0-89 e ‘ Barnett, H. L. and V. G. Lilly. 1955. The effects of humidity, temperature and carbon dioxide on sporulation of Choanephora cucurbitarum. Mycologia 41:26-29. Barnett, H. L. and v. G. Lilly. 1956. Factors affecting the production of zygospores by Choanephora cucurbitarum. Mycologia g§:6l7-627. Bodine, J. M. and L. R. Fitzgerald. l9u9. 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Bog. ,3g:320. Robinson, W. 1925. On Some features of growth and re- e production in Sporodinia grandis Link. Brit. Myc. Soc. ; j Trans. 1Q:307-3fl1. ‘L Stanier, R. Y. l9h2. The Cytophaga group: a contribution tJ to the biology of mycobacteria. Bact. Reus.‘é:1u3-l96. Yudkin, J. 1953. Reduction potential of bacterial sus- pensions. “Biochem. J. 22;1130.