IN VITRO STUDIES ON SPECIES AND MUTANTS OF AGARICUS, CANTHARELLUS, LEPISTA. PLEUROTUS, AND VOLVARIELLA Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY Paul Albert VoIz 19.6.6 - ~ vuw-or-v» .‘«.- . qo—“—.‘-‘ ' “$2.- ' ' f. 'b I LIBRARY Michigan Sn. University This is to certify that the thesis entitled IN VITRO STUDIES ON SPECIES AND MUTANTS OF AGARICUS, CANTHARELLUS, LEPISTA, PLEUROTUS, AND VOLVARIELLA presented by Paul Albert Volz has been accepted towards fulfillment of the requirements for PhoDo degree in MYCOlogy Department of Botany and Plant Pathology :3 , ,. - 7/7 $594.? JCK’a cal—C" (.4 K 2/(_‘) Major professor 0-169 ABSTRACT IN VITRO STUDIES ON SPECIES AND MUTANTS OF AGARICUS, CANTHARELLUS, LEPISTA, PLEUROTUS, AND VOLVARIELLA by Paul Albert Volz Twelve representatives of the Agaricales were selected for nutritional work and related studies. Those included were Cantharellus clavatus Fries, three strains of Q. cibarius Fries, Agaricus campestris L. ex. Fries, A. bisporus (Lange) Singer plus two of its mutants, Lepista nuda (Bull. ex. Fries) Cooke, Pleurotus ostreatus (Jacq. ex. Fries) Kummer, Volvariella volvacea (Bull. ex. Fries) Singer and one of its mutants. The fungi were grown in Humfeld's medium with variations of carbon and nitrogen sources. A total of 11 monosaccharides, 7 disaccharides, 1 trisaccharide, 7 polysaccharides, 9 organic acids, 2 sugar alcohols, and miscellaneous compounds were added singly to Humfeld's medium as carbon sources at an equiva- lent carbon concentration. Urea was the nitrogen source. A few carbohydrates were added to the basal medium at three different carbon concentrations, and glucose was selected for growth curve studies. Paul Albert Volz Fourteen ammonium and nitrate nitrogen sources, sixteen amino acids or related compounds were added singly to the basal medium in place of urea. Mycelial growth com- parisons were made for each of the 12 selected fungi. Vitamin and growth hormone studies were also made. Observations by electron and light micrOSCOpy were made on mycelium grown in Humfeld's media and hyphae from fructifications formed in culture. Four of the 12 fungi were grown on 15 spawn media. The same four were subjected to various temperatures while growing on rye grain. Morphological characteristics and colony measurements were made of the 12 fungi when grown on 20 selected agar media. IN VITRO STUDIES ON SPECIES AND MUTANTS OF AGARICUS, CANTHARELLUS, LEPISTA, PLEUROTUS, AND VOLVARIELLA By Paul Albert Volz A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1966 Dedication to a patient parent Mrs. Frieda C. Volz and to a pertinent professor Dr. Arthur G. McQuate whose persuasion formulated this work. ACKNOWLEDGMENTS Deep appreciation is expressed to Dr. Everett S. Beneke for his guidance and assistance throughout this study, and to Dr. William B. Drew, Dr. Henry A. Imshaug, Dr. Irving w. Knobloch, Dr. Gerald w. Prescott and Mr. Samuel T. Bass for their helpful criticism of this work. Many suggestions, specimen identifications and col- lection sites were given by Dr. Alexander H. Smith, University of Michigan, Ann Arbor; and Mrs. Ingrid Bartelli, Upper Pen- insula Extension Center, Marquette, Michigan. Their help was most valuable. Appreciation is also extended to Dr. W. James Merry, Northern Michigan University, Marquette, for laboratory usage during part of the study. Grateful recognition is extended to Mr. Ivan and Mrs. Betty Ivanovich, both of the Spawn and Research Company, Utica, Michigan, for laboratory space and equipment. Materials, equipment, mushroom house space, and labor were kindly donated by Mr. Al and Mr. Pete Vannini, Rochester, Michigan. Valuable materials and cultures were received from Dr. Flordeliz R. Uyenco, University of the Philippines; and from Dr. M. San Juan, and Mr. A. I. Alicbusan, College of Agriculture, Central Experiment Station, Philippine Islands. iii Sincere appreciation is also extended to Dr. G. Bernard Wilson, Dr. Gordon C. Spink, and Mrs. June P. Mack for use of the electron micrOSCOpe and accompanying equip- ment, and to Mr. Philip G. Coleman for his photographic assistance. This work was supported, in part, by grant funds from the National Institutes of Health, Grant No. CA-OAIOS— OH, and the Michigan Cancer Foundation. iv TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . ix LIST OF GRAPHS . . . . . . . . . . . . . . . . . . xii INTRODUCTION . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . 3 MATERIALS AND METHODS . . . . . . . . . . . . . . . 28 RESULTS . . . . . . . . . . . . . . . . . . . . . . 51 DISCUSSION . . . . . . . . . . . . . . . . . . . . 189 SUMMARY . . . . . . . . . . . . . . . . . . . . . . 209 APPENDIX . . . . . . . . . . . . . . . . . . . . . 213 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . 222 LIST OF TABLES Table l. The utilization of monosaccharides by twelve fungi grown in submerged culture reported as averaged mycelial dry weight in milli- grams of three replicates at the end of both 10 and 20 days . . . . . . . . . 2. The utilization of disaccharides plus one trisaccharide by twelve fungi grown in sub- merged culture, reported as averaged my- celial dry weight in milligrams of three replicates at the end of both 10 and 20 days . . . . . . . . . . . . . . . . 3. The utilization of polysaccharides by twelve fungi grown in submerged culture, reported as averaged mycelial dry weight in milli- grams of three replicates at the end of both 10 and 20 days . . . . . . . . . A. The utilization of organic acids by twelve fungi grown in submerged culture, reported as averaged mycelial dry weight in milli- grams of three replicates at the end of both 10 and 20 days . . . . . . . . . . . 5. The utilization of miscellaneous carbon sources by twelve fungi grown in submerged Culture, reported as averaged mycelial dry weight in milligrams of three replicates at the end of both 10 and 20 days 6. The utilization of carbon sources at concen- trations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates at the end of both 10 and 20 days for g. clavatus, and three strains of g. cibarius . . . . . . vi Page 58 63 69 7A 80 91 Table 10. 11. 12. 13. 1A. 15. The utilization of carbon sources at concen- trations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates at the end of both 10 and 20 days for A. campestris, and three strains of A. bisporus . . . . . The utilization of carbon sources at concen— trations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams "of two replicates at the end of both 10 and 20 days for L. nuda, P. ostreatus, and and two strains of V. volvacea . The utilization of glucose by twelve fungi grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates at the end of 10,20, 30 and 40 days . . . . . . . . . . . . . The utilization of amino acid and related nitrogen sources by twelve fungi grown in submerged culture, reported as averaged mycelial dry weight in milligrams of three replicates at the end of IA days . . . . The utilization of ammonium and nitrate nitro- gen sources in comparison to urea by twelve fungi grown in submerged culture, reported as averaged mycelial dry weight in milli- grams of three replicates at the end of 1A days . . . . . . . . . . . . . . . . . . Hyphal growth in millimeters at two day inter- vals after inoculation on agar media con- taining vitamins . . . . . . . . . . . . Suitability of spawn media for growth of four selected fungal cultures . . . . . . . . . Colony morphology of Cantharellus clavatus (C-63-8) grown on twenty agar media for one month 0 O I O O O O O O O C O O O O O 0 O O Colony morphology of Cantharellus cibarius (C-63-15) grown on twenty agar media for one month 0 O O O O I O O O O C O O O vii Page 93 95 97 116 12A 129 133 I“? 150 Table 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Colony morphology of Cantharellus cibarius (0—63-86) grown on twenty agar media for one month I I O O 0 I O O O O O O I O O C O O O Colony morphology of Cantharellus cibarius (C- 63- 96) grown on twenty agar media for one month . O C O O C O C U C O O ‘ O O O Colony morphology of Agaricus campestris (A- 63-136) grown on twenty agar media for one month I O I O O O O O O ‘ O O O O O O O O O O Colony morphology of Agaricus bisporus (A- 63-137) grown on twenty agar media for one month 0 O O O O O O O O O O l O O O O ‘ O O Colony morphology of Agaricus bisporus pleo- morph (A-63-138) grown on twenty agar media for one month 0 O C O O O O O O O O O C C O Colony morphology of Agaricus bisporus rhizo— morph (A— 63- 139) grown on twenty agar media for one month 0 o o ' o o o o o o o Colony morphology of Lepista nuda (L-63-133) grown on twenty agar media for one month . Colony morphology of Pleurotus ostreatus (P- 63-51) grown on twenty agar media for one month 0 O 0 O O O o O O O O O O I ' O O O O O Colony morphology of Volvariella volvacea (V-63— 135) grown on twenty agar media for one month . . . . ,,. . . . . . Colony morphology of Volvariella volvacea mutant (V-63-13u) grown on twenty agar media for one month . . . . . . . . . . . . viii Page 153 156 159 161 164 167 170 173 176 179 Figure 1. 10. 11. 12. LIST OF FIGURES Drawing of Cantharellus clavatus . . . . . . Drawing of Cantharellus cibarius Drawing of Agaricus campestris . . . . . . . Drawing of Lepista nuda . Drawing of Pleurotus ostreatus . . . . . . Drawing of Volvariella volvacea Cantharellus cibarius (C-63-86) hyphae with clamp connections at all cross walls, grown 20 days in submerged culture, using Humfeld's medium with L-sorbose . . . . . . . . . . . Volvariella volvacea (V-63-135) grown on 2% ’malt agar for 15 days, showing aerial hyphae with characteristic spherical chlamydospores Lepista nuda (L—63-133) hypha with two domi- nant nuclei, from a submerged culture, grown 20 days in Humfeld's medium with glucose . . Cantharellus cibarius (C-63-86) hyphae showing several vacuoles, from a submerged culture, grown 20 days in Humfeld's medium wit glucose . . . . . . . . . . . . . .-. .4. Volvariella volvacea (V-63-135) hyphal cross section, showing three mitochondria with cristae, grown for 20 days in Humfeld's medium and glucose . . . . . . . . . . . . . Cantharellus cibarigs (0-63-96) hyphae with numerous vacuoles and crystal formation, . from a submerged culture, grown 20 days in Humfeld's medium with glucose . . . . . . . ix Page 3A 35 36 37 38 39 8H 8H 85 85 86 87 Figure Page 13. Crystal formation with Pleurotus ostreatus (P-63—51) hyphae grown 20 days in sub- merged culture, using Humfeld's medium with L-sorbose . . .p. ... . . . . . . . . . 88 14. Cantharellus cibarius (CF63-86) production of crystals along hyphae grown 20 days in sub— merged culture, using Hemfeld's medium with casein . . . . . . . . . . . . . . . . . . . 88 15. Cantharellus cibarius (0-63—86) exudate for- mation with a three month old culture, grow- ing on millet grain . . . . . . . . . . . . 136 l6. Cantharellus cibarius (C-63-86) fructifications on rye grain with Humfeld's medium minus carbohydrate, three month old culture . . . 136 17. Pleurotus ostreatus (P-63-51) basidiocarp for- mation on ground corn cobs, six weeks . . . 138 18. Pleurotus ostreatus (P-63-51) fructification on a 2% malt agar slant, six weeks old . . . 138 19. Pleurotus ostreatus (P-63-51) basidiocarp initials on 2% malt agar, five weeks old . . 139 20. Pleurotus ostreatus (P-63-51) development of rhizomorphs on 2% malt agar in a six week old culture . . . . . . . . . . . . . . . . 139 21. Agaricus bisporus rhizomorph (A-63-139) showing early formation of rhizomorphs . . . . . . . 1A1 22. Hymenium and basidiospores of Pleurotus ostrea- tus (P-63-51) grown on 2% malt agar slant . 1A1 23. Pleurotus ostreatus (P-63-51) showing one dolipore septum in cross section from a fruiting body . . . . . . . . . . . . . . . 1A2 2A. Pleurotus ostreatus (P-63-51) cell with doli- pore septa on adjacent walls, from a fructi- fication grown on ground corn cob spawn medium . . . . . . . . . . . . . . . . . . . 143 25. Cantharellus cibarius (C- 63— 86) adjacent cells isolated from a fruiting body showing a simple pore septum . . . . . . . 1AA Figure 26. 27. 28. 29. Cantharellus cibarius (C-63-86) growing on the following media: (a) Potato dextrose agar; (b) Mycological agar; (c) Micro assay cul— ture agar; (d) Violet-red bile agar . . . Lepista nuda (L-63-133) growing on the follow- ing media: (a) Chlamydospore agar; (b) Tomato juice agar special; (c) Violet-red bile agar; (d) Thioglycollate medium . . Pleurotus ostreatus (P-63-51) growing on the following media: (a) Tomato juice agar special; (b) Brewer anaerobic agar without carbohydrate; (0) Potato dextrose agar; (d) Chlamydospore agar . . . . . . Volvariella volvacea (V—63-135) growing on the following media: (a) Mycological agar; (b) Violet-red bile agar; (0) Micro assay culture agar; (d) Prune agar . . . . . . . . xi Page 182 183 184 185 Graph LIST OF GRAPHS Cantharellus clavatus (C-63—8) utilization of glucosejat concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and 40 days incubation . Cantharellus cibarius (0-63-15) utilization of glucose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and 40 days incubation . . Cantharellus cibarius (0—63-86) utilization of glucose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and 40 days incubation . . . . Cantharellus cibarius (0-63-96) utilization of glucose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and “0 days incubation . . . Agaricus campestris (A-63—136) utilization of glucose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and U0 days incubation . . . Agaricus bisporus (A-63-137) utilization of glucose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and H0 days incubation xii Page 98 99 101 102 10“ 105 Graph Page 7. Agaricus bisporus pleomorph (A-63—l38) utili- zation of glucose at concentrations of 25, 50 and 75 grams per liter grown in sub— merged culture, reported as averaged myceli- al dry weight in milligrams of two repli- cates each at the end of 10, 20, 30 and A0 days incubation . . . . . . . . . . . . . . 107 8. Agaricus bisporus rhizomorph (A-63-l39) utilization of glucose at concentrations of 25, 50 and 75 Grams per liter grown in sub- merged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and 40 days incubation . . . . . . . . . . . 108 9. Lepista nuda (L-63-133) utilization of glucose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milli- grams of two replicates each at the end of 10, 20, 30 and “0 days incubation . . . . . 110 lO. Pleurotus ostreatus (P-63-51) utilization of glucose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and A0 days incubation . . 111 ll. Volvariella volvacea (V-63—135) utilization of glucose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and A0 days incubation . . 113 12. Volvariella volvacea mutant (V—63—13U) utili- zation of glucose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and 40 days incu- bation . . . . . . . . . . . . . . . . . . 11A xiii INTRODUCTION The purpose of this work was to determine how well various sources of carbon, nitrogen, vitamins, and growth hormones were utilized by some Agaricales species. The fungi were further subjected to variations in carbohy- drate concentrations for possible substantial mycelial growth differences in submerged culture. Fungi selected for the studies include: Cantharellus clavatus Fries, Q. cibarius Fries, Lepista nuda (Bull. ex. Fries) Cooke, Pleurotus ostreatus (Jacq. ex Fries) Kummer, and Agaricus campestris L. ex. Fries collected in nature. The culti- vated mushrooms, Volvariella volvacea (Bull. ex Fries) Singer and Agaricus bisporus (Lange) Singer, were also em- ployed in these nutrient studies, along with pleomorphic and rhizomorphic mutants of A. bisporus, and one mutant of V. volvacea. Three strains of g, cibarius were selected to determine if nutritional requirement differences occur for that species. Various materials were selected as possible sources of spawn media, and Optimal temperatures were de- termined for rye grain spawn. Different agar media were utilized for morphological descriptions of the colonies. Representatives of the wild genera and V. volvacea were subjected to environmental conditions suited for mushroom production of A. bisporus to learn if fructifications would Occur under such conditions. Some anatomical work was done on hyphae grown in submerged culture and on hyphae removed from fruit bodies grown in culture. A11 fungi selected were edible and had pleasing flavors; the wild species have a potential use in commer- cial production. LITERATURE REVIEW Ultrastructural details of electron micrographs were identified according to nomenclature of current lit— erature (18, 30, 61, 85, 87, 168, 187, 188, 277, 28“). Moore and McAlear (187) were first to identify the septal pores of Basidiomycetes; structural details were further .clarified by other workers. The dolipore septum or septal pore apparatus consists of the pore bounded by the discon- tinuous septal pore cap. The latter term has replaced the earlier two dimensional concept of the parenthesome, now the cap is considered to be a dome-shaped, perforated membrane, surrounding the septal pore. When first described, the structural complex of the pore was thought to prevent inter- change of cellular organelles. Various intermediate stages interpreted as steps in the breakdown of complex to simple. pores have been seen by Giesy and Day (85) which may facili- tate migration of cell parts. Structures frequently found within the hyphal cells include mitochondria, nuclei, vacuoles, and endoplasmic reticulum. Among growth promoting substances, gibberellin has been a widely used material with Agaricales species. An increase of mycelial growth and basidiocarp production was noted when gibberellin was added to Agaricus compestris compost (3). Gibberellin in any concentration hindered growth of FomitOpsis annosa (191), Amanita caesaria, A; rubes- cens, A; muscaria, Boletus lutens and E; bicolor (2A8). High concentrations of 3-indoleacetic acid (heteroauxin) proved toxic and low concentrations failed to induce a growth stimu- lation for Pleurotus corticatus (137), Lentinus omphalodes (74), Collybia velutipes (9), and SchiZOphyllum commune (230), but markedly increased growth of Psalliota hortensis f. albida in the presence of vitamins (29, 62). Schizophyllum commune mycelium was strongly inhibited by 2,A-dichlorophenoxyacetic acid while Pleurotus ostreatus and Marasmius rotula had an increase of spore germination and hyphal growth (129). Inhi— bition responses were noted with 3-indolepr0pionic acid (7A), a naphthalene acetic acid, and 2,“,5-trichlorophenoxyacetic acid (230). Of 53 phenolic compounds tested on Polyporus schweinitzii, vanillin, vanillic acid, isoeugenol, ferulic acid and 3,H-dimethoxycinnamic acid showed high growth pro- moting activities (243). Increased mycelial production was noted with: three unidentified growth promoting substances for Poria ambigua isolated in accordance to light or dark conditions (238); Norit A filtrates of tomatoes or beech wood for Stereum fasciatum, S. subileatum, Hydnum coralloides and Peniophora coccineofulva (2H1); an M-factor isolated from roots of tomato and pea for RhiZOpogon roseolus, roots of tomato and wheat for Boletus variegatus and B. ele ans, and roots of only pea for Russula xerampelina (182); and two unidentified growth promoting substances in malt extract for Polyporus schweinitzii (239) and Fomes igniarius var. popu- _l__1_n_1_1_§_(89). Growth of representatives of the Agaricales was first developed with media composed of extracts and wastes of plant or animal material. Yeast and malt extract in con— centrations of 2 to 8% in agar or added to other nutrients have increased mycelial growth. Using malt or yeast extract, good mycelial growth was obtained with Poria ambigua (238), Formes lignosus (231), F. igniarius (89), F. annosus (A5), Pellicularia koleroga (170), Coprinus fimetarius (66), P01 - porus abietinus (79), P. schweinitzii (239, 2A3), P. cinna— barinus and P. sanguineus (163), Pholiota aurea (13), Stereum gausapatum (98), Agaricus blazei (23), Lenzites sepiaria, L. tigrinus and Polystictus versicolor (153, 198). The two ex- tracts also produced good mycelial growth in mass scale studies involving several genera of the Agaricales conducted by Robbins and Kavanagh (2A5), Law (135), Leonian and Lilly who included the species Pleurotus corticatus (138), Jennison (113), Robbins and Hervey (237), Fries who used three Tri- choloma species (70), Humphrey and Siggers who included Pleurotus ostreatus (110), Yusef (287), and Mikola (183). Fries noted that species of Boletus, Craterellus and Hydnum produced better mycelial growth in 2.5% malt extract agar in the presence of living Torulopsis sanguinea (72). Robbins and Hervey (235,237) found only A1 of 82 species, mostly Agaricales, were not stimulated by malt extract and 11 had injurious effects. For Polyporus tulipiferus and P. vul- plnus, Yusef (287) found the beneficial effect of malt ex- tract was largely the result of thiamine and amino acids in the natural product. Malt and yeast extracts at times retarded fruiting for Schizophyllum commune (220), but typical fructifications occurred in test tubes with the agar surfaces facing down- wards instead of upwards as did cultures of Lenzites sagpiaria, Hohenbuehelia reniformis, and Pyconoporus cinnabarius. Long (70) found no relationship offructificationsvmth horizon- tally and vertically placed cultural tubes. Coprinus sassii fruited well on yeast extract (19) while malt extract pro- vided a suitable medium for Collybia velutipes (8, 216, 279), Polyporus fumosus but not P. adustus or Lenzites trabea (38), Paxillus panuoides (59), Polyporus brumalis (216, 217), Fomes ulmarius, Poria ambigua (15A), and several other bracket fungi in experiments by Papazian (209) and Etter (55). Other substrates and extracts have been used for hyphal growth of the Agaricales. In addition to malt, Block et a1. (23) used orange juice, corn steep liquor, and citrus press water for submerged growth of Agaricus blazei while Mathew (170) selected onion, carrot, oat, bean, and potato extracts for growth of Pellicularia koleroga. Styer (267) utilized peach gum and pectin, wheat bran, wheat straw, peat moss and lignin for growth of Agaricus bisporus; in addition to various leaves, soils, beans, sugar beets, potato and corn meal selected by Duggar (51) for growth of A. bisporus as well as Pleurotus ostreatus, Tricholoma personatum and 16 other species. Stereum gausapatum (99) grew well on potato extracts, Coprinus and Boletus species on string bean or sugar beet decoctions (50), Polyporus amarus on incense-cedar heartwood extract (36), P. zonatus, P. fuliginosus, P. annosus on pine saw dust (233), Stereum sanguinolentum on balsam fir heartwood and less on sapwood (5A), Fomes annosus on various wood extracts (88) and extracts of a bacterium or Trichoderma when added to a solution of various salts (A6), Fomes pini on extracts of pine xylem dead several years or recently killed by weevils and not other agents (A8). La Fuze (130) made extracts of several woods and obtained good growth with Polyporus betulinus, Fomes pinicola and Polystictus versicolor, while Melin (172, 173), cultured several mycor- rhizal fungi isolated from pine and spruce, Badcock (l5) utilized sawdust for sporocarp production of Pleurotus ostreatus, P. euosmus, P. lingalilis, and 77 other wood-rotting species. Bechmann (l7) grewluxuriantlnycelial cultures of Agaricus campestris on orange, banana and tomato extracts while Humfeld (106) obtained good growth on wart agar, liquid media of asparagus-butt juice or press juice from pear waste. Potato dextrose agar produced dense hyphal colonies of many Agaricales, including Pleurotus ostreatus (98, 122). Robbins and Hervey (237) selected 82 species, mostly Agaricales, and obtained good growth with beech wood extracts except with Polyporus rutilans and Poria carbonica, and tomato extract, and with tomato extract except with Fomes ulmarius and Stereum rufum. The authors (239) also obtained growth increases of Polyporus schweinitzii by using tomato juice, coconut milk, casein, cork, agar and gelatine. In experiments involving many Agaricales species, Jennison (113) obtained good growth with extracts of potato, soybean meal, bran, corn steep liquor, molasses and gluten; Ferguson (58) utilized bean stems or pods, sugar beets, beef broth for Pleurotus ostreatus, Tricholoma personatum, Agaricus campestris plus A0 other Agaricales; in addition Long (156) used celery, alfalfa, parsnip, prune, beet, and corn meal extracts in agar. Mycelia of large numbers of Agaricales were grown on various media and decoctions of fresh vegetables and dried fruits by Robbins and Hervey (2A1), Zeller et al. who includ- ed Pleurotus sapidus (290), Robak (23A), Lyman (161), Falanghe et al. who selected Cantharellus cibarius and Tricholoma nudum plus other species (57), and Cartwright and Findley who selected Pleurotus ostreatus among their isolates (39). The earliest studies of mycelial growth were carried out by Eidam (52) in 1875 with Agaricus coprophilus on manure decoction, Wehmer (283) in 1895 with Pleurotus ostreatus grown in a sugar solution, Brefeld between 1877 and 1889 who grew over 160 species of Agaricales in various decoctions (58). The first purely synthetic medium for Agaricales mycelium was developed by Raulin in 1869 composed of ammonium nitrate, ammonium phosphate, ammonium sulfate, zinc sulfate, iron sulfate, magnesium carbonate, potassium carbonate, potassium silicate, sucrose, tartaric acid, and water (222). Before 1860, Woronin grew Exobasidium, and Hoffman worked with growth of Agaricus campestris on unde— fined media (58), while Constantin grew Polyporus squamosus in culture around 1890 (A3). Various decoctions served as media for fructifi— cations of several Agaricales. Findley (59) fruited Lenzites saepiaria on apple juice agar while Hawker (95) produced fruit bodies of Armillaria mellea, Collybia velutipes, Fomes fraxineus, Hydnum coralloides, Schizophyllum commune and Sphaerobolus stellatus on media with extracts of potatoes or lentils. Sporophores of Coprinus cubensis (119) grew on potato dextrose agar, beef extract or peptone; PDA also proved beneficial for fruiting of Agaricus leucotrichus (186) and Lentinus.tuber-regium (80). The earliest sporocarp production studies on undefined media include those done by 10 Constantin in 1891 with Nyctalis lyCOperdoides (A2), Reess in 1876 with two Coprinus species (223), VanTieghem in 1875 with Collybia velutipes (275) and Labourdette in 1861 with Agaricus campestris (131). When powdered pine, spruce or juniper wood was added to a medium containing malt extract, corn starch, Sphagnum moss, and corn meal, sporOphores of Pleurotus ostreatus and species of Polyporus, Cogrinus, Xerotus, Pholiota, Ganoderma, Lentinus and Trametes occurred (55). Beech sawdust with additions of maize meal, bone meal, or cotton wool produced fructifications of Lentinus, Phellinus, and Polystictus (1A). Alicbusan and Ela (6) produced Volvari— ella volvacea sporocarps on numerous media including ipil- ipil leaves, coir dust, wheat bran, brewer's dried grain, coffee pulp, bean leaves, corn cobs, leaflets of madre de cacao, tobacco midribs, unhulled rice, cow and horse manure. A mixture of horse dung and sawdust covered with a 6 inch soil layer produced fruit bodies of Cgprinus comatus after 10 months hyphal growth (190). Borzini utilized synthetic media to fructificate Agaricus campestris, Tricholoma species, Volvaria volvacea, and Tuber melanogaster (28). Block et a1. cultivated Agaricus campestris on composted gum-wood sawdust and Pleurotus ostreatus on balsa—wood sawdust fortified with oatmeal or soybean meal (25). P. ostreatus is able to grow on other woods (125, 198, 259) and on dead receptacles of that species (228). 11 Various garden soils influenced fructification of Tricholoma nudum (AA) and Volvaria volvacea (31), while comp- 081:; or horse dung extracts and salts in the medium developed fruit bodies of Agaricus leucotrichus (186), 00prinus sassii (19 ) , and _C_3_. sterquilinus (189). A nutrient depleted medium is generally an inductive agent for fungal fructifications or a medium suited for less mycelial growth (9), or portions of a. colony where aerial growth is the least (156). Carbon dioxide appears to play a role in basidio— carp develOpment for several species of Agaricales. Res- PiI‘atory C02 in sealed chambers arrests fruiting of Schizo- W commune at the primordia stage (102, 193, 19“), redues-s pilei expansion of Collybia velutipes (155, 215): inhibits fruit body production of Polyporus palustris (60), and produces shorter stipes and small pilei of Agaricus w (132). Wherever Agaricus campestr’is is grown, substances of volatile nature accumulate and become detri- mental to fructification (167). Respiration of §_. commune was increased with sucrose, certain hexoses, D-xylose, aCEtate and ethanol whereas certain other pentoses and amino acids had little effect, and D—glucose or acetate produced incomplete oxidation thus varying 002 release and fruit body forms (193). Certain nitrogen sources activate homodikaryotic fruiting in P. commune (102). 12 The temperature and pH influence production of fruit bodies. The optimum temperature for best fruiting was 250 C for COprinus lagOpus (27, 166, 276), Cyathus stercoreus (15 7), Armillaria mellea (225), Poria ambigua (2A0), Schizo- phy llum commune (93), 3O-A5O C for Volvariella volvacea (2 , 6), 26-310 c for Pleurotus ostreatus on PDA and 22-29° c on rye grain (132), 31° C for Agaricus campestris on PDA and and 22-26° Con rye grain (2A), 22-270 C for Polystictus Ersicolor, Polyporus annosus, Pleurotus ostreatus and Lsalliota bispora grown on a basal medium of glucose, vita- ruins and salts (125), 150 C for Polystictus versicolor on male extract and peptone agar (l6). Aschan-Aberg learned that Collybia velutipes developed good fruit bodies at 25° c in darkness for one week followed by 15° C in light (8, 9), OP 20° 0 without temperature fluctuations (10), while Kinugawa stated that a minimal duration for fruiting was 2 days at 50 to 100 0 followed by 150 c or at alternating temperatures of 50 and 200 c (123). The optimal initial hB’Citr'oogen ion concentrations were recorded as follows for a few species: 9. velutipes, 5.2-7.2 (8, 9, 215); E. ostreatus, 5-6.2 (2A); Armillaria mellea, 5.0 (225); Agaricus campestris, 5-5-6.0 (6A). The terminal pH was generally somewhat lower. The presence of light functions in fruit body formation of several species. Long (156) believed the presence of light is essential to the production of sporophores when grown on artificial media while the character of the substra- tum plays only a very minor role in sporOphore initiation. 13 He obtained 629 sporophores in the light and only 11 in dark- ne SS from the following genera: Coprinus, Daedalea, Exidea, Formes, Ganoderma, Irpex, Lentinus, Lenzites, Marulius, Panus, Pleurotus, Polyporus, Polystictus, Poria, Stereum and Trametes In darkness, Polyporus farlowii, P. cinnabarius, and Trametes serialis developed sporophores but the latter produced no spores. Stipe growth occurs in darkness but light is required for pilei expansion and normal spore production of Pleurotus, Polystictus (125), Collybia velutipes (2, 8, 155, 216), and _P_Silocybe COprOphila (86). The blue light of the spectrum is positive while red light is negative for sporocarp deve- lODment of Poria ambigua (238, 2A0), Comrinus lagopus (27, 253) , and Collybia velutipes (11). Long stipes, small caps and no gills form with Coprinus lagopus in yellow green light as in the dark (27). In contrast to Long, Block (2A) fC’l-ltt'ld that Pleurotus ostreatus fruited quite normally in I316: dark, while Koch (125) stated that mycelial growth of §§é1lliota bispora was inhibited by light. Koch (125) also shOwed light was not required by Polyporus and Psalliota for r“Dismal develOpment and maturity, and Ferguson (58) believed light inhibited spore germination of Agaricus campestris. Stipe elongation of Collybia velutipes and Polyporus brumalis occurs in pure culture as light intensity is reduced (216). Other authors also suggested light was needed for normal fruiting body formation of Collybia velutipes (9, 215), COprinus lagopus (276), Polyporus pglustris (60), Schizo- phyllum commune (102, 19A, 220), Lentinus lepideus (33), 1A I><3>21yporus brumalis (217), Pleurotus ostreatus (15,210), Poly- 551:.:ictus versicolor, Trametes serialis and Fomes pinicola, I?<:>:1yporus schweinitzii (l5), and Lentinus tuber-regium (80). c3<:>1:rinus lagopus will fruit in continuous light or in brief exposures to light of one second at 250 ft c or 5 seconds at (3.. J. ft 0, but not in darkness (166). The amount of light €3r1€317gy required for fruiting of Cyathus stercoreus is a con— :st:zirit 17200 ft 0 hours at a light intensity of 2A0 ft c (157). (31:1163r conditions needed for fruiting are: genetic inheritance ( 86) , rich, well aeriated medium, high relative humidity (15), and accumulation of metabolic products (220). Gravity was shown to effect sporocarps in culture. 'rrhe stipe of Polyporus brumalis (217) and sporocarp of Pleu— rotu —-._~§ ostreatus (2A) are - geotrOpic and + phototrOpic, the phototrOpism can mask the geotrOpism. Amanita phalloides and A. sgrenulata stipes are - geotrOpic while the caps are + geo- Iac: . euro us os rea us ru ng on pOpu ar ogs “‘0 i (265) P1 t t t f iti l 1 is tnot modified by position of log but by direction of gravity I271). When grown on a defined medium, variations of nutri— ents have shown variations in the ability of fruiting. The following carbohydrates produced fructifications in media with the following Agaricales: Schizophyllum commune - sucrose, maltose, trehalose, cellobiose, glucose, fructose, mannose, galactose, xylose, mannitol, glycerol, ethanol, while acetate and citrate appeared inhibitory (195); Coprinus sassii, C. 15 rlgggggtemerus, g. congregatus - maltose, sucrose, but glucose (31:1:1y developed a luxurant mycelial growth (20); Coprinus EL£§E£3£22§.' cellulose, glucose, maltose, fructose, starch, vv1:1:1le negative results develOped with galactose, lactose, £521.31cerol, sodium oleate, sodium potassium tartrate (165,278); Pleurotus ostreatus — corn steep liquor, molasses, glucose, (zeajlglulose, peptone, while starch was negative (2A,l2A); g;;>:1§;ybia velutipes - sucrose, glucose, while maltose PVEiES poor (121,215); Polystictus versicolor - fructose, gly- cerin, mannitol, maltose, xylose, glucose, saccharose, and Starch (125). Glucose was the carbohydrate source singly Selected by authors for sporocarp production media along with El Iligtrogen source and a variety of salts (96,220,2A0) for Phleb \ Opus lignicola (207), P. sulphureus (206), COprinus .Ee hemerus (96), Xerocomus badius and A. illudens (208), Sui -\11 us rubinellus (16A), Polystictus versicolor (125), and % ambigua (238,2A0). The following fungi were able to utilize the listed n1trogen sources and produce sporocarps in culture; Schizo- PIEzllum commune - asparagine, urea, glutamine, glutamic acid, Serine, alanine, arginine, tryptophan, ammonium phosphate, Complex nitrogen sources and ammonium salts (195); Coprinus lagopus - organic, ammonium, and nitrate nitrogen (165,250,278); Pleurotus ostreatus - casamino acids, pep- tone, sodium nitrate, ammonium sulfate (2A,l25); Collybia l6 ‘Vregeilutipes - asparagine, ammonium tartrate (8,215); Poly- 5515;:1ctus versicolor - ammonium chloride, ammonium tartrate, Elly-gainine, asparagine, glycine, glutamic acid (125). Vitamins have increased fruit body production of several Agaricales. Those affected and the vitamins in- vo 1ved are: SchiZOphyllum commune - thiamine (93,220); IngzaJricus campestris - thiamine, biotin (273); Poria ambigua 1:11e known B vitamins (2M0); Polystictus versicolor - thia- n13.r1€3 (125); and thiamine for Coprinus ephemerus (96,21“), Sl- Ilagogus (165,278), g. sassii (l9), Pleurotus ostreatus (3311,2125), Poria ambigua (238), and Polyporus palustris (60) IIr1 ciecreasing order, nicotinic acid, pantothenic acid, bio- txir1,. thiamine, riboflavin, and pyridoxine increased yields Of‘ Ikgaricus bisporus when added to mushroom beds (J~C)3,13H,22u) and a 78% greater yield occurred with a com- bir(lation of riboflavin, nicotinic acid and pantothenic acid (]~C)3). None of the B vitamins had an effect on fruiting of C<3\:Llybia velutipes (8). Miscellaneous substances increasing fructifications °i7.Agarica1es include four fatty acids (linoleic, palmitic, Oleic and stearic acid) for Polyporus schweinitzii (2M3). Mycelium inhibitors, rose bengal, tergitol-7, aerosol, and sodium lauryl sulfate, initiates earlier and more profuse fruiting of SchiZOphyllum commune (220). l7 Vitamins added to basal media composed of carbon, r1;i,‘trogen and salts greatly increased mycelial growth and dry ‘gweazjghts of the Agaricales. The organisms heterotrophic for t:]:1:1amine or the pyrimidine component of thiamine include c>1r1£e report of “1 species or strains of Polyporus and Fomes (.22i37,288), Fomes lignosus (231), E. annosus (226), Polyporus £1t3:ixetinus (79), Polystictus versicolor (125), Paxillus Igzrtzziulus, Amanita pantherina, Tricholoma albobrunneum, T Llavobrunneum, T. vaccinum, T. fumosum, T. pessundatum, T_. Ambricatum, Boletus luteus, _B_. variegatus, g. piperatus, B. granulatus, Cenococcum graniforme (68,176,177,178,201), SeVezr'al species of Polyporaceae (2514), Stereum murraii (236) (3011 -_i__gybia tuberosa (138,245), Cogrinus sp. (245), and Maras- EEELig; fulvobulbillosus (148). Thiazole is probably synthe- Sized by the fungi but pyrimidine must be supplied by other SO11:!rces. 6 If both pyrimidine and thiazole are present, thia mihe can be synthesized by Polyporus zonatus (25M), Amanita E§J1therina (178), Marasmius alliaceus, M, rotula, M. scoro- dOnius (68,1u8). In experiments involving many fungi, thiamine deficiency has also been noted by Lindeberg (109) for species of Clitocybe, Collybia, Lepiota, Mycena, flypholoma, Pholiota, Flammula, Panus, fiydnum, Clavaria, Stropharia, Tubaria; by Fries (75) for species of Peniophora, Panaeolus, Panus, Polyporus, Lentinus, flyctalis, Omphalia, Trametes, 18 Strgpharia, Hydnum, Collybia, and Sistrotrema; and by Jennison et al. (115) for species of Daedalea, Fomes, Hydnum, Lentinus, Lenzites, Polyporus, Peniophora, Poria, Ptycho- ga ster, and Trametes. Several other authors have noted thiamine deficiency for: 8 Coprinus sp. (65), 15 Marasmius Sp . (148), 12 Polyporus sp. (70), 19 Mycena sp. (73), 4 Polyporus sp. (140), Collybia velutipes (140,215), 6 Clava- ria sp., 4 Boletus sp., Lactarius deliciosus, Tricholoma _i_I_n_- 31 catum, Psalliota arvensis, Collybia dryOphila, Mycena eEiEterygia, Stropharia aeruginosa (174), Marasmius foetidus, Boletus variegatus (151), __B_. granulatus (181), E; m, Clestc) \ pilus prunulus, Tricholoma imbricatum, T. pessundatum (177,180,245), Lenzites betulina, Fomes annosus (35,53), F- 1—Sl'liarius, Stereum frustulosum, Hydnum erinaceus, Polyporus W (199). E. was (212). B- mils, B- dim, QOphora olivacea (204), Coprinus lagopus, ijctalis sp., lM‘\Qllrotus corticatus (138), T’. ostreatus (24,129,274), Lenti- nu\S omphalodes (74), Cortinarius glaucgms (176), Lenzites tr \— Eibea (141), Stereum gausapatum (101), Schizophyllum commune (2’44), and Armillaria mellea (84). Essentially no requirements for vitamins, parti- cularly thiamine autotrOphy, occurred with Lactarius delicio- §u§, Tricholoma albobrunneum (177), Coprinus curtus (68), Poria monticola, Polyporus balsameus (204), Lenzites seapiaria (126,262), Agaricus campestris (244), Psalliota bispora (125), l9 Pleurotus ostreatus and Lenzites trabea (140). In contrast other workers found thiamine beneficial for strains of Agaricus bisporus (63), T. ostreatus and T. trabea (24,129,14l,274). The thiazole moiety of the thiamine molecule is required and pyrimidine therefore is synthesized for light emission of Collybia velutipes (l), and for mycelial growth of g. velutipes (169), Stereum frustulosum (200), Fomes noxius (232). Members of the Agaricales found to be hetero- trOphic for thiamine and nicotinic acid include Polyporus abietinus (79), Pholiota aurea (l3). Polyporus texanus is heterotrOphic for thiamine and pantothenic acid, and T. immitus is heterotrOphic for thiamine and adenine (288). Best mycelial growth occurred with thiamine and biotin for Marasmius foetidus, M. androsaceus, and M, perforans, very poor growth developed while vitamins were used singly or with inositol (145). A combination of thiamine and biotin best stimu- lated hyphal growth of Psalliota hortensis f. albida (62), Polyporus palustris, T. tulipiferus (114), and Pellicularia koleroga if thiamine was added to glucose or sucrose, or if biotin was added to sucrose medium but not glucose (170). Collybia dryOphila (150), g. velutipes (169) also had in- creased mycelial growth on riboflavin (280),along with Trametes serialis (262), Psalliota campestris (124,273). Marasmius androsaceus (148), and Tricholoma albobrunneum (177). 20 Thiamine, inositol and biotin benefitted Poly- pgrus adustus, B. abietinus, Fomes pinicola, Tricholoma nudum (126,262), and Pellicularia filamentosa (256). Biotin alone in media was heterotrOphic for hyphal growth of Fomitopsis annosa (191), Marasmius androsaceus (147), and Daedalea quercina (140). Tricholoma imbricatum is partially heterotro- phic for panthotenic acid (201) while T. fumosum and Lactarius deliciosus are partially deficient for nicotinic acid (175,176,201). A mixture of 12 vitamins added to a basal medium increased hyphal growth of 41 Polyporus and Fomes genera (235). Lactarius species were unable to utilize thiamine or biotin but had excellent growth in— creases with inositol, calcium, pantothenate, p—aminoben- zoic acid and good growth with folic acid, nicotinic acid, pyridoxamine, pyridoxine and riboflavin (112). Khudiakov and Vozniakovskaia (121) obtained good hyphal growth with Boletus edulus, B. luteus, B. luridus, and B. variegatus inra medium containing nicotinic acid, p-aminobenizoic acid, riboflavin, calcium pantothenate, biotin, and thia- mine. Pyridoxine, inositol and biotin singly act as accessory factors for growth of Ganoderma lucidum (140). Lilly and Barnett (140) believe it is entirely possible under varying environmental conditions that some fungi would be deficient for one or more growth substances and that other isolates of the same species would differ in their nutrient requirements. 21 Hydrogen ion concentration of liquid media for mycelial production of Agaricales has been investigated by a few authors. The Optimal pH for several cultures was observed as follows: Pleurotus ostreatus, 5.5 (274); Polyporus zonatus, 5.5; B. fuliginosus, 4.5; B. annosus, 5.5 (233); Psalliota bispora f. avellanea, 6.0—6.8 depending on substrate (273); 8 Tricholoma sp., 4.0-5.0 (201); Fomes annosus, 4.6-5.6 (53, 272); Pholiota aggga, 5.0-6.4 (13); Coprinus cubensis, 6.9 (119); Polyporus betulinus, Fomes pinicola, Polystictus versi- BBTBB, 3-4.5 (130); Collybia velutipes, 6.5-6.9 (279), and 6.0 for maximum luminescence (1); Agaricus campestris, 4.5 (109); Boletus elegans, 4.5 (105); Marasmius alliaceus, 5.2; M. foetidus, 3.1; B. graminum, 6.9 (146); a Cogrinus sp., 5-7 and 8.0 (69); Stereum murraii, 5.3-6.1 (236); 8 Coprinus sp., 7.8-8.2 (65); Agaricus blazei, 5.5 (23); COprinus cubensis, 6.9 (119); Psalliota bispora, 6.0 (64), 6.8 (273), 6.3-6.7 (133). Early pH investigations by Schmitz (251), Wolpert (285), Rumbolt (247), and Meacham (171) indicated Optimum growth range between 3.5 and 6.5 for Polystictus versi- color, B. adustus, SchiZOphyllum commune, Lenzites sepieria, Pleurotus ostreatus, Pholiota adiposa, Armillaria mellea and Fomes pinicola, B. roseus. Except for B. commune and B. ostreatus, mycelial growth was checked in alkaline solutions and inhibited at pH 3.0. 22 Hacskaylo et a1. (91) found that the greater the relative amount of growth of an isolate at succeeding temperatures, the lower the final pH of the corresponding solution. Of 135 Agaricales species, the raising, lowering or stabilization Of the initial pH occurs according to individual species, strain, even isolate, and media (184, 273). Wang and Miles (282) learned with Schizor phyllum commune that changes in pH and total acidity cor- responded roughly to the growth of mycelium, an increase in mycelial dry weight was accompanied by a decrease in pH and an increase in the total acidity of the culture medium. 4 Optimum temperatures for mycelial growth were determined for several Agaricales by various workers. Amanita rubescens, and Russula emetica grew best in sub- merged culture at 290 C, Suillus punctipes between 24—29° C, and B. luteus 18-24° C (91). Other temperature Optima are: Amanita muscaria 25° C (205), Fomes annosus 23° C (53), COprinus cubensis 350 C (119), Paxillus panuoides 25°.C (59), Agaricus campestris 25° C (109, 266), Schizo- phyllum commune, Polyporus Oblique, B. albellus, B. tulipi- fera, Lenzites trabea 250 C (144), a Coprinus sp. 250 C (69), Polyporus abietinus 250 C, Mycena flavo-alba 15° C (77), Armillaria mellea 25° C (225), 19 Mycena sp. 20° C (73), 25 and 31° c for different Polyporus abietinus strains (79), Psalliota bispora 24° C (273), 26.70 C (51), 25° C (132), 13-210 C recorded by various authors reported in early literature (273). 23 Nutrient media for Agaricales usually have glucose as the carbonsource if physiological studies are involved (1, 21, 35, 63, 69, 92, 94, 105, 108, 109, 112, 116, 121, 125, 146, 196, 197, 199, 201, 202, 203, 205, 212, 225, 236, 264, 268, 269, 288) while a few authors selected sucrose (101, 215, 279) and maltose (19, 130, 160, 250). In carbon studies, some researchers identified useful carbon sources by analysizing the presence of sugars or enzymes in basidiocarp fractions (34, 128, 135, 136, 221, 289) or liquid cultures (252), however, all others carried on such experiments by varying nutrient media. The monosaccharides glucose, fructose, mannose, or xylose provided greatest hyphal growth for species of Coprinus (68, 119), Lactarius (112), Collybia (1, 215, 279), Stereum, Hydnum, Polyporus, Polystictus, Fomes (77, 90, 100, 125, 130, 143, 199, 212), Pholiota (13), SoniZOpnyllum (143, 196, 269, 282), Lenzites (143), Boletus (105, 175), Mycena (77), Armillaria (225, 252), Pleurotus (125, 274), and Agaricus (26, 81, 107, 109, 266). Fructose-1,6-diphos- phate had a very striking growth promoting effect on Boletus and Collybia (197), and all species of Agaricales used by Lilly and Barnett grew more rapidly and produced more mycelium on L- compared to D- arabinose (26, 144, 269). Among disaccharides, maltose, lactose or sucrose were found most usable by several authors for Agaricales 24 (13, 26, 77, 81, 100, 105, 107, 109, 112, 119, 130, 143, 196, 212, 215, 225, 252, 266, 267, 269, 279, 289). Collybia was usually not able to utilize disaccharides (1), but Plunkett (215) noted that sucrose equalled glucose as the best carbon source in hyphal production. Raffinose was usable for Polyporus, Fomes, Armillaria, Daedalea (130, 143, 252), Lenzites (143, 289), Stereum (100), SchiZOphyllum, Collybia (143, 196, 269), Coprinus (119), Polystictus (130). According to Stoller (264), there should be an application of agricultural practices to mushroom culture Of establishing NPK and C/N ratios due to the principle of limiting factors. Depending upon what nitrogen source is selected, the carbon source is then utilized in B. bisporus cultures (26). A review of literature indicates the polysac- charides dextrin, starch, and xylan proved good carbon sources for the Agaricales Boletus, Lepiota (94, 105), Polyporus, Daedalea, Armillaria (130, 212, 252), Lenzites (289), Stereum (100), Fomes, Polystictus (125, 130), COprinus (68, 119), Pholiota (13), Lactarius (112), Pleurotus (125, 274), Agaricus (81, 94, 107, 109, 133, 266, 267). Cellulose or lignin was utilized by Armillaria (225, 252, 285), Lenzites (285, 289), Stereum (100), COprinus (68), Polyporus, Polystictus (37, 83, 285), 25 Paxillus (59), Fomes (53), Marasmius (148), Tricholoma (175, 201, 227), Pleurotus, Pholiota (37, 285), and Agaricus (26, 133, 266, 267, 281). Moderate or poor mycelial growth developed with organic acids (26, 51, 77, 105, 112, 274), and sugar alcohols (13, 26, 27, 143, 196, 227, 269). Nitrogen utilization studies for hyphal growth are limited in comparison to other nutrient studies with species of Agaricales. Amino acids, proteins, ammonia, ammonium salts, peptone and urea all serve as useful nitrogen sources for Agaricales species (22,23,26,51,63, 82, 107, 109, 111, 133, 266, 267, 273). Ammonium salts and nitrates induced poor growth with Pleurotus while peptone, urea and amino acids were best (92, 125, 138, 139, 262, 274). Peptone also was very useful as a nitrogen source for other Agaricales (13, 84, 100, 101, 105, 119, 130, 199, 225). Greatest hyphal growth was Obtained with amino acids and urea while ammonium salts increased growth, nitrates were slightly or not utilized by species of PoTystictus (125), Tricholoma (175, 201, 202, 227), Armillaria (84, 225), Mycena (73, 77), Polyporus (77, 115, 130) Stereum (100, 101, 236), Fomes (115, 130, 232), Bydnum, Lentinus, Lenzites, PeniOphora, Poria, Ptychogaster, and Trametes (115). NOecker (199) found an ammonium source for nitrogen was slightly better for hyphal growth than an amino acid for species of Stereum, Hydnum, Polyporus, and Fomes. 26 It was assumed by most workers that only the L- isomers in the racemic amino acid mixtures were utilized, although some of the D— isomers may be utilized also (117, 227), the L- form was more useful than the D- form (116). D-alanine was used by Lenzites trabea (117), and Psalliota campestris can hydrolize both Optical isomers of proline, alanine and leucine (219). Ammonium salts produced Optimum growth while amino acids and nitrates were of less use for species of Boletus (105), Collybia (l, 8, 138, 215, 279, 280), Marasmius (148). Several amino acids were found useful as the sole nitrogen source for Marasmius (152). Ammonium salts also proved productive for Lactarius species (112), however, 18 amino acids used singly or in combination increased the hyphal yield (179). Vinasse, a fermentation product Of sugar beets, high in ammonium salts was useful for Boletus, Agaricus, Tricholoma but useless to Cantharel- lus and Collybia (56). Having an unknown metabolic role, ferulic acid greatly increased hyphal growth Of species of Corticium, Fomes, Hydnum, Hymenochaete, Peniophora, Poly— porus, Poria, and Stereum (242). In nucleic acid nutritional studies with fungi, Fries found an adenine deficiency for Lenzites betulina, B. abietina, Polyporus abietinus, B. cervinus, B. annosus (75), adenosine deficiency for Mycena species, and a slight growth promoting effect by hypoxanthine, guanine and grano- sine for species of Polyporus (76). 27 Amino acids and ammonium salts induced greatest hyphal growth while nitrates were poor for SchiZOphyllum (196, 269). Nitrates, nitrites and urea were more useful than amino acids to COprinus (119, 138) while amino acids were superior to ammonium salts (68, 139, 262), particu- larly useful was asparagine (214)and DL-methionine (67). Using 8 COprinus species, Fries found that ammonium salts seemed to give better growth than asparagine (65), and obtained physiological mutations induced by nitrogen mustard that had differing amino acid requirements (66). Physiological mutants produced from oidia, basidiospores, by use of ultra-violet light, and nitrogen mustard developed arginine - less, methionine — less, adenine - less, cytidine - less, uridine - less, amino acid - less, and nucleic acid - less strains of Collybia velutipes (7). MATERIALS AND METHODS Nine homobasidiomycetes were used in this study. Initial transfers of eight of them were made aseptically from pileus tissue. The ninth, Pleurotus ostreatus was grown from spores streaked on agar media. The two mutants Of Agaricus bisporus and one of Volvariella volvacea occur- red in stock cultures. All stocks were transferred every ten weeks. The collection sites and dates for the Cantharel- ABE specimens are as follows: 9. clavatus (C-63-8) on the Michigan State University campus, July 8, 1963; B, cibarius (C-63-15) from a forest east of Marquette, Michigan, August 2, 1963; B, cibarius (C-63-86) in the University of Michigan Savoy Forest near Pellston, Michigan, August 26, 1963; and B. cibarius (0-63-96) in a Jack pine grove west of Marquette, September 21, 1963. The cultures were maintained on 2% malt agar (see appendix for media contents). Agaricus campestris (A-63-l36) was collected on the grounds of Walnut Hills Golf Club, East Lansing, Michigan, September 25, 1963. A white strain of A. bisporus (A-63-l37) was Obtained from the Mushroom Growers Association, Niles, Michigan, July 12, 1963. The mutant forms occurred naturally 28 29 TB vitro from this culture. Agaricus bisporus rhizo— morph (A-63-139), a mutant, produced abnormal numbers of rhizomorphic strands in comparison to the parent colony. A second mutant, B. bisporus pleomorph (A-63—138) developed a cottony aerial culture. Three of the Agaricus cultures were maintained on medium A while the rhizomorphic form was kept in stock on 2% malt agar. The latter originated from a parent culture grown on medium A. Lepista nuda (L-63-133) was collected at the University Of Michigan Biological Station, Pellston, Michigan on September 14, 1963. The spore isolates of Pleurotus ostreatus (P-63-51) were from a spore print of a basidiocarp grown on beech near Tahquamenon, Michigan, August 8, 1963. Volvariella volvacea (V-63-l35) was obtained from the University Of the Philippines; 2, volvacea mutant (V—63-134) is a white colony form of the commercial Philippine mushroom. All were kept on 2% malt agar in stock. The description of the fungi used follows: Cantharellus clavatus Fries, the pig's ear mushroom (218, 259, 260), has a broad pileus, which becomes funnel- shaped and lobed (Figure 1). The stipe and pileus are in distinguishingly fused, both tapering downward, with a dry surface which later becomes scaly. The pileus surface remains smooth. Ridge-like gills are decurrent, extending almost to the stipe base, freely anastomosing by means of 30 interconnecting "veins". The tissue is firm and white to yellowish brown. The spore print is ochraceous, from four-spored basidia. The hymenium is without cystidia. The immature grayish to purplish tinted basidiocarp fades to a dull brown with age. The stipe is compound, fusing together at the base, and solid becoming hollow. In Michigan the species is found during late summer and fall, in a conifer forest habitat. Cantharellus cibarius Fries, the chanterelle mushroom (218, 259, 260), has a broad, flat to slightly depressed pileus with an inrolled margin which becomes wavy and lobed (FigureED. The surface is dry, covered with a thin coat of fine fibrils. The flesh is thick, white to pale yellow. then young, the basidiocarp is yellow, becoming bright yellow to orange with age. Lamellae are decurrent, dichotomously forked, narrow and have interconnecting "veins". The basidia have four spores, and the spore print is yellow. The stipe is thick, solid and white. The species is found in summer and early fall, singly or in clumps, and generally in a conifer forest. Agaricus campestris L. ex. Fries is the common pink gilled field mushroom (120, 185, 218, 259). The glabrous pileus is convex with a flattened center (Figure 3). It is white colored, later turning slightly yellow to light brown. A slightly fibrillose condition was noted. The flesh is white. The gills are free, thin, crowded, wide, 31 rosy when young and turning pink to dark brown at maturity. The basidia are four-spored, clavate, and have dominant sterigmata. The spore print is dark brown. The cylindrical stipe is thick, solid, tapered downward, smooth, becoming hollow. A ring is found slightly higher than the stipe center; its margin is ragged. In late summer to fall, the species is found in Open areas such as pastures or meadows. Its smell is acidulous. Bgaricus bisporus (Lange) Singer (185, 257) is the cultivated mushroom most commonly grown in the western hemisphere. The cap is convex with a slightly flattened center, white or cream, finely squamulose, with a thick margin slightly fibrillose to striate or crenate. The gills are free, crowded, narrow, fimbriate edged, pink to brown at maturity. Numerous cystidia are on the gill edge. The spore print is sepia to dark brown and the basidia are two-spored. The flesh is white to light tan in color, at times slightly pink. The stipe is white to tan, solid to fibrous to hollow with age, and slightly flOcculose below annulus. The ring is white, thick, narrow and striate above. The stipe base is slightly expanded, tapering upwards. The Odor at first is pleasant and mild, becoming acidulous with age. Wild strains Of B. bisporus are found during summer and fall in rich soil near manure heaps, in gardens and parks. 32 Lepista nuda (Bull. ex Fries) Cooke (162, 218, 258, 259), the wood blewit, has a smooth, moist cap which is a slightly convex disc or plane, thick, very solid, pale blue gray to purple (Figure 4). Some have a viola- ceus margin and a brownish disc with fine hairs present. The flesh is tinted lavender, white or gray. The free lamellae are crowded, broad, violaceous to white to brown with age and are easily separated from the cap. The spore print is white with a slight salmon tint. The stem is short, solid, slightly tapered at the apex from a thickened base. The surface possesses fine hairs and is blue to violet in color. It is found in late summer and fall in hardwood forests, in piles of decaying leaves or soils with a high organic content. Pleurotus ostreatus (Jacq. ex. Fries) Kummer, the oyster mushroom (162, 218, 259), is found on aspen, beech and sometimes elm in Michigan (Figure 5). The pileus is oyster shell-shaped, smooth, moist, white becoming brown or gray. The flesh is thick, white and soft. The gills are decurrent, broad, quite distant, continuing to stem base, white becoming light yellow. The stipe is lateral, short or nearly absent, large, solid, and white. The spore print is lilac gray. Pleurotus is found in Spring or fall, during extended moist weather. Volvariella volvacea (Bull. ex. Fries) Singer (97, 162, 255, 257) is the paddy straw mushroom, and is 33 cultivated on a small scale in the Philippine Islands and other tropical areas (Figure 6). The pileus is broad, ovoid, convex, whitish to gray brown with dark gray silky fibrils. It has a subumbonate disc and the flesh is white. The gills are broad, free, close, and white becoming salmon. Conspicuous cystidia are present. The basidia are four- spored. The spore print is dull pink to brownish pink. The stipe is off white to dull brown, firm but soon becoming hollow, fibrillose, very slightly attenuated upwards. A distinctly large volva is present which is irregularly lobed, membranous, floccose, brownish. Its natural habitat is wood, soil, dung, where it spOrulates from June to Septem- ber singly or in tufts throughout trOpical Asia. All experiments were done in replicates Of two or three, occasionally as many as five. Submerged culture experiments were carried out in a constant temperature room of 740 F. The inoculum grew on stock plate cultures for three weeks, after which a 1.5 inch square section Of the colony was removed and blended one minute with 55 ml distilled water in a 350 ml Monel metal semi-micro Waring Blendor or a Barocilicate glass Jap Blendor. Five ml of the blended mycelium was pipetted into 250 ml Erlenmeyer flasks or 250 ml DeLong Culture Flasks containing 50 ml test medium and then placed on New Brunswick Gyrotary Shakers at 180 RPM. Flasks were stoppered with cotton, covered with aluminum foil, 34 Cantharellus clavatus Figure l. 35 Cantharellus cibarius Figure 2. 36 /’/ ,k\\ J r Agaricus campestris Figure 3. 37 Lepista. nuda Figure 4. 38 efifi‘i 75? = _~ / . , ,/ , F‘s‘wfi’fiw 1 2117/7 +~ “ga:§wf‘MfiMflfj Pleurotus ostreatus Figure 5. 39 Volvariella volvacea Figure 6. 40 or Morton culture tube closures. The flasks were auto— claved twenty minutes at fifteen pounds pressure per square inch, 1210 C before inoculations were made. Ex- periments were carried out under aseptic conditions. A modified Humfeld's basal medium (109) was used for the shake cultures and substitutions were made according to the experiments involved. The ingredients of Humfeld's medium are: glucose, 50 g; KHZPOu, 0.87 g; MgHPOu, 0.40 g; CaC12 - H20, 0.37 g; H2SOu(2N), 5.1 ml.; trace element solution, 20.0 ml (FeC13 ' 6H20, 0.5 g; MnC12 - 4H20, 0.36 g; ZnC12, 0.20 g; CuSOu, 0.05 8; distilled H2O to make one liter), 1,000 ml distilled H2O.) One g urea was added per liter for the nitrogen source. Of 1, 2 or 3 g urea per liter, one g produced the greatest mycelial weight with most of the twelve fungal cultures. Triplicate petri dish cultures of the twelve fungi were also made using Humfeld's medium plus 30 g agar per liter, with l, 2 or 3 g urea per liter. Greatest colony growth also occurred with one g urea per liter. Potassium phosphate was dissolved in the sulfuric acid and 50 ml distilled H2O before being added to the Humfeld medium. The pH was adjusted to 4.5 with NaOH (1N). Using NO. 501 7.5 cm diameter Sargents filter paper upon harvest, the mycelium was vacuum filtered, washed, then dried twelve hours or more in an 85° C oven. Filter papers with the mycelium were individually suspended in 25 or 50 m1 beakers in the oven to enhance drying. 41 TO determine the most advantageous pH level with Humfeld's medium for all the cultures, a range of 4.5 to 7.2 was used. Duplicates of the twelve fungi were run in Humfeld's medium at pH 4.5, 5.5, 6.5 and 7.2, ten and twenty days each. The density Of the inoculum was checked using a Howard mold count chamber, and the average positive percentage of 25 fields. One field was considered positive if two or more hyphal strands were within that field. The percentages of the twelve fungal inocula were as follows: C-63—8, 76%; C-63-15, 84%; C-63-86, 80%; c-63—96, 90%; A-63-l36, 84%; A—63-137, 76%; A-63-139, 80%; A-63-138, 84%; L-63-133, 84%; P-63-51, 76%; V-63-135, 76%; V-63-134, 84%. Hyphal diameters of the inocula were within the following micron ranges: C-63—8, 1.73-3.46u; C-63-15, 1.73-3.46u; C—63-86, 1.73-3.46u; C-63-96, 1.73-3.46u; A-63-l36, 1.73-3.46u; A—63—l37, 2.5—3.46u; A-63-l39, 3.46u; 4-63-138, 1.73-5-19u; L-53-l33, 1.73-3.46u; P-53-51, 1.73u3 V-63-l35, 3.46-6.92u; V-63-134, 1.73-3.46u. The fungi were also checked for frequency of clamp connections with various carbon sources in Humfeld's medium. Using various carbohydrates, eight grams carbon per liter was substituted for glucose in Humfeld's medium. Fifty ml medium adjusted to pH 4.5 with NaOH(lN), and 5 ml blended inoculum were added to the 250 m1 Erlenmeyer flasks. 42 Triplicate cultures of the twelve test organisms were incu— bated on rotary shakers for ten and twenty days each. Data were recorded as gram weight of the dried mycelium. The car- bon sources included eleven monosaccharides: D-arabinose, DL—arabinose, L-arabinose, D-fructose, D-galactose, D—glu- cose, D-mannose, L-rhamnose, D-ribose, L-sorbose, and D— xylose; seven disaccharides: D-cellobiose, alactose, B lactose, D-maltose, and sucrose; one trisaccharide: raf- finose; seven polysaccharides: cellulose, dextrin, glyco- gen, inulin, pectin, starch, and xylan; and nine organic acids: citric acid, galacturonic acid, DL-malic acid, oxalic acid, pyrogallic acid, 8 resorcylic acid, succinic acid, tannic acid, and D-tartaric acid. DL—malic acid was Seitz filtered and pipetted aseptically to the individual sterile rotary shaker culture flasks. Water filtrates Of casein and xylan were used and one gram cellulose was measured indivi— dually for each flask. Other carbon sources used included i-inositol, D-mannitol, pectinol A (a commercially pre- pared substance Of di- and polygalacturonic acid), D- sorbitol, D-glucosamine hydrochloride, and fructose-1,6- diphosphate. Several carbohydrates were used at 25, 50 and 75 g per liter. D-galactose, D-fructose, a lactose, sucrose, starch, i-inositol and D—sorbitol were added as the carbon source to Humfeld's medium adjusted to pH 4.5. Averaged duplicate dry mycelial weights of the twelve_fungi were taken at the end of both ten and twenty days. 43 Glucose utilization by the twelve fungi in Humfeld's medium at pH 4.5 were recorded as averaged mycelial dry weight of duplicate cultures at the end of ten, twenty, thirty and forty days each. Growth rate studies were also included for these data. Fourteen day dry mycelial weights of the twelve fungi in replicates of three were averaged for several am— monium and nitrate nitrogen sources: ammonium bicarbonate, ammonium chloride, ammonium citrate diabasic, ammonium ni- trate, ammonium sulfate, ammonium tartrate, calcium nitrate, cobaltous ammonium sulfate, cobaltous nitrate, cupric nitrate, magnesium nitrate, potassium nitrate, sodium nitrate and uranium nitrate. With a nitrogen equivalent based on one gram urea per liter, 0.47 g nitrogen per liter was used for each of the above compounds. The pH was adjusted to 4.5 for Humfeld's medium, with glucose as the carbon source at con- centrations of 8 g carbon per liter. The pH was also taken at the end of fourteen days. Several nitrogen sources were not autoclaved with the medium, but were added aseptically to each 250 ml culture flask after Humfeld's medium was sterilized. The inorganic nitrogen sources Seitz filtered included ammonium bicarbonate, ammonium citrate diabasic, ammonium tartrate, cobaltous nitrate, cupric nitrate, mag- nesium nitrate and uranium nitrate. Urea was also Seitz filtered. 44 Seventeen amino acid and related nitrogen sources at 0.47 g nitrogen per liter were used in Humfeld's medium, pH4.5. Dry mycelial weight of the twelve fungi were aver— aged from replicates of three rotary shaker culture flasks each, grown in 50 ml of medium using 5 m1 inoculum. At the end of fourteen days the pH was taken. Amino acid compounds for this study included DL-alanine, L-asparagine, L-aspartic acid, betaine hydrochloride, creatine, cysteine hydrochloride, L-cystine, L-glutamic acid, glycine, D-histidine, DL-isoleu- cine, DL—methionine, L-proline, DL-threonine, DL-trypto— phane, DL-valine and peptone. Volvariella volvacea (V-63-l35), Lepista nuda (L- 63-133), Cantharellus cibarius (C—63-86) and Pleurotus 2E? treatus (P-63—51) were selected from the twelve fungal cul- tures for vitamin studies. The vitamins selected include p-aminobenzoic acid, ascorbic acid, biotin, calcium panto- thenate, niacin, niacinamide, pyridoxine hydrochloride, riboflavin, rutin and thiamine. Replicates of three were made Of each vitamin at 0.5 mg per liter Of glucose-casein hydrolysate with agar (104, 142). The media contained: glucose, 25 g; casein (vitamin free), 2 g; MgSOu - 7H2O, 0.5 8; KHZPOu, 1.0 g; fumaric acid, 1.32 g; Na2CO3 ' H20, 1.12 g; FeSOu, 0.2 mg; ZnSOu, 0.2 mg; MnSOu, 0.1 mg; highly purified oxoid code NO. L13 agar No. 3, 20 g; distilled H20, 1,000 ml. Twenty ml media per 25 mm test tube were 45 used as agar slants and hyphal growth was measured in mm, six times at two day intervals, from point of inoculation to colony margin. Colony characteristics were compared at two weeks- The four fungi selected for vitamin studies were also grown in 2% malt agar with 0.1%, 0.01% and 0.001% gib- berellic acid, kinetin, 2, 4-dichlorOphenoxy acetic acid, indoleacetic acid and indolebutyric acid each in replicates of three.1 The growth hormones were dissolved in three m1 acetone before adding to the 2% malt agar. Twenty m1 agar was poured into plastic petri plates, and the colony radius was measured every two days from point of inoculation to the margin. Due to difficulty in controlling pH, the results were negative except at the(1001% concentration. Volvariella volvacea (V-63-l35), Lepista nuda (L-63-l33), Cantharellus cibarius (C-63-86) and Pleurotus ostreatus (P-63-51) were chosen for the spawn experiment. The following substrates were used: flax, lupine, millet, milo, orange cane, rape, sorghum, spelt, sudan grass, vetch,. wheat, rye and sunflower seeds. Other media included ground corn cobs, spruce needles, rye plus 2 g glucose per flask and rye plus 30 m1 Humfeld's medium without glucose per flask. The spawn was incubated at 24° 0; additional rye spawn was grown at 12, 18, 30 and 36° C. Twenty-five grams of grain, or enough to fill flasks one—fourth full, 30 ml distilled H20, or enough water to adequately cover the 46 grain, and 0.3 g calcium carbonate were added to 125 ml Erlenmeyer flasks in replicates of four. The initial pH reading was 5.7. Larger quantities of spawn medium in pro- portions to the above were placed in 500 ml Erlenmeyer flasks and milk bottles, in replicates of two to five. The media were autoclaved twenty minutes, vigorously shaken to loosen and mix grain with the calcium carbonate, then inoculated. The hyphal inoculations were made from mycelium growing on 2% malt agar blocks cut out from cultures. One two cm square agar block was transferred aseptically to each flask. A two week period was allotted for spawn growth; rapid develOping cultures were allowed to grow up to three months for possible fructifications. Sterile distilled water was added periodically to the three month old cultures to prevent drying. Using fourteen day old rye spawn, the same four fungal strains mentioned above were sown in mushroom houses suited to the conditions of Bgaricus bisporus. Wooden boxes one foot square and eight inches in depth were con- structed and made in replicates of five for each of the four fungi. Mushroom production methods were similar to those described by Atkins (l2) and Richards (229). The horse manure and wheat straw was composted ten days and underwent three turns for aeration and thorough mixing with the 28 pounds calcium carbonate per ton compost. 47 At the end of ten days, the compost was placed in the wooden boxes, autoclaved forty minutes at 1210 C, fifteen pounds pressure per square inch. Boxes were placed in a mushroom house just completing sweat out, the final fermentation to reduce the ammonia concentration, reduce contamination, lower the insect pOpulation and eliminate excess moisture. Inoculum was added and allowed to grow three weeks at 650 F before one-half inch layer of casing soil was spread over the compost surface to supposedly enhance fructification. Beds were watered occasionally with a fine spray to prevent drying of casing soil and compost. In another experiment, two strains of Pleurotus ostreatus (P-63-51) and (P-64-6) each were inoculated on 2% malt agar petri plates, and placed in dark, light, inverted and upright positions. Half the inverted plates contained 10% KOH in the petri dish cover. They were allowed to grow and checked for fruiting initials. The electron micrographs were taken with a Philips EM-lOOB. Material was fixed a few seconds in 5% KMnOu then dehydrated for five minutes each in 30, 50, 70 and 85% ethanol, for ten minutes each in 95% ETOH and three changes of absolute ETOH. Two fifteen minute changes of prOpylene oxide followed, then material remained for one hour in a mixture of prOpylene oxide and an Epon 812 resin mixture. The Epon 812 resin mixture was composed of equal amounts of 48 mixture A: Epon 812 (Shell Oil Company), 62 ml; dodecenyl succinic-anhydride (DDSA hardener), 100 m1; and Mixture B: Epon 812, 100 ml; methyl nadic anhydride (MNA accelerator), 89 ml. The prOpylene oxide and Epon 812 mixture was drained and replaced by the Epon 812 mixture. After six hours, the first Epon change was centrifuged and pipetted Off. A sec- ond change of Epon was added to the material and remained overnight. Material was then transferred with an inocula- tion lOOp to NO. l gelatin capsules which were half filled with a resin mixture of five ml each of mixtures A and B plus 0.15 ml of tridimethylaminomethylphenol (DMP accelera- tor). Capsules were polymerized twelve hours each at 35, 45 and 60° C. Sections were cut on a Porter-Blum Ultramicro- tome using glass knives, then were placed on specimen grids covered with a parlodion film. The procedures were formu- lated according to Luft (158, 159) and Pease (211). Light microscOpy procedures were carried out ac- cording to Sass (249) and Johansen (118) with modifications. Using a suction pump, material was killed and fixed twenty- four hours in Craf (equal parts of solution A: chromic acid, 1 g; glacial acetic acid, 7 ml; distilled H20, 92 ml; and solution B: neutral formalin, 30 ml; distilled H20, 70 ml). The dehydration method was as follows: 50% ethanol and tertiary butyl alcohol (distilled H20, 50 ml; 95% ETOH, 40 m1; TBA, 10 ml), two hours; 70% ETOH and TBA (distilled H O, 30 ml; 95% ETOH, 50 m1; TBA, 20 m1), twelve hours; 2 49 85% ETOH and TBA (distilled H20, 15 ml; 95% ETOH, 50 ml; TBA, 35 ml), two hours; 95% ETOH and TBA (distilled H20, 5 ml; 95% ETOH, 40 ml; TBA, 55 ml) 2 hours; 100% ETOH and TBA (absolute ETOH, 25 ml; TBA, 75 ml) plus a few grains of erythrosin, 2 hours; three changes of TBA, 3 hours each; TBA and paraffin oil, equal parts, 12 hours. Vials were then filled one-third with tissuemat (56.5° C melting point), allowed to cool, then filled another one-third with material to be imbedded plus the TBA and paraffin oil. Four changes of tissuemat were made in four hour intervals with the paraf— fin oven at 700 C. Embedding was done from the fifth change of tissuemat. Sectioning was done on a rotary microtome. Paraffin ribbons of the material were floated on microscOpe slides using a modified Sass (249) adhesive 3 (solution A: gelatin, 1 g; sodium benzoate, 0.5 g; dis- tilled H20, 100 ml; solution B: chromium potassium sulfate, 1 g; formalin, 10 ml; distilled H20, 90 ml) mixing one part of solution A with six parts of solution B. A modified Conant's quadruple staining procedure was used for the sections Of fruiting bodies grown in cul- ture. Slides were passed through the following COplin jar series in three minute intervals: two changes of xylene, 100%, 95%, 70%, 50% ETOH; 1% safranin in 50% ETOH, two hours; 30% ETOH, distilled H2O, three minutes each; 1% aqueous crystal violet, one-half minute; distilled H20, one minute, 30%, 50%, 70%, 95%, 100%, 100% ETOH, two minutes each; nine 50 parts clove oil saturated with orange gold and one part 1% fast green in absolute ETOH, two minutes; four baths of clove oil and orange G, two minutes each; three changes of xylene, three minutes each. Clarite was the mounting medium. Mycelium from submerged cultures were directly mounted in lactophenol cotton blue for light microscopy. The twelve fungal cultures were grown on various Difco media (49) for identification purposes. The following were used: tryptic soy agar, blood agar base, nutrient agar, rice agar, Russian medium, Russian medium minus casein and tryptOphan, Russian medium minus vitamins, potato dex- trose agar, prune agar, tomato juice agar special, mycolo- gical agar, brewer anaerobic agar, corn meal agar, mycosel agar, liver spleen glucose agar, chlamydospore agar, thio— glycollate medium, micro assay culture agar, violet red bile agar, O. Modess (12) agar; see appendix. Replicates of three were made of each media for the four fungi and allowed to grow three weeks in petri plates. RESULTS Effect of pH on the Cultures The pH of Humfeld's medium was varied to deter- mine the most acceptable level for all 12 fungal cultures. The greatest mycelial dry weight with Lepista at ten days was with pH 7.2; 4.5 was Optimum at twenty days. Pleurotus was best at pH 6.5 at ten days, and 7.2 at twenty days. Volvariella had good growth at pH 4.5, ten and twenty days, as did all four Cantharellus species. A pH of 6.5 or 7.2 produced the best growth with the Agaricus species. Dry mycelial weight differences within the pH range tested were not great, a pH of 4.5 was selected for the later performed submerged culture experiments. Utilization of Monosaccharides The experimental results in Table 1 present the utilization of eleven monosaccharides by the twelve fungi selected for study. Each fungal strain had certain sub- merged growth characteristics in Humfeld's medium that were described with the corresponding carbohydrate, and nitrogen sources. Cantharellus clavatus (C-63-8) produced no great increase in growth with monosaccharides in comparison to the control (Table 1). Each sugar did produce slightly 51 52 higher dry weights than Humfeld's medium minus carbohy- drate. The mycelium in shake culture did not form compact pellets, instead only individual strands and occasionally loosely interwoven clumps formed. D-arabinose, DL-arabi- nose and D-fructose produced similar low dry weight read- ings, slightly higher than control. L-arabinose, D-glucose and D—xylose gave the next highest dry weights followed in succession by D—ribose, D-galactose, D-mannose, L-rhamnose and L-sorbose. The latter gave the highest dry weight reading at 20 days. A one mg decrease in dry weight be- tween the 10 and 20 day readings occurred only with DL- arabinose and D-fructose, all others gave higher readings at 20 days than 10 days. Wide variations in dry weight readings occurred with B. cibarius (C—63-l5) using monosaccharides as the carbon source in comparison to B. clavatus. Good pellet formation develOped particularly at 20 days for several cultures having high dry weights. D-ribose and D-galactose produced the least hyphal growth; D—arabinose, L—arabinose, D-fructose, L-rhamnose and L-sorbose gave moderate growth results within the same dry weight range at 10 and 20 days. D-mannose and D-xylose had good mycelial growth while DL— arabinose and D-glucose produced nearly identical results, and had the highest dry weights. Hyphal growth was loosely organized, no pellets formed. 53 The B. cibarius strain (C-63-86) was quite com— parable to B. clavatus in utilizing monosaccharides. Growth with D-arabinose, DL-arabinose, and D-mannose produced slight autolysis as did the control. No growth occurred with D-fructose. A group of monosaccharides which produced a moderate amount of growth include D-arabinose, DL-arabinose L-arabinose,D-glucose,D-mannosa,D-ribose and L—sorbose. In increaseing order, L-rhamnose, Djxylose and D-galactose produced the highest dry mycelial weights. Few pellets formed in submerged growth. B. cibarius (C—63-96) was consistently much better in hyphal production compared to the other Cantharellus species; approximately six to eight times greater dry weights were recorded with the monosaccharides. L-rhamnose was poor- ly utilized by the tenth day, and was the only monosaccharide to show a weight increase between 10 and 20 days; autolysis occurred in all other carbon cultures as the peak in the growth phase must have been passed. D-arabinose, DL-arabi- nose, and D-ribose had dry mycelial weights within 200 to 300 mg. Ten day mycelial weights were above 300 mg and 20 day weights were above 200 mg for D-fructose, D-galactose, D—glucose, D-mannose, L-sorbose and D-xylose. Maximum mycelial growth recorded was at 10 days with L-arabinose as the carbohydrate source in Humfeld's medium. Excellent hyphal pellet formation occurred in each monosaccharide medium at both 10 and 20 days, and a large mycelial ring grew 54 on the flask side at the height of the media splash levels produced by the shaker motion. All cultures also developed a water soluble Yellow-green pigment in solution, a common characteristic of only the B. cibarius (C-63-96) strain. The greater the mycelial growth, the more pronounced was the pigment coloration. D—fructose, D-glucose and D—mannose were excep— tionally good monosaccharide carbon sources for Agaricus campestris (A-63-l36), while the other sources were not as good from a concentration of ngcarbon per liter of media. Hyphae grew in submerged culture as well formed pellets and occasionally a mycelial ring develOped on the flask wall if good growth occurred. Poor growth occurred with B. bisporus (A-63-l37) grown in Humfeld's medium using D-arabinose, DL-arabinose, L-rhamnose or D-ribose; moderate growth with L-arabinose, D-galactose and D-xylose; and slightly better growth with D—glucose, D—mannose and L-sorbose. The best growth formed in the medium with D-fructose, particuarly at 20 days. Hyphae did not form pellets; only occasional flask rings grew, while hyphae in submerged culture remained single stranded. The pleomorphic form of A-63-l37, B. bisporus pleomorph (A-63-l38) showed similarities to its parent cul- ture. D-arabinose, DL-arabinose, D-galactose, L—rhamnose, D-ribose and L-sorbose produced poor growth. L-arabinose 55 and D-xylose gave similar results while D—fructose produced good growth. These were all under 100 mg dry weight at both 10 and 20 days. Best growth occurred with D-glucose and D- mannose, both resulted in over 20 mg of mycelial dry weights at 20 days incubation. NO pellet formation resulted in the hyphal growth, but frequent flask mycelial rings occurred. Most of the carbohydrate cultures having poor growth also showed autolysis or reduced mycelial dry weight at 20 days compared to 10 days incubation. The rhizomorphic form of A—63-l37, B. bisgorus rhizomorph (A-63-l39) also basically followed its parent in carbohydrate utilization. D—arabinose had the least mycelial growth Of the monosaccharides. DL—arabinose, L- arabinose, D-galactose, L-rhamnose, and L-sorbose gave moderate growth; cultures grown in D-fructose, and D-xylose showed good growth at 20 days. The same monosaccharides, D-glucose and D-mannose, produced the highest dry weight readings particularly at 20 days with the pleomorphic strain resembling that of the parent strain but at a much lower value. D-fructose and L-ribose were not as readily utilized as by the parent strain. NO distinctive submerged culture growth was evident, hyphae remained single stranded both at 10 and 20 days growth. Lepista nuda (L-63-133) had poor growth at the end of both 10 and 20 days with D-arabinose, D—ribose and D—xylose as the monosaccharide carbon source at 8 g carbon 56 per liter of Humfeld's medium. L-arabinose, D-galactose, L-rhamnose and L-sorbose formed fair growths of B, 2292 in submerged culture while DL-arabinose showed good growth, particularly at 20 days incubation. Excellent dry weights, three to five times greater than other monosaccharides, were obtained by using D—fructose, D-glucose and D-mannose as the carbon source at 8 g per liter in Humfeld's medium. The latter three carbohydrates formed good pellets in sub— merged growth while mycelium in other carbon media tended to remain single stranded. Pleurotus ostreatus (P-63-51) grew poorly in D- arabinose, DL-arabinose, D-galactose and D-glucose, but slightly better with D-mannose and D-ribose. Fair growth occurred with L-arabinose and D-xylose while D-fructose, D-sorbose and L-rhamnose produced the most mycelium. No significant hyphal weight differences occurred between the monosaccharides giving the poorest and best results with B. ostreatus, the difference was no greater than 25 mg. A few loosely woven pellets formed in submerged culture and some flask mycelial rings develOped. Like Pleurotus, Volvariella volvacea (V-63—135) showed no significant growth increases with monosaccharides in Humfeld's medium. Quite poor growth occurred with D- arabinose, DL-arabinose, D-fructose, D—ribose and D-xylose. Slightly higher dry mycelial weights were Obtained with L- arabinose, D-galactose, D-glucose and D-mannose. Hyphal 57 growth was fair with D—sorbose while L-rhamnose was best. Submerged growth was quite distinct compared to other genera grown in shake culture. One large loosely interwoven mass of hyphae formed. Chlamydospore masses produced the charac- teristic gold—brown to tan color. B. volvacea mutant (V-63-134) developed smaller masses of hyphae but no chlamydospores compared with B. volvacea (V—63-135). Growth differences indicated by the poorest and greatest dry mycelial weight were not great. Cultures grown in D-arabinose, D-fructose or D-sorbose pro— duced poor growth while DL-arabinose, L-arabinose and D- glucose were only slightly better. D-galactose, D—mannose and D-ribose showed fair dry mycelial weights. L-rhamnose and D-xylose were the best monosaccharides for carbon utilization. Utilization of Disaccharides and a Trisaccharide Seven disaccharides and one trisaccharide were substituted in Humfeld's medium as the carbohydrate source at 8 g carbon per liter (Table 2). With Cantharellus clava- BEE (C-63—8), the eight sugars produced similar dry weight levels, all apparently equal‘in usability, 20 to 30 mg dry weight above control. No pellets formed; some loose masses ofhyphae organized in submerged culture. 9. cibarius (C-63-15) produced the least growth with a lactose and raffinose while B lactose, D-maltose 58 3.3 was: 9% Tom H.Hm 2.1mm m.mm m.mm afimm w.mm OOm TON om mmHum 1a I O.OH O.mm O.Om s.OO m.Om m.Om O.Om O.am m.Hm m.mm O.Om O.mm OH sonoEONHrs mOsOOOHO .a H.mH O.OO s.mm m.gm m.mm H.Hmm O.smm 0.0m O.HH m.OO m.em H.Om Om OmHum 1a II O.mH m.mm H.sm H.OO m.Hm 0.0a 0.00H m.em 0.0m H.ez 0.0m m.Om OH rososooHo .nsso mHo .a O.sH m.HH O.Om s.sm O.Hm O.mm O.mm O.ms O.HO O.ms N.Om O.mm Om AMH1O 1a 11 m.OH m.Hm m.Om s.Om O.Om O.OO m.Hm m.Om 0.0m m.em m.Om m.Hm OH msso WHO .a m.mm m.mm Oam Tan 93 THOH m.mHm m.mm THE :.mm O.mm Tam om mmHummna m.Om H.Om m.mm s.Om m.mm A.HO m.OHH O.mm m.mOm O.m O.mm s.Hm OH mHsomooemo nsOHsomm H.OH O.mmm O.OHN 0.0mm O.OO O.mam O.OOm O.mO O.Hmm 0.0Hm 0.0Hm m.sOH Om Oaumm1O 1| 0.0H m.mmm O OHM m.OOm O.Oe H.eOm m.Omm A.MOm O.OHm O.mOm O.OOm O.OON OH nsHsmoHo .O N.OH H.sm s.Om g.mm m.Om H.Om 0.0m s.mO O.Hm H.sm m.Om 3.3m Om Om1mm1O O.Hm H.Om O.mm H.Hm s.gm O.mm O.mm O.mm s.OH O.Om O.sm O.mm OH msHseoHo 4m O.mm O.mO O.HO O.Om O.ma m.OO H.OOH m.mm H.ms m.Hm O.mOH O.mO Om mH1mm1O II H.OH O.Am s.Om m.mH m.mm O.Om H.Os O.mm O.mm H.Om O.Os H.3m OH mthsOHo .O m.OH O.Om m.Om O.hm O.Om O.mm m.Om O.Om O.Om O.Hm m.mm m.Hm Om O1MO1O 0.0H s.Om s.am O.mm m.Om O.Om O.Om e.gm O.Hm O.Om s.mm N.OH OH msomseHo msHHosmeoemO 33 G n1 G O1 G G G G n1 G G G 08 _ _ . _ . _ _ _ . n1 _ B UJ X S J J m on on IO 2 _ B K 1.0. «NA 0 I. U. B I E J J B J 8 J0 T. J O. E u n T. n B J 9 OH O O. O m u 0 B 0 O. B O. I s o s u o o O 4 I q I H. e S a O S S 1. O u I. u J a S a a O S O u 0 8 8 S 8 S O S e a e % a .mzmo om one OH 5909 no use on» ad mmmeHHomp moan» mo memnwfiaafie 2H pnwfimz ago Hmfiaoome Ommmpo>m mm nmufinmnoommocoe mo coapmufiafius ocean.a maome coupoomn whopaso OownoEnsm cfi ozopw chsm O>Hmzp an 59 .HOOHH Loo w w coflpmpocmocoo coopmo a. m.ma m.m: m.mm ©.Hm >.mm m.mm m.:m m.Hm m.©H O.mm :.mm H.0m om :malm |> II O.OH O.sm m.Om O.mm O.mm 0.0m O.Om m.mm O.mH m.OH m.mm H.mm OH echoes .moomsHo> .> m.mm m.mm ~.mm m.zm o.~m H.m: H.m: m.mm m.mm O.HO m.mm H.mm om mMHumm1> O.AH O.Om O.mm H.Om O.OO O.ms 0.0m O.HO O.Om O.Om O.Om O.em OH moom>Ho> mHHoHsmsHo> m.wm o.wm m.m: m.Om :.H: :.mm m.Hm m.am 3.3m m.mm w.mm O.mm om Hmnmmlm m.OH m.©m w.m: :.mm m.Hm >.mm >.Om m.mm m.:: m.wm O.wm m.©m 0H msummpomo m5pOL3OHm H.mm :.mm m.m: m.Hm m.mm H.o:m w.m©m ©.H: m.mOH H.0m :.mm 0.0m om mmaummnq H.mm O.Hm m.mm m.mm 5.3: O.mam m.oma H.am ©.me m.o> m.Om :.mm 0H mono mpmfiomq 00 Q Fl 0 n1 G G G G W. G G G 08 _ . _ _ . . _ _ _ OI. _ E UJ X S J J m on as J B _ B K 10. K O I. U. B I B J J B J 8 J0 T1 J O. B u n _L n B J 8 OH O G. O m U 0 E O O. B G. T. S O S u 0 O O 3 I. O. I. d a S a O S S 1 O u I. u J a S a a O S O u 0 a a S e S O S a a a w a .UOSCHpCooII.H OHQMB 60 developed fair growth. Sucrose, D—cellobiose in that order produced better growths with the C-63—15 strain. NO pel— lets formed; only isolated hyphae grew in submerged shake culture. C—63—86 strain of B. cibarius also showed moder- ate growth with most carbohydrates listed in Table 2. All disaccharides were utilized at about the same rate; dry mycelial weights at 10 and 20 days were similar for each carbon source. The trisaccharide, raffinose, produced the highest growth reading,particularly at the 20 day reading. A few small pellets grew in culture. The third strain Of B. cibarius, C-63-96, again produced much greater dry weights than did the other two strains. Numerous compact pellets and hyphal flask rings formed in each culture. In the presence of B lactose, 3 to 5 times less hyphal growth occurred than with a lactose, D-cellobiose, D-maltose, sucrose or raffinose. Sugars Of the latter group had dry weights exceeding 200 or 300 mg and all exhibited autolysis by a decreased hyphal weight at 20 days compared to 10 days. A water soluble yellow- green pigment was present in most media. Best growth for Agaricus campestris (A-63-l36) was achieved with raffinose, the disaccharides all produced less than half the mycelial dry weight of the trisaccharide. Only a small number of loose pellets grew in the cultures 61 except in Humfeld's media using raffinose or a lactose, D- cellobiose and sucrose. Raffinose and D-cellobiose produced excellent hyphal growth with A- bisporus (A-63-l37), particularly at 20 days incubation. Sucrose, followed by a lactose, B lactose, and D-maltose produced moderate amounts of growth at both 10 and 20 days. With the best carbohydrate sources, some pellets formed in submerged culture and mycelial rings on sides of flasks develOped. The pleomorphic strain of A-63-l37, B. bisporus pleomorph (A—63—l38) was very similar in its usage of car- bohydrates of high molecular weights to its parent except with D-cellobiose which produced more than twice the amount of mycelial dry weight, particularly in 20 days. Hyphae grew in submerged culture as a loose mass. B. bisporus rhizomorph (A-63-l39) differed slightly in carbon utilization of carbohydrates (Table 2) in compar- ison to the parent culture, A-63-l37. D-cellobiose was still the best carbon source in this sugar group, but dry weight results with D-cellobiose were not nearly as great as were the results of the other B. bisporus strains. Sub- merged cultures grew in Humfeld's media as large loosely woven pellets. Lgpista nuda (L-63-l33) pellet formation was much greater in disaccharide media than monosaccharide media. Sucrose, a lactose and 8 lactose produced the least amount 62 of hyphal growth while D-maltose was a moderately good source of carbon. Raffinose was good and D-cellobiose was excellent as carbon sources at both 10 and 20 days incubation. Best mycelial growth for Pleurotus ostreatus (P-63-5l) was produced on D-cellobiose, B lactose and D- maltose. Moderate growth resulted when cultures were grown in Humfeld's media with 8 g carbon per liter of a lactose, sucrose or raffinose. Pellet formation was poor except for D-cellobiose; other media produced only hyphal flask rings or submerged isolated strands. Volvariella volvacea (V-63—l35) grew best on D- cellobiose while D-maltose was a fairly good growth pro- moter for both V-63-135 and B. volvacea mutant (V-63-l34). Raffinose and other disaccharides listed in Table 2 showed a moderate growth pattern. D-cellobiose was only of moder— ate value tO the mutant form, V-63-l34. In comparison to B. volvacea, the growths of the mutant were less with each respective carbon source except D-cellobiose. Utilization of Polysaccharides Seven polysaccharides were selected for carbon utilization by the 12 fungi, and the results are tabulated in Table 3. Cantharellus clavatus (C-63-8) was readily able to utilize cellulose in Humfeld's medium while only moderate growth was achieved with dextrin, glycogen,inulin, pectin, starch or xylan, although pectin was better than other carbohydrates of this group. Hyphae grew singly as submerged strands and did not produce pellets. 63 O.OH O.Om O.Om O.Hm H.mm m.sm m.:: Om OmH1MO1a 1| 0.0H O.Om H.mO O.ms m.Om m.Om s.Om OH canoEONHcs .mmmmmmmm .O H.mH H.Og H.Om O.me O.mm 0.0m O.Osm Om OMHIMO1< o.ma m.mm m.m: m.mm ~.mm H.mm m.wm OH SmuoEooHQ .mSLommHo .< O.OH m.mO O.HO m.sm m.mm O.Om O.mOH Om smH1m 1a .II m.OH A.mm m.HO m.Om m.Om O.Hm m.mm OH mono nHo .< O.mm O.mHH m.m: O.Hm s.Om H.:: 0.0m Om OmH1mO1< O.:m H.OHH m.Os O.Om O.mm H.Om s.OO OH anomoosoO neOHsmme H.sH m.Omm s.Osm s.OOm s.:m m.eOH O.OHm Om OmummnO II OJOH H.Omm O.OOm :.mmm O.H: H.smm O.mOm OH nOHsOOHo .O m.OH m.OOH O.Om m.:m m.Om O.mm O.mm Om OO1mm1O O.Hm H.3m H.mm m.mm O.Om m.Hm m.Om OH neHsmoHo .O O.mm O.Hm m.mm H.mm 0.0m O.Om H.AO Om mHummuo II H.OH m.Om O.Om H.Om H.mm H.Om m.O: OH nsHsmoHo .O O.OH H.Om O.mm O.Om O1Hm m.Om H.mm Om mmmOuo O.OH m.Om s.sm m.Om m.Om m.Hm O.mm OH msom>mHo msHHcsmcocmO )0 Tu S mu 9 D mu W %W moo m w T. T. a nA 9,4 .1 J B e B a 8 JJ I. 0 TL 0 0 TL 010 u S 1. 3 3 T. OT. 0 a O O O O U S S S S O. .I\ a a a a T... O S a .mmmo om pom OH Spoo mo oco ozp pm mOOmOHHOOL ompnp mo mEmeHHHHE CH osmfioz app Hmfiamome Oowmpo>m mm poutoomp .mpspHSO Omwnmeosm CH ozonw chsm o>ao3p an oofipmcoommflpp moo msHo *noofinmnoommwo mo COHpmNOHHp: ozeul.m magma 64 .nmpHH LOO w m coapmapcmocoo coonmo x. (uoqaeo—) m.mH 0.0H a.mm 0.0m a.mm m.mm O.wm Om OMHum 1> In 0.0H O.mm m.mm m.sm O.mm p.3m O.:m OH peruse .moos>Ho> .> m.mm s.om O.me 0.00 O.mm m.Om 0.0m Om mmHan1> O.AH m.Hz m.:m s.H: H.mm O.am O.OO OH moom>Ho> mHHoHsm>Ho> m.em 0.0m m.Om O.mm s.m: 0.0m s.Hm Om Hmumw1a m.>H m.Hm H.om m.am m.Om o.wm m.mz OH mspmoppwo mzoosonm H.mm H.AOH H.mm m.mO m.mm O.Om m.mmm Om mmHummuH H.mm m.sHH O.OO O.mm 0.0m O.me O.HmH OH moss eonHocH O J S U do n G G O B n _ _ B u J O m .L .L O ...A 1. Io J B E E a S J I. O T. O 0 TL 0 U S n4 ,4 1. T. .L O a 0 O O O S S S S 0.. a a a 9 T.. O S a .eoscHocoouu.m oHome 65 B. cibarius (C-63—l5) grew fairly well on inulin and xylan, and well at 20 days with dextrin, glycogen, pec- tin and starch. Greatest growth occurred with cellulose as the carbon source in 10 days. No pellets formed; submerged hyphae and thin hyphal flask rings developed. Similarly with the other Cantharellus strains, B. cibarius (C-63-86) grew best in Humfeld's medium with l g of cellulose per flask. Moderately good growth was obtained with pectin, starch, dextrin, glycogen, and inulin. Xylan produced the poorest results similar to the latter two species. Pellet formation commonly occurred; hyphae were loosely woven, however. Polysaccharide utilization was a bit modified with B. cibarius (C-63-96) in comparison to the other Cantharellus species. Dextrin and starch were excellent carbon sources; glycogen and cellulose were also good. In contrast inulin, pectin and xylan were fair carbon sources. Dense pellet formation was common and the yellow-green water soluble pig- ment occurred frequently in the Humfeld and polysaccharide media. Agaricus campestris (A-63-l36) grew well on cellu- lose, particularly at 10 days incubation. Good hyphal pro— duction was Obtained with dextrin, pectin or starch with 20 days growth while only moderate growth occurred with glyco- gen, inulin and xylan. Mycelium grew as isolated strands in submerged culture. 66 B. bisporus (A-63—l37) and one of the two mutants from this culture, B. bisporus pleomorph (A—63-l38) were similar in carbon utilization of polysaccharides. Cellu- lose and xylan produced excellent hyphal growth, however cellulose was not as readily utilized; xylan doubled the dry mycelial weight between 10 and 20 days incubation. Pectin produced good growth, but only moderate growth oc- curred with dextrin, glycogen, inulin or starch. Some pellet production resulted in the submerged cultures of A-63-l37, but A-63-138 hyphae grown in Humfeld's media formed only a singular dense interwoven mass of mycelium in each flask. I The second mutant of A-63-l37, B. bisporus rhizo— morph (A-63—l39) differed slightly from its parent and the pleomorphic mutant in utilization of polysaccharides. Cel- lulose was readily used by the rhizomorph mutant, but no hyphal growth with xylan as the carbon source in Humfeld's medium occurred. Mycelium remained single stranded in sub- merged culture at both 10 and 20 days incubation, no pellets formed. Lepista nuda (L-63-133) had excellent growth of pellet and flask ring formation with pectin and xylan, par- ticularly at 20 days incubation. Cellulose gave good my- celial growth at 20 days but was quite poor with only 10 days incubation. Starch resulted in moderate growth at 67 both incubation times. Moderate growth developed from dextrin, glycogen or inulin in Humfeld's medium at 8 g car- bon per liter. Cellulose produced the best growth in Pleurotus ostreatus (P—63-51) shaker cultures. Good growth was Obtain- edvdjfllglycogen, pectin, or xylan as carbon sources in Humfeld's medium. Moderate growth resulted with dextrin and starch while inulin was poor for hyphal growth in sub- merged culture. Loose pellets deveIOped in media with glycogen or pectin; other cultures grew as isolated myce— lial strands. Growth was extremely good with Volvariella vol— 22222 (V-63-l35) in Humfeld's media and 8 g carbon per liter glycogen, at both 10 and 20 days incubation. Cellu- lose and starch produced good hyphal growth but proved to be only about a third as much mycelium as glycogen. Mode- rate growth occurred with dextrin, inulin and pectin while xylan was not utilized. Xylan was also not utilized with'B. volvacea mutant (V-63-l34) in mycelium production. Best growth at both 10 and 20 days incubation was with cellulose in Hum- feld's medium. Moderately poor growth resulted with dex- trin, glycogen, inulin, pectin or starch. One g of cellulose, insoluble in water, was added to each flask of 50 ml Humfeld's medium then 1 g was sub- tracted from each dry weight reading of mycelium obtained 68 at both 10 and 20 day incubations. Inaccurate data inter- pretation may have resulted with cellulose as a carbon source. All other polysaccharides as other carbon sources were in concentrations of 8 g carbon per liter of Humfeld's medium. Utilization of Organic Acids The utilization Of nine organic acids was compared for each of the twelve fungal strains by dry mycelial weights at the end of 10 and 20 days incubation (Table 4). A pH reading at the end Of 20 days for each culture was taken; the initial pH was 4.5. In comparison to the other fungal cultures, B. BT- barius (C-63-96) showed the most variation in the pH of the medium after 20 days incubation. Terminal pH readings with citric acid for eleven fungal strains were 2.0, while one (C-63-96) was 7.0; DL-malic acid used in the medium had a pH of 4.0 for 11 cultures, 9.6 for one (C-63-96); oxalic acid terminal pH was 8.0 for one culture (C-63-96), 7.6 for other cultures; succinic acid in the medium for B. cibarius (0-63-96) had a 20 day pH of 10.6, about 6.0 for other strains; a pH of 9.6 for D-tartaric acid was recorded for C-63—96, 3.0 for the other cultures. Pyrogallic acid and tannic acid had terminal pH readings for all 12 cultures equal to the 4.5 initial readings. Pyrogallic acid medium was 8.0 at 20 days while galacturonic acid medium had varied terminal pH readings. 69 s.OH :.sm O.Om m.eO 0.0m O.ms m.sm O.OMH Om OmHum 1a II O.OH O.OH m.mm O.ms m.Hm m.Om :.sm O.mO OH cososooHcs .msso nHo .a H.mH O.ONH 3.3m O.mm 0.0m O.sm O.sm O.OO Om OmHumm1< O.MH m.sO H.mm O.mO s.mm O.Om m.sm O.OO OH canoEocHo .nsso WHO .a O.OH O.OMH O.mm m.OO A.Hm H.5m s.mm m.mMH Om smHum 1< I: m.OH O.Om O.mm H.OO O.mm m.mm O.Om O.OO OH mesa mHo .a O.mm m.mm s.OO m.ms s.Hm m.Om H.ms O.mm Om OOH1MO1< O.Om N.OH O.mm O.Om H.mm A.Hm O.Om O.mO OH mHsomcoeeo meOHsmma H.sH O.Hs O.msm m.mm N.Om s.OO A.Omm O.mmH Om Omum 1O II O.OH m.sm O.mOm s.mm O.mm m.OmH O.mOm O.OO OH meHsmOHo .O m.OH 0.0H O.mO m.mO 0.0m m.Om O.mm m.AHH om Om1m 1O II O.Hm m.mH m.Om O.ms O.Om m.am s.mm s.mm OH msHsmoHc .O O.mm O.ms s.Om m.HO s.Om m.mm O.Om O.ms Om mH1m 1O :1 H.OH O.mm H.OO H.mm m.Om m.Om s.Os O.MOH OH msHsmOHo .O m.OH m.mH O.Hm s.ms m.Om m.Hm m.mm m.OHH Om mummuo O.sH m.Om 0.0m O.Os 0.0m O.Om O.Om m.HOH OH msom>cHo msHHoseeocmO )3 X S d I. on D. O G 1% m O O m a O O. a. Donn». by J n4 .I. O 1, TL 8 .uJ u 0 I I o J n nwo q u u S I. H1 OTL a u 0 w. u % .mxmo om com OH noon mo com on» um mOmeHHomp moan» mo mEmanHHHE CO unwfioz map Hmfiamome Oowmnm>m mm Omppoo not .msspaso Omwsmsosm Ca czogm Hmong O>Ho3p an *mmofipmcoommzaoo mo COHpmNHHHp: onenn.m canoe 70 .noufia LOO m m cofipmmpcoocoo coommo * m.mH m.OH H.mm 0.0m H.Om 0.0m m.Om O.mHH Om OmHumm1> II O.OH m.=H O.mm ~.Om m.om O.Hm O.mm H.Om OH scenes .ocomsHo> .> m.Om m.OO O.mm 0.0m m.sm O.aOm O.mO 0.0HH Om mmH1mO1> O.eH O.mm 0.00 0.0m s.mm m.OOm O.mm m.Hm OH moom>Ho> mHHoHsm>Ho> m.sm s.mm H.Os 0.0m 0.0m O.mO 0.0m m.mHH Om Hmummwa m.sH 0.00 m.sm 0.0m 0.0m O.OO m.ms O.ss OH nsomosono msoossoHa H.mm m.ww O.Hs :.HmH O.m: :.NO m.om m.mHH Om mmHummuH H.mm u.mm o.mo o.wm m.mm 0.2: m.~: :.HH 0H mos: mpmwmoq )3 X S d I. on D. O G .O 0A 1. a U T. a 8 9 OH HL 9 O n R X .L K 84+ 9 J 3 .L O 3 .L S JJ u 0 I. I. O J n QHO q u u S I I 01 a u 0 w u H .eoseHoeoO11.m oHoee 71 Cantharellus clavatus (C-63—8) had poor to fair mycelial growth in citric acid, DL-malic acid, oxalic acid, pyrogallic acid, Bresorcylic acid, succinic acid and D— tartaric acid. Good growth was obtained with galacturonic acid and tannic acid. Mycelial growth with g, cibarius (C-63—15) was poor to fair in Humfeld's media containing pyrogallic acid, D-tartaric acid, citric acid, oxalic acid, Bresorcylic acid or tannic acid while moderately good growth was ob- ' tained with galacturonic acid, DL-malic acid or succinic acid. B. cibarius (C-63-86) developed poor hyphal growth in media with citric acid, DL-malic acid, Bresorcylic acid and pyrogallic acid. It showed good growth with galactu- ronic acid and succinic acid. B. cibarius (C—63—96) grew well with citric acid, DL-malic acid, succinic acid or D- tartaric acid. Hyphal density in the shaker cultures with these media reduced nutrient movement greatly. Humfeld's medium with galacturonic acid, pyrogallic acid or tannic acid produced moderate mycelial growth; little growth dev- elOped in media containing oxalic acid or eresorcylic acid. Mycelium of the four Cantharellus species developed good hyphal pellets only in cultures with high mycelial dry weights; other media produced only isolated hyphal strands. Agaricus campestris (A-63-l36) grew fairly well with galacturonic acid or tannic acid. It had moderately 72 poor growth with citric acid, oxalic acid or D-tartaric acid, but grew quite poorly with DL-malic acid, pyrogal- lic acid, Bresorcylic acid or succinic acid. B. bisporus (A-63-13T) had moderately poor growth in Humfeld's media containing citric acid, DL— malic acid, pyrogallic acid, succinic acid, oxalic acid, Bresorcyllicacid,or D-tartaric acid media while good growth formed in media with galacturonic acid or tannic acid. The pleomorphic strain of B. bis orus, B. bisporus pleomorph (A-63-l38), grew poorly in DL-malic acid in Hum- feld's medium, slightly better in all the other organic acids, with good growth in tannic acid. The rhizomorphic strain, B. bisporus rhizomorph (A-63-l39) had little or no growth in media containing citric acid, DL-malic acid, pyro- gallic acid, succinic acid or D-tartaric acid; fair growth develOped in galacturonic acid,8 resorcylic acid or tannic acid; good growth occurred in oxalic acid particularly at 10 days incubation. Only the succinic acid medium had good pellet formation for the Agaricus cultures. Lepista nuda (L-63-133) grew exceptionally well in Humfeld's medium containing galacturonic acid both at 10 and 20 days incubation. Moderate hyphal growth develOped in media with citric acid, pyrogallic acid, Bresorcylic acid, tannic acid or D-tartaric acid, and poor or no growth oc- curred with DL—malic acid, oxalic acid or succinic acid. Pleurotus ostreatus (P-63-51) showed only moderate mycelial 73 growth with organic acids, with tannic acid giving the highest mycelial weights both at 10 and 20 days incubation and pyrogallic acid showing the least growth. D-galactu- ronic acid and succinic acid were the only media to support pellet formation for both Lepista and Pleurotus. Volvariella volvacea (V-63-135) grew moderately well in the medium containing galacturonic acid, and quite poorly or not at all in pyrogallic acid or D-tartaric acid. Dry mycelial weights Obtained with citric acid, DL-malic acid, oxalic acid,e resorcylic acid, succinic acid or tannic acid were only moderately good. The mutant form, B. volvacea mutant (V-63-l34) showed no growth in media containing DL-malic acid, pyrogallic acid or succinic acid, moderate growth with citric acid, galacturonic acid, oxalic acid,§3resorcylic acid or D-tartaric acid, and best growth with tannic acid. Volvariella mycelium grew in submerged culture as masses of interwoven hyphae. Miscellaneous Carbon Sources Miscellaneous carbon sources, including two sugar alcohols, were substituted for glucose at 8 g carbon per liter in Humfeld's medium,and hyphal dry weights were taken of the 12 fungi at 10 and 20 days incubation (Table 5). With Cantharellus clavatus (C-63-8) and two of the three 9. cibarius isolates, C-63-15 and C-63-86, the ability to use these carbon sources was nearly the same, except B. Table 4.-The utilization of organic acids by twelve fungi grown in submerged culture, reported as averaged mycelial dry weight in milligrams of three replicates at the end of both 10 and 20 days. (uoqan-) toaiuoo DIOR OTJelJel-q ptoe OTuuel 0109 OIUIOOHS DID? OIIKOJOSBJ 9 DIDB OIIIBBOJKd ptoe OIIBXO 0:09 OIIBw—Tq DIOB OTuoanioeIeB 0:09 OIJQIG *9!- Days :(YWO mmoo 20 pH 10 Cantharellus clavatus C-63—8 LOCO C1.) L".::r :T' \ mmxo :TGDH om: 20.4 10 20 pH cibarius C— 3—15 C. 5. 10.4 1:. r-ir—{O (IDS-If Ln-II' 10 20 pH cibarius C- 3- 6 C. 18.8 7. 223.9 9.6 46.5 302.3 4.5 92.3 32-5 10.6 22.9 119.9 21.4 4.5 8.0 33-2 26.5 25.0 20.3 8.0 9.6 275.3 206.7 4 1 5 26 35 0 277.3 222.8 7.0 10 20 pH C- 3-96 cibarius C. A-63-136 Agaricus campestris 16.3 16 5 4. (ONO CNZ'M r-iN Ob—Lfi mo:- Hr-i WWI—1V3 meow NH [‘00 :rFL: rid LDLDO HKOM m: 10 20 pH A- 3-137 bis orus A. Table 4.—-Continued. (uoqan-) toaluoo PIG? otaeqaeq-q pros OTuueq p109 otutoons PIG? OIIKOJOSSJ 9 PI09 OIIIESOJKd pros OIIBXO DIG? OIIem-TG PIG? OIuoanloeIes 0102 Grants if Days 0. 20.8 3 =w~uw moo 3 Ln: \DNO MNO MH CTN-{O mmoo MN man o o o 3&3 NN (“b-\D o O o CDNN NN COO 10 20 pH bis orus, pleomorph A— 3-138 1. :30 find“) ch 05m 0 o 0 I‘m: HH \DNW \Oafi =‘r-I' FOO NH: t-Ic-i mac 0 O O \me MN 20.9 9. 2 10 20 pH bis orus, rhizomorph A- 3-139 11 Ht—i mmo mam NN m :rzrm Odom: om: Nwfio 0N0 NN c~r~o O O O QHQ NN mom 0 I O MONS’ MN HNQ 3'05 NH FNO O O 0 NF: t-‘lc-i 195.6 202. 2. 20. 27.7 2. 10 20 pH L-63-l33 Lepista nuda mmm oo=ro O C 0 NW“) NN FQO O O O :‘NCD NN ouduw I O O coax: “Md Guam: >435- Ncu Ina-o O O 0 3L“: MN Pleurotus ostreatus P-63—51 16 14.9 4. 6 23.6 7. COO LGMH mm MNO 10 20 pH V-63-135 Volvariella volvacea NkDLfi \ozrxo o o o fiONN NH \DOC o o 0 I‘m: H 21.0 7. 2.0 10 20 pH volvacea, mutant V-63-l34 V. Carbon concentration 8 g per liter. pH was taken at 20 days. 76 cibarius (C—63-l5) grew much better than the rest with fructose-1,6-diphosphate. Casein was poorly utilized as were i-inositol, D-mannitol, D-sorbitol and D-glucosamine hydrochloride. Pectinol A was fairly good while fructose-1,6- diphosphate produced excellent mycelial growth, the latter far surpassing dry weight readings of hyphae grown in the other carbohydrates at 8 g carbon per liter of Humfeld's medium. Pellet formation developed in submerged growth with all three culture strains when grown in pectinol A, casein and fructose-1,6—diphosphate. The other nutrient flasks had only isolated hyphal growth. Cantharellus cibarius (C-63-96) differed from the other Cantharellus culture strains in that again large quantities of mycelium were produced. All carbon sources produced well formed hyphal pellets, and the best sources also developed hyphal flask rings at media splash levels. The yellow-green pigment in sOlution was easily recognized. Hyphae grown in Humfeld's medium with D-mannitol or D- sorbitol grew extremely fast. Mycelial production in pec- tinol A or fructose-1,6—diphosphate was also very great. Moderately large amounts Of mycelium were obtained from ieinositol and D—glucosamine hydrochloride while casein was a poor source. Additional carbon sources were selected for utilization by B. cibarius (C-63-96). The 10 and 20 day dry mycelial weights in mg for the carbon sources not recorded 77 ixlTable41wereIRM?lOand 20 days respectively: D-trehalose, 299.2, 217.0 mg; glucose-l-phosphate, 49.5, 67.9 mg; glu- curonolactone, 179.5, 232.3 mg. Agaricus campestris (A-63-l36) grew poorly in Humfeld's media with casein or D—glucosamine hydrochloride and moderately in i-inositol. D-mannitol, pectinol A and D—sorbitol produced good mycelial growth while fructose-1,6- diphosphate yielded excellent hyphal production at both 10 and 20 days incubation. Pellets formed only in cultures containing D-mannitol or fructose-l,6-diphosphate; hyphae grew in strands in submerged cultures of the other miscel- laneous carbon sources. Abundant mycelium was produced in media containing pectinol A or fructose-1,6-diphosphate, while good growth with D-mannitol or casein develOped as submerged hyphae for B. bisporus (A-63-137). Moderate growth was Obtained from Humfeld's media with 8 g carbon per liter i-inositol or D- sorbitol and poor growth developed when casein was used. Hyphae grew as a mass in submerged culture at times pro— ducing rings on flask sides. The pleomorphic mutant of A— 63-137, B. bisporus pleomorph (A-63-l38), had growth re- sponses similar to the parent strain although pectinol A was not utilized as well. B. bisporus rhizomorph (A-63-139), a mutant of A— 63-137, had a similar growth pattern to the parent strain except pectinol A and casein were able to be utilized nearly as well. 78 Both pectinol A and fructose-1,6-diphosphate were excellent carbon sources for growth of Lepista nuda (L-63—133) at 8 g carbon per liter. D—mannitol and casein produced good yields Of mycelium at 10 and 20 days incu- bation while only moderate results were Obtained with i- inositol, D—sorbitol or D-glucosamine hydrochloride. Good pellet formation was obtained with pectinol A and fructose- l,6-diphosphate; other hyphal cultures produced only sub- merged mycelial masses. Pleurotus ostreatus (P-63-51) grew exceptionally well in Humfeld's medium with 8 g fructose-1,6-diphosphate carbon per liter. Good hyphal growth was produced with pectinol A while moderate mycelial production occurred with i—inositol, D-mannitol, D-sorbitol or casein. D-glucosa- mine hydrochloride was not utilized. Good pellets formed if the carbohydrates were readily usable by the mycelium. Volvariella volvacea (V-63-l35) and B. volvacea mutant (V—63-134) showed differences in abilities to uti- lize carbohydrates listed in Table 5. Fructose-1,6- diphosphate was the best carbon source for both cultures; pectinol A was also an excellent carbon source for V-63-135 but was only moderately useful for V-63-l34, the mutated colony. For both strains, i-inositol, D-mannitol and D- sorbitol produced some hyphal growth in submerged culture while D-glucosamine hydrochloride and casein were poor 79 carbon sources. Minute hyphal pellets grew in cultures using the best two carbon sources; other miscellaneous carbon sources produced submerged masses of interwoven hyphae. 80 O.ON O.mO m.NN O.Nm N.Om N.es m.ms ON OmHum 1a II O.OH O.mO H.Hm O.ON m.OO N.Nm m.NN OH rososooHes .nsso nHo .a H.N: O.NOH m.OH 0.0N O.HHH O.ms =.mm ON OMHum 1a O.mm m.OO O.OH m.Om N.mm 0.0m O.NN OH nososocHo .msso mHo 4m H.Hm m.ONH O.mN H.mm H.OmH m.HO m.sm ON AMHummua II m.OO N.OHH H.OH m.Om O.HO N.HO N.ON OH mono nHo .< m.HN N.OOH H.NN N.OO H.Om m.HO H.Om ON mmHumO1a O.OH m.mHH N.ON O.NO O.NO O.s= O.Om 1 OH mHsoncosco sOOHnmwO O.Om m.OOm N.OHN O.NOm N.OON H.OOm m.=OH ON omummuu II m.mm :.ONm O.OmN m.ONO N.Ozm m.OHO m.smN OH nsHHOOHo .O H.m= O.mOH O.OO N.mm 0.0NH N.N: N.Om ON omummuo .: II z.NH m.mHH H.OH H.Hm O.NHH O.Hm 0.0m OH nsHseoHo .O 0.0H O.NHm H.Om N.NO m.OHH N.O: 0.00 ON mHummuo .u.|1 O.ON H.mHm O.mN 0.0m m.HN m.Nm O.Om OH msHseoHo .O m.sm O.HOH O.N: 0.0m O.OOH . O.Hm m.NH ON man1O O.Om H.OmH O.MN O.mN N.mO O.mm O.mm OH msoe>eHo nsHHcsmeoeeO 0 DJ U.G G d O I. G H... mm Ma .1. w m .H. m a U10 JT. 0 3. B u S Tr 0.4 non J I. u o u SO 00 O. U u S d8 U.O I. O I. I. "He .LS 4 T. 3. 3. B 09 O O O 1. Jw T. V T. T. e.L HIT. c pu n.) 88 .nsmo ON one OH coon no new coo on noose IHHOOH oops» no mEmeHHHHE Ca ucmfioz ago Hmaamoze Omwmno>m mm OcuHOQOH .onsuazo nowsos loam 2H ozonw chsm O>Hozp mo swoonsom coonmo msomcmaawomfie mo coapmufiafip: ocell.m canoe Table 5.-Continued. UIGSEO aqeudsoudtp -9‘I- asoqonJJ apTJOIuOOJqu autwnsoonIB—q Tourqaos-q v {outqoad toqtuuem—Q IOQISOUI-I sfleq 39. 55- 10 ° ta nuda 18 Le 290.0 13.8 234.1 76.5 44.5 51.4 20 L-53-133 r—IO HQ mm 3:)“ our HN 19.9 21. 32. 32. NC\ (13G) (IDKO NCID moo If: 10 20 Pleurotus ostreatus P -63-51 40.3 109.4 44.9 9. 121.1 50.9 23. 38.9 10 20 Volvariella volvacea V-53-l35 10 20 volvacea, mutant V- 3-134 V. Carbon concentration 8 g per liter. 82 Clamp Connections in Hyphae in Submerged Cultures The frequency variation of clamp connections with various carbon sources in Humfeld's medium was checked for the twelve fungal cultures. L—arabinose, casein, D- mannitol, pectin, L-sorbose produced no clamp frequency differences in the inocula. Agaricus and Volvariella were without clamp connections, Lepista.had a moderate number, Pleurotus had a slightly higher frequency of clamps while Cantharellus produced clamps at almost all cross walls. When mycelium was transferred from the above carbon source media to 2% malt agar for Cantharellus, Pleurotus and BBB: variella species, medium A for Lepista, and allowed to grow three weeks, no change in clamp connection numbers was noted. Hyphal clamp connections can be seen in mycelium of B. cibarius (C-63-86) at all cross walls when grown in Hum- feld's medium and L-sorbose (Figure 7). A characteristic of Volvariella in either agar or liquid media is the numerous spherical chlamydospores which gives the cultures the common tan color (Figure 8). Cell anatomy of some mycelium grown in submerged culture was further studied with the aid Of the electron and light microscOpes. Distinct nuclei were seen in Lepista £293 (L-63-l33) hyphae grown for 20 days in submerged cul- ture of Humfeld's medium with glucose (Figure 9). Numerous vacuoles were present in Cantharellus cibarius (C—63-86) 83 myceltium grown in Humfeld's medium with glucose, at 20 days inculbation (Figure 10). Mitochondria were identified in hyphal cells of Volvariella volvacea (V-63—135), grown in Humfeld's medium with glucose (Figure 11). It is well known that fungi belonging to the Agaricales are able to produce crystals of calcium oxalate in culture (47). Crystal formation was seen on mycelium of Cantharellus cibarius (C-63-96) when grown in Humfeld's medium with glucose (Figure 12), mycelium of Pleurotus ostreatus (P—63-51) when grown in Humfeld's medium with L- sorbose (Figure 13), and Cantharellus cibarius (C-63-86) mycelium when grown in Humfeld's medium with casein (Figure 14). 84 Figure 7.--Cantharellus cibarius (C-63—86) hyphae with clamp connections at all cross walls, grown 20 days in submerged culture, using Humfeld's medi- um with L-sorbose (cc, clamp connection). x 1,400 Figure 8.--Volvariella volvacea (V-63-l35) grown on 2% malt agar for 15 days, showing aerial hyphae with characteristic spherical chlamydospores. x 4,000 85 Figure 9.--Lepista nuda (L-63-l33) hypha with two dominant nuclei, from a submerged culture, grown 20 days in Humfeld's medium with glucose (n,nuclei). x 17,500 Figure 10.--Cantharellus cibarius (C-63—86) hyphae showing several vacuoles, from a submerged culture, grown 20 days in Humfeld's medium with glucose (v, vacuoles). x 14,800 86 Figure ll.—-Volvariella volvacea (V-63-l35) hyphal cross section, showing three mitochondria with cristae, grown for 20 days in Humfeld's medium with glucose (m, mitochondria; c, cristae). x 27,400 87 Figure l2.--Cantharellus cibarius (C-63—96) hyphae with numerous vacuoles and crystal formation, from a submerged culture, grown 20 days in Humfeld's medium with glucose (or, crystal; v, vacuole). x 12,600 88 Figure l3.——Crystal formation with Pleurotus ostreatus (P—63—5l) hyphae grown 20 days in submerged culture, using Humfeld's medium with L-sorbose. x 900 Figure l4.-—Cantharellus cibarius (C-63-86) production of cryStals along hyphae grown 20 days in submerged culture, using Humfeld's medium with casein (h, hypha without crystals; h or, hyphae bordered by crystals). x 1,000 89 Utilization of Carbohydrates at Various Concentrations Cantharellus cultures were grown in 25, 50 and 75 g of selected carbohydrates per liter of Humfeld's medium as an indication of concentration tolerance levels. As indicated in Table 6, media were made of D-galactose, D-fructose, olactose, sucrose, starch, i-inositol and D- sorbitol. Cantharellus clavatus (Ce63-8) produced the greatest mycelial dry weight at 75 g per liter with D- galactose, D-fructose, starch, sucrose and i—inositol. Mycelial production leveled Off at 50 g per liter and showed little variation between 50 and 75 g carbohydrate per liter if alactose, sucrose or D-sorbitol were used. 9. cibarius (C-63-15) grew most successfully at 75 g per liter of sucrose, starch, with moderate growth for alactose, i-inositol or D-sorbitol. There was little dif- ference in dry mycelial weight at 50 and 75 g per liter with D-galactose or D-fructose. Cantharellus cibarius (C-63-96) grew much more profoundly with most carbon sources used at concentrations of both 50 and 75.g carbohydrate per liter at both 10 and 20 days incubation (Table 6). There is generally a leveling off of hyphal growth with many Of the carbon sources between 50 and 75 g per liter at 10 days incubation as compared with 25 and 50 g per liter. In some cases a surge of growth can also be seen at 20 days incubation. A large increase in mycelial 90 weight occurs with the majority of carbohydrates between 50 zand 75 g per liter. The mycelial weight increase be- tween 25 and 50 g at 20 days incubation is generally not qUite as substantial as between 25 and 50 g. Table 7 records utilization of the 7 carbohydrate sources at 25, 50 and 75 g per liter selected for the ESE? BTBB§_species. The four Agaric cultures were grown under the same experimental conditions for 10 and 20 days. Bg- aricus campestris (A—63-136) has greater hyphal growth at 75 g per liter of Humfeld's medium for all eight carbon sources at 20 days incubation, while sucrose was best at 10 days. Mycelial dry weights at 10 days were higher at 50 instead of 75 g per liter with sucrose and starch. B. bisporus (A—63-l37) was quite inconsistent in carbohydrate concentration requirements. D-fructose, alac— tose, starch and D-sorbitol produced the greatest hyphal growth at 75 g per liter at both 10 and 20 days incubation. I—inositol at 50 g per liter gave the best hyphal yields at both 10 and 20 days, while 50 g per liter at 20 days and 75 g per liter at 10 days gave the best results for D-galac- tose. Sucrose at 20 days with 25 g per liter was the best concentration. The pleomorphic mutant of A-63-l37, B. bisporus pleomorph (A—63—l38) had greater growth consistency at similar carbohydrate concentrations than its parent. 91 N.mwm ©.m3w m.owm w.m3 N.®m ©.ON N.»@ 5.33 m.3m N.M3 3.53 H.Om ON m.mmw m.mm~ O.mm3 w.mm O.mm m.om o.mm O.mm O.Hm m.mm m.Hm m.Om OH HOOHOHOOIO O.m3m 0.0N3 N.M3N 0.00 O.Nm O.Hm 0.0m m.om m.Hm m.mo m.m3 m.Om om 0.0~m 3.33m H.Hmm 3.H3 w.mm o.om w.HO O.Hm 3.0m H.O3 0.0m H.mm OH HOpHmocHIH 0.00MH O.mmm w.3mm 0.00©H O.meH m.HH© N.OMH m.MOH 9.0m H.mm O.mo w.mm ON H.®ONH m.NOOH.3.HOm H.mmma 3.HOOH ©.>mm O.mOm m.3w 5.3m m.3m H.mw O.mm OH SULMum m.Omm O.maw N.mw3 H.0maa w.mmm 3.m©3 H.0Nm H.NOH m.3m 0.0m m.m3 3.5m ON w.mom m.omm m.w3m 0.3NOH H.0mw m.mmm m.wm 3.00 m.O3 m.HO H.m3 m.m3 OH mmoposm m.woo m.MM3 m.mmm 3.wma m.3m o.mm m.mm p.03 m.3m m.m3 0.3m w.mm om m.mm m.OMH N.Hma H.3m O.om m.Om m.m3 m.mm m.ON O.mm m.m: m.mm OH mwOpomH O ~.$ama O.mwm w.©mm O.M3ma 3.moo m.mmm m.3a m.m© O.m3 w.Om m.mm m.03 ON 3.4mma m.@>oa “.303 m.ooma 3.noma m.3Hm m.ow H.mm w.mm m.HO O.m3 m.33 OH Om0possmlO 3.mm® w.omo w.mom m.amw m.bao O.mw3 m.3O m.mm m.m© m.®3 m.mm m.3m ON O.mOO O.OOO O.NOO 3.OON N.HOH O.N3O m.Om H.Om 0.0N O.H3 0.0m m.m. OH omoOomHOmsa OH OO ON ON OO ON ON OO ON ON OO ON OHOO omlmmlo mmlnmnu malmmlo mummlo msfiumowo .o mzflhmpfio .O mOOLOOHO.O msum>mHO mzaaonmnpcmo .OOHOOOHO .0 mo mcfimnpm oops» new .mzpm>mHO 4m pom mmwo om new 0H npoo wo OOO one pm mmmeHHOms 03o wo msmthHHHE ca unwamz mac Hmflaoome Omwmmo>m mm Oopnoaop .mpzpaso OmwponOm CH czopw HOOHH LOO mempw mp com om .mm pm mcoflpmppcoocoo pm moonsom coonmo OO COOOONHHHo: OQBII.O OHome 92 Seventy—five g per liter gave the best growth at 20 days incubation with D-fructose, alactose, sucrose, starch, i- inositol and D—sorbitol. D-galactose produced nearly the same dry mycelial weights at 50 and 75 g per liter at 20 days incubation. B. bisporus rhizomorph (A—63-l39) had some car- bohydrate utilization concentration tolerances in growth patterns similar to the parent culture, B. bisporus (A-63— 137). Starch, alactose and D-sorbitol cultures grew the best mycelium at 75 g per liten, both 10 and 20 days. Su- crose in Humfeld's medium cultures produced greatest hyphal growth at 25 g carbohydrate per liter at 20 days incuba- tion. I-inositol at 50 g per liter, 20 days incubation, produced high mycelial yields. Compared to the parent strain, the rhizomorphic form differed with the following mycelial dry weight results: D-galactose had little varia- tion between 25, 50 or 75 g per liter both 10 and 20 days growth; hyphal formation was best in D-fructose at 50 g per liter, 10 days and 75 8 per liter, 20 days; olactose produced highest yields at 75 8 per liter, both 10 and 20 days incubation, and did not utilize sucrose as readily. The utilization of carbon sources at concentra- tions of 25, 50 and 75 g per liter in Humfeld's medium by Lepista, Pleurotus and Volvariella are reported in Table 8. Lepista nuda (L-63-l33) had greater hyphal growth both at 10 and 20 days incubation using D—fructose, alactose, starch, 93 OHNO O.Om O.Om O.OO O.Os H.Om H.OO N.Hs H.Hm N.OO O.OO N.NO ON m H3 0.mm 3.0m m.03 0.0m 3.am 0.m3 m.mm H.0m m.m3 0.03 0.0m 0H HOpHopomIO O.HO O.NNN H.Om 0.00 O.NO m.O3 0.0N O.HO N.OO 3.NO 3.OO O.OO ON O.OO N.OO O.Hm O.OO H.NO O.NN N.NO H.OO 0.0m O.mN O.OO O.Om OH HOOHmocHuH O.NOH 3.mOH O.3m 0.0HO O.NOH N.O3 H.NHH m.OO N.mO m.ONH N.ON O.OO ON N.OO O.HN O.Nm N.OHN N.33 O.OO O.ONH O.ON m.O3 H.NO H.3O O.O3 OH cosmom O.NO N.OO O.OO O.NON N.OO O.OO O.HO 0.0s 0.00H 0.00 s.NO 0.00 ON N.H3 m.m3 N.om H.30 m.mm 0.00 N.HN H.N0 m.03 H.H0m 0.H0 0.m3 0H Omogosn N.3OH O.WH N.ON O.OOH O.OO O.Om O.OO H.ON O.HO O.OO O.m3 O.NN ON O.NN O.OO O.Hm O.OO O.NO N.Om N.OO N.3m m.Nm O.HO m.NO O.ON OH OmoooOH a N.3OH O.OO O.Om O.m3N N.OOH H.OOH N.NOH N.ONH H.OO O.NON m.OON O.ON ON H.OO N.ON O.NO O.HON O.NOH H.OHH O.ONH m.mO O.OO O.OON 0.0N N.OO OH OmooossmuO 0.0. O.Nm O.NO m.ON N.ON O.JN H.OO 0.00 N.NN O.HOH O.OHH O.NO ON a H.Om O.ON 0.0N N.Nm O.NH O.OO 0.0N 0.00 O.Om 0.00 O.OHH N.NO OH OmoooOHOsuo ON OO . ON 1N OO ON ON OO ON ON OO ON ONOO OOH1OO1< OOH1OO1< NOH1OO1< .OmHumOua LOLOEONOQL :aLoEomHQ mzsommHo .< mwspmomemo .mspoqmfio .< .mssommfio .< mOOHme< .mspommflo 4O Oo mcfimppm mogcp Ocm .mwppmmmEmo .< mom mzmb 0m 0:0 0H Lpon mo OCO ng pm mOmeHHOOL 030 no mEmeHHHHE OH pcwfloz SHO HONHOOOE Omwmpm>m mm OOOLOQOL .OHOOHOO Ommsoeosm CO czosw HOONH LOO memsw mp new 0m .mm mo mCOHumsucoocoo pm mmopzom consmo mo OOHOMNHHHOS O£B|I.O oaome 94 i—inositol, or D-sorbitol at 75 g per liter. D—galactose was most effective at 50 g per liter, 20 days. Sucrose at 50 g per liter, both 10 and 20 days produced the highest mycelial dry weights for that particular carbohydrate. Pleurotus ostreatus (P-63-51) showed maximum growth at 20 days incubation, 75 g per liter with D-galac- tose and alactose, and in 10 days for starch. D-fructose was moderately good in 10 days at 75 g per liter. Moderate hyphal growth differences were not great at 50 and 75 g for i-inositol, 20 days growth. The Volvariella strains showed some data simi- larities in Table8. In both cases sucrose and starch were utilized much more rapidly by the parent strain, maxi- mum growth occurred at 75 g per liter at 10 instead of 20 days incubation for the mutant. The V-63-134 mutant had good yields at 75 g per liter with i—inositol, D-sorbitol, alactose, D-fructose and D-galactose. Fifty g per liter, 10 days; and 75 g per liter, 20 days produced fairly good hyphal growth with V-63-l35 in D-galactose, D-fructose, alactose and i-inositol. V-63-l35 growing in D-sorbitol developed the peak in a moderate amount of hyphal growth at 10 days incubation in 75 g per liter, and 50 g per liter at 20 days incubation. In general, mycelium showing poor growth in various carbohydrate media frequently produced cytOplasmic coagula- tion. Also, chlamydospores were more frequent, shorter hyphal 95 0.00 0.0m 0.N3 3.HO 0.00 0.00 3.Hm 0.03 0.00 0.H0 0.0m m.HO 0N 3.00 N.H3 0.0m m.Hm 0.03 0.00 m.Hm 0.Nm 0.0m N.NO 0.0m 0.03 0H HOOHOOOOID 0.00 0.03 m.mm N.Nm 0.03 m.Nm m.H0 O.N0 0.00 0.00 0.03 H.0m 0N N.03 O.HO O.NN N.OO O.OO O.ON O.3O O.HO H.Om 3.0N N.Nm 0.Om OH HOOHmoeHuH H.OO O.mO O.mm N.N3N 0.00 O.HO H.3OH O.NOH 3.ON O.NNH O.ONH 0.00 ON O.NN H.NO N.mm H.ONm 3.NOH 0.0N O.N3N 0.0m 0.0N O.ONH O.OO O.O3 OH cosmom N.Om O.Nm O.mN O.OO O.OO O.33 O.OO O.Om H.03 N.30 O.OO O.OO ON O.O3 0.3m O.ON O.OO N.mO O.HO O.mN N.OO N.Nm 3.3HH O.OHH O.OO OH 898:O O.OO O.Om 3.ON O.OHH O.ON H.33 3.ONH N.Om O.N3 O.HO O.OO 3.0O ON 3.33 0.Nm 0.0m N.00 0.00 0.00 N.03 0.0m 3.H3 0.00 0.00 3.Nm 0H OmOpoOH O O.OO O.Om O.ON N.HN O.HO N.03 N.3N O.HO O.HO O.NO3 0.0N3 O.ONO ON 0 0.00 N.03 3.0m O.OO O.HO O.O3 m.OOH 0.0O 0.00 m.0mm H.3mm 0.000 OH O-oooscO1O 0.0m N.ON m.NN O.OHH N.ON N.Om N.HON 0.00 H.0m 0.3O O.OO 3.0O ON a n m.mm m.ON O.NN m.OH O.3O O.HO 0.00 H.N3 O.Nm 3.O3 O.0m N.Nm OH o.ooomHmteO ON 00 mm ON 00 mm 0N om ON ON om mm OOOO 3mH1m01> mmHnmmu> Hmum0um mmHummnq OCOOSE OOOm>HO> OOOOOLOOO Obs: .OOOO>HO> .> OHHOHLO>H0> OOOOLOOHO OOOHOOA r LOO OEOLw mm 0:0 00 .OOoO>Ho> 4% mo OCHOLOO 03» 0:0 .OOOOOLOOO .m .003: 4m LOO OOOO 00 com 0a 0000 mo OOO O00 HO mOpOOHHOOL oz» mo OEOLwHHHHE CH pnwfiOz >00 HOHHOOSE OOOOLO>O mm OOOLOOOL .OHOOHSO OOOLOEOOO CH C3000 LOOHH .00 mo OOOHOOLOCOOcOO pm mOOssom :oommo mo OONOONHHHOO Oceul.0 OHOOB 96 cells were present, there were more frequent cross walls, and hyphal walls were less parallel. Utilization of Glucose at Various Concentrations Growth curves were made of the twelve fungi grown in 25, 50 and 75 g glucose per liter of Humfeld's medium and incubated for 10, 20, 30 and 40 days each. The data, averages of two replicates each (Table 9), were plotted on graphs 1 through l2° Cantharellus clavatus (C-63-8) grown in submerged culture of 25 g glucose per liter produced little hyphal growth over a 40 day period, dry weight remained about equal to the dry weight Of the inoculum (Graph 1). At 50 g glucose per liter, a rapid growth increase occurred between 20 and 30 days then leveled Off after 30 days incubation. A steady hyphal growth in- crease developed in flasks of 75 g glucose per liter until a steep growth decrease (autolysis) occurred after 30 days after reaching a growth peak of about 95 g dry mycelial weight. A maximum growth of 155 g dry weight occurred at 50 g glucose per liter, 40 days incubation and the minimum growth of 30 g dry weight occurred at 10 days incubation, 25 g glucose per liter. B. cibarius (C-63-15) mycelium grown in submerged culture at 25 and 50 g glucose per liter produced similar results at 10, 20, 30 or 40 days incubation (Graph 2). Dry 97 Table 9.eeThe utilization of glucose by twelve fungi grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates at the end of 10, 20, 30, and 40 days. CultUre Grams Days per Liter 10 20 3O 40 Cantharellus glavatus 25 . 29.4 28.5 35.4 34.0 C-53-8 50 27.7 32.1 134.7 155-7 75 38.9 61.3 96.7 40.9 QT cibarius 25 35.2 68.0 83.2 47.8 C~63al5 50 34.8 88.0 79.3 56.6 75 295.4 214.6 104.6 55.5 C5 Clbarius 25 24.3 22.3 38.8 35.9 ”" 0-63-86 50 28.5 39.5 46.4 42.1 75 35.5 54.5 4213 52-2 Q; cibarius 25 433.2 372.7 340.1 317.5 w5333—75956 50 805.7 704.3 603.1 535.9 75 1031.2 938.5 851.3 778.4 Agaricus ggmpestris 25 111.8 247.1 259.2 238.5 A- 3-136 50 470.2 253.8 310.3 224.6 75 432.5 408.9 291.4 314.9 5; bis orus 25 36.5 44.3 81.4 90.5 ‘“78%6§TI37 50 32.9 51.2 67.2 213.9 75 37.7 45.1 8517 87.7 8; bis orus pleomorph 25 - 35.8 100.8 171.9 307.5 ‘Ip‘m - 38 50 31.2 121.8 364.6 363.2 75 46.5 46.8 283.1 361.0 8: bis orus, rhizomorph 25 29.0 27.6 36.4 40.7 - 3-139 50 34.6 39.0 44.3 146.9 - 75 41.4 32.4 121.6 108.5 Le ista nuda 25 155-3 241.2 322.7 397-3 f-63-133 50 115.3 240.9 390.8 461.9 75 114.8 155.4 386.1 451.8 Pleurotus ostreatus 25 35.7 37.2 27.2 28.7 -63-51 50 38.9 99.2 86.9 175.7 75 47.7 71.3 282.0 216.9 Volvariella volvacea 25 34.5 30.9 39.3 39.8 V-63-135 50 57.6 54.6 44.5 33.2 75 89.2 97.2 65.2 54.5 X; volvacea mutant 25 20.7 21.9 33.7 19.3 8-63-134 50 24.2 31.1 60.9 23.0 ‘ 75 32.7 51.3 180.0 268.7 Milligrams 98 Graph 1.-—Cantharellus clavatus (C—63-8) utilization of glu- cose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and 40 days incubation. 1601- 150 .. 140- A 25 g per liter 0 50 g per liter 0 130 1. U 75 g per liter 1201- 110‘“ H O O \D O 80*- 70" 601' 50‘1 30" 1 \ 10 JD Days Milligrams 99 Graph 2.—-Cantharellus cibarius (C-63-15) utilization of glu- cose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and_40 days incubation. ‘ 306 u 288 a 270 .. 252 «v- 234 1 216 .1 a 198 a H m 0 I A 25 g per liter 0 50 g per liter D 75 g per liter 144 126 + 108 T 90 l” o . >A O 72 .. A 5’4 41- [>0 36 GD 18 7 Days 100 mycelial weights increased to 20 days, leveled Off between 20 to 30 days, then decreased after 30 days growth. At 75 g glucose per liter, hyphal growth decreased from a max- imum Of 300 mg dry weight at 10 days to a minimum dry weight at 40 days incubation that was similar to the 40 day re- sults of the other two glucose concentrations. A tremen- dous surge of growth probably occurred during the first 10 days incubation at 75 8 glucose per liter. The mathmngrowth for C—63-l5 at all three concentrations occurred at 10 days. B. cibarius (C-63-86) grown in 25 g glucose per liter had a slight growth decrease at 20 days incuba- tion but increased to 30 days when another decrease followed to 40 days incubation (Graph 3). At 50 g glucose per liter, growth steadily increased up to 30 days then decreased as with 25 g glucose per liter. Hyphal growth in 75 g glucose per liter greatly increased between 10 and 20 days incuba- tion, decreased between 20 and 30 then increased once again at 40 days incubation. The similar growth decrease then occurred both in 25 and 75 8 glucose media, however, at 75 g this phenomoma occurred 10 days later than in the 25 g medium. The greatest growth for C-63—86 strain occurred at 20 days incubation, 75 8 glucose while the least growth occurred at 20 days, 25 g glucose per liter. In Graph 4, dry mycelial weight of B. cibarius (C—63-96) is plotted to determine the growth curves at various glucose concentrations during 10, 20, 30 or 40 days Milligrams 101 Graph 3.--Cantharellus cibarius (C-63—86) utilization Of glu- cose at concentrations Of 25, 50 and 75 grams per liter grown in submerged cultures, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and 40 days incubation. v 60 +- 56 ” E! 52 .. D 148 T1- ‘ 44 u I: O 401. O A\ 36 4’ D A 32 + 281- ‘0 2” h A\ 20 w 16 t A. 25 g per liter 12 w 0 50 g per liter 8 n D 75 g per liter 4 4 0 1'0 20 30 4'0 102 Graph 4.--Cantharellus cibarius (C-63-96) utilization of glu- cose at concentrations Of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and 40 days incubation. 1040 1 a 975 “ 910 a 845 T 780 f t: 715 " O 650 n 585 ” ~“‘---\~ 520 " Milligrams O 455 7 1A 390 “ -~\\0\‘\“‘A _A\ 325 1' A 260 w 25 g per liter 195 a O 50 g per liter 0 75 g per liter 130 n 65 -- 0 10 20 30 40 Days 103 incubation. At each glucose concentration of 25, 50 and 75 g per liter, hyphal growth reached a high at 10 days incubation which was followed by a gradual but steady de- crease at 20, 30 and 40 days. The greatest dry mycelial weight was at 10 days, 75 g glucose per liter, and the smallest dry weight occurred at 40 days incubation, 25 g glucose media. Mycelial growth for Agaricus campestris (A-63- 136) in varied glucose media is pictured in Graph 5. A great increase of growth in media containing 25 g glucose per liter occurred between 10 and 20 days incubation then leveled between 20 and 30 days followed by a decrease of dry weight at 40 days incubation. At 50 g glucose per liter, the highest growth at 10 days was followed by a rapid decrease to 20 days, then a slight hyphal dry weight increase to 30 days and again a decrease at 40 days. Growth in 75 g glucose per liter was somewhat similar to growth in the 50 g per liter medium, with a growth peak at 10 days followed by a slight decrease at 20 days, then a greater decrease at 30 days. A slight increase of growth occurred at 40 days in the 75 g medium. The least growth of submerged hyphae occurred at 25 g carbohydrate per liter, 10 days incubation; the greatest was at 10 days 75 g glucose per liter. The utilization of glucose for B. bisporus (A-63- 137) is diagramed in Graph 6. At 25 and 75 g, dry mycelial Milligrams 104 Graph 5.--Agaricus campestris (A-63-136) utilization of glu- cose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and 40 days incubation. 48 ' O 0 450‘L CI 420” . D 390*" 360‘- 3304. O D 3OO‘P D 270" 1 A O/ 240«- A 4 0' 210'r 1804 150.. 120‘" A A 25 g per liter 90.. 0 50 g per liter 0 75 g per liter 60... 3o4 0 10 20 3O 40 Days Milligrams 105 Graph 6.--Agaricus bisporus (A-63-l37) utilization of glucose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams of two replicates each at the end of 10, 20, 30 and 40 days incubation. 224 .. 210 H 0 196 4r A 25 g per liter 182 J» O 50 g per liter 168 1. U 75 g per liter 154 1. 140 ~- 126 4 112 .. 98 4.- E3t> 84 <- A 70 a- 56 1» 42 .. a C) 28 1p 14 ‘- nOYYG 106 weights were almost identical. A slight increase in growth occurred between 10 and 20 days incubation, a greater in- crease followed at 30 days, and a lesser increase occurred at 40 days incubation. Submerged growth in 50 g glucose per liter gradually increased up to 30 days incubation then greatly increased to a maximum dry weight reading at 40 days of over 200 mg. The minimum growth occurred at 10 days incubation, 50 g glucose per liter. B. bisporus pleomorph (A-63—l38) growth curves in glucose and Humfeld's media are plotted in Graph 7. At 25 g per liter, hyphal growth steadily increased from 10 to 40 days incubation; the greatest increase occurred be- tween 30 and 40 days. Mycelial growth at 50 g per liter showed a rapid increase from 10 to 30 days but then re- mained stationary between 30 and 40 days. Dry mycelial weights were also stationary when grown in 75 g glucose per liter, 10 and 20 days incubation. After 20 days, growth rapidly increased up to 30 days, then slightly lessened. Mycelial dry weight was the smallest at 10 days, 50 g glu— cose per liter, and the greatest at 30 days, 50 g. Graph 8 represents submerged hyphal growth Of B. bisporus rhizomorph (A-63-l39) in Humfeld's glucose media at the three selected carbohydrate concentrations and four growth periods. In 25 and 50 g glucose per liter, growth slightly increased between 10 and 30 days incubation, a slight decrease was registered in the 25 g medium between Milligrams 107 Graph 7.--Agaricus bisporus pleomorph (A-63-l38) utilization of glucose at concentrations of 25, 50 and 75 grams per liter grown in submerged culture, reported as averaged mycelial dry weight in milligrams Of two replicates each at the end of 10, 20, 30 and 40 days incubation. 368 " L ‘30 345 1r 3223 040 310 O10 O10 O10 O10 FIQ (U Q .C U) Q. 3 SH 12 (n O O .H E :> 94 0 CU 4-9 (D r—4 (0 r—-{ 0 <1) Qoo Q,\O (\- m :0 Ln 0 \0 gm m «N4 300 (Dr—1 moo mm (Or—4 car—i (0| v—il 23 I 5| :5 l O I 3| 3m r—im Hm won or—im (‘0 SHOW g, (UNI) 1.0 1.0 :4 HDTV.)- 0 o. 54 I (0| (0| (0| 3| I <1: (00 .00 .OO .00 oc> am one 08 - 33 mm It I I'm N \0 mun .3<3 GUTPTQSTq-G \03 003 H H a s m 3:: 33:: >3 HQ. HQ cu Q m :3 4..) m > «3 r1 0 m tn :Soo 02H HI :5! Hrn Hrn (no $-. LI cut CUE.) .00 .C: H p o s: m -| O O 78.2 4.5 355.8 320.6 6.8 21.5 “.5 70.7 “.0 lu.6 1“ pH 1“ pH 1n C- 3- 6 A-63-136w A- 3-137 A- 3—138 A- 3-139 C- 3-96 bis orus, pleomorph bis orus, rhizomorph cibarius cibarius bis orus Agaricus campestris g_._ g; g; g; L Table lO.-Continued. aquKIS PIG? otmeanS-q autquo-q aptaotqooapfiq eutaqsfio eutqeaao aptaotqooapflq auteqaq p109 otqaedse-q outfieaedse-q autueIe-qq Days** 118 00 m3 157. 4. in pH L-63-l33 Lepista nuda Pleurotus ostreatus P-63-51 Volvariella volvacea V-53-l35 :TO om3 \om volvacea, mutant V- 3-13u V. Table lO.—-Continued. Toaguoo auoidad GUIIEA-TG aueqdoqdfidq-qq autuoaauq-qq auttoad—q SUIUOanew -id autonatosI—qq GUIptnstu—d Days** “6.2 102.“ 20.6 in Lepista nuda pH L-63-133 mm (YT-IT OWL.“ Pleurotus ostreatus P-b3-Sl O\O m3 1“ pH V-63-l35 Volvariella volvacea “.5 17.6 1U pH volvacea, mutant V— 3-134 V. Nitrogen content based on 0.47 g per liter, the amount in one g of urea. * pH was taken at 1U days. ** 120 and 4.0 to 4.5 for all other amino acid media. 9. cibarius (C-63-86) had growth comparable to control in Humfeld's media for a number of the amino acids, and other related compounds. Moderate hyphal growth resulted with creatine, glycine, DL-isoleucine, L-proline, and DL-tryptophane while the best growth was obtained with peptone at 1“ days incubation. The pH at 14 days was between 3.5 and ”.5 with all cultures except peptone which was 6.8. Excellent mycelial growth for g. cibarius (C-63- 96) resulted in media containing 0.47 g nitrogen per liter of DL-alanine, L-aspartic acid, betain hydrochloride, L- glutamic acid, glycine, DL-isoleucine, L-proline, DL- threonine, DL-tryptophane, DL-valine or peptone. Moderately heavy growth develOped in media with L—asparagine, cysteine hydrochloride, D-histidine, DL-methionine while little or no growth occurred in cultures with creatine or L-cystine. The terminal pH was “.0 or below for cysteine hydrochloride, L-cystine, D-histidine and DL-methionine, 4.5 for peptone, and 5.0 to 7.0 for the remaining amino acid media. 9. gige agius (C-63-96) produced loosely organized pellets and hy- phal flask rings in the media containing amino acids. The other Cantharellus strains develOped minute pellets in media containing L-glutamic acid, L-asparagine, betaine hy- drochloride, L-proline or peptone. Agaricus campestris (A-63-l36) had exceptionally good growth in media containing L-glutamic acid, dry mycelial 121 weight at 1“ days was above 600 mg. Dry weights were be- tween 180 to “80 in media with DL-alanine, L-asparagine, L-aspartic acid, glycine or peptone. Moderate growth was obtained with DL-threonine or DL-valine, all other amino acids produced dry weights equal to control or less. The pH remained between “.0 and “.5 for all cultures except for drops to 3.0 for L-cystine, 5.0 for creatine and 6.“ for L-aspartic acid and L—glutamic acid. A. bisporus (A-63-137) could not utilize any amino acids except DL-alanine, creatine, L-proline or the related betaine hydrochloride and peptone. A moderate my- celial growth at the end of 1“ days occurred. Terminal pH was “.0 to “.5 except for creatine at 5.0 and L-cystine at 3.0. A. bisporus pleomorph (A-63-l38) grew best in the medium with peptone, moderately well only in creatine or L- proline and equal to control in all other amino acid media. A. bisporus rhizomorph (A-63-139) had moderate growth with glycine and L—proline, the best growth with peptone and no growth in the remaining amino acid media. The two Agaricus mutants had final pH readings of “.0 to “.5 except 5.6 for both mutants in the medium with creatine, and 6.0 for the pleomorphic strain in L-asparagine and glycine. A. campes— tris (A-63-l36) formed hyphal pellets in most amino acid cultures while 5. bisporus (A-63-137) and its two mutants produced few pellets. 122 Glycine and peptone both produced excellent hy- phal growth in submerged culture for Lepista nuda (L-63-l33) when added in concentrations of 0.“? g nitrogen per liter. Good growth at 1“ days incubation was also obtained with DL-alanine, L—aspartic acid, L-glutamic acid and DL- methionine. The remaining amino acid nitrogen sources produced growth near to Control Or less: The pH remained between “.0 to “.5 for all cultures except pH 5.0 for creatine and 3.5 for cysteine hydrochloride. Q. nuda in submerged culture containing peptone produced a purple color characteristic of the basidiocarp. In other cultures, pellet and flask rings were very common. Pleurotus ostreatus (P-63-51) had good growth in media containing DL-alanine or peptone, and no growth with L-cystine, glycine or D-histidine. Moderate mycelial de- velOpment was obtained with the other amino acids added individually to Humfeld's media. The pH at 1“ days re- mained at “.0 to “.5 for all media except 3.5 for cysteine hydrochloride, and 5.6 for creatine. Pleurotus grew as loose interwoven hyphal masses in amino acid media. Volvariella volvacea (V-63-135) and y, volvacea mutant (V-63—l3“) both grew best in peptone, while V-63- 135 also grew well in L-asparagine. Moderate growth for both strains resulted with cysteine hydrochloride, L- proline and DL-valine; V-63-135 produced moderate growth 123 also in DL—alanine, L-glutamic acid, glycine and DL- isoleucine; while mutant strain V—63-l3“ grew moderately in media containing betaine hydrochloride, creatine or DL- tryptophane when results were compared to control. No growth equivalent to control was obtained with L—aspartic acid, L-cystine, D-histidine and DL-methionine for both Volvariella strains. Poor growth for Y, volvacea (V-63- 135) also develOped in betaine hydrochloride, DL-threonine or DL-tryptOphane, and for 1. volvacea mutant (V-63-l3“) in DL-alanine, L-asparagine, L-glutamic acid, glycine and DL—isoleucine. The pH remained at “.0 to “.5 except for 3.5 for cysteine hydrochloride and 5.6 for creatine. Hyphae of the Volvariella cultures grew as submerged mycelial masses in the amino acid media. Several ammonium and nitrate compounds were se— lected for nitrogen utilization in Humfeld's medium by the twelve fungi and compared to urea; results were recorded in Table 11. Dry mycelial weights of three replicates for each nitrogen source and culture were averaged at the end of 1“ days incubation. Only compounds producing hyphal growth greater or equal to that of urea medium are consid- ered here, and terminal pH changes from the initial “.5 are mentioned. The nitrogen content for each medium was 0.“? g per liter, or the equivalent amount of nitrogen in one g urea. by twelve mycelial dry weight in milligrams of three repli- wn in submerged culture, reported as averaged cates at the end of 1“ days. Table ll.-The utilization of ammonium and nitrate nitrogen sources in comparison to urea fungi gro 12“ 33 003N3MOC‘-.00 c3003 \03 OO ,3 H3 93‘1“ HO (Hooo'cmrHOxo meow \0\0 \CQ HO O\\O M L0 L0 0 ‘O Ln M M 00 3 O; H (\1 QQBJQIU Ho 00040200000 000.0. 000\ 000*. NO 01m umrumm L0H HH\0HOH3H 00HHH NH NH LnH L0H r-1 H H m m N (\J H (“-1 H MRIpOS L03 0.353pr3 N3Ln3 L03 M3 M3 Ox: N m m Ln 3 (H N 3 3 3 m 339,1un mm NLflMLfiONNLfl Manxm mm 3L0 0m \00 mntsseqod [\3 H3cfi3tflt~x03 0333 H3 O3 H3 {\Ln m m m 04 W M M m 3 M 3 M 00m 3Inmmom00m Chum—{O b-Ln Hm Mm Mm QQBJQIU . 0 . I O O O I O O O C O O O Q Q 0 O O O O 33 0.3M3Otxm3 03mm O3 M3 O3 O3 wntsaufiem N 3 b_ 3- N, F_ N m\ (n M (Hm MUNNLfl—ZIOMLR MLnxom tHLn 00m \0Ln OLn aqedqtu O O I I O I I I I C I O O C O O I D O O 0 O OIJan HM 3ML0MMLnMM 3MNM OM OM (\M NM (0 m m M m H m m M \ - \m', \ M O 00 om 919.1un “C“? ‘9”. .‘T‘i‘iofi‘? “T“?‘YL‘? .L‘? .L‘? “3 . .. 0 33 \O-ZTK):TDH\O-:f-:T H3003 003 \03 H3 03 SROQIBQ 0 H ,3 ,._4 N (\J (\J N H r-4 (\1 4—1 on 3211231118 OLn 3urxmmooxmm ommm mm mm Hm own ShOQI’QQOD N m M 3 M M m m (\J m SQBJQIU LDLO C'xmmmm33m (DLDKOLO mm OLD LDLD mm wnIOI’BO \03 M333\0\0\03 N3M3 M3 33 0.3 \03 m m M [H o. M M m M M m 9312.13.19; \om mommwmxom HO3O coo Hm Hm mm wnIUOUIUIB M3 mmm3m3xom mmxom mm 003 M3 33 M (H m 31831103 00m 00OML00\0\00O 3M3Ln 3L0 00m 00m Hm mnIUOWB \03 mMH3OH003 0.3ON3 33 (\3 m3 M3 aQBJQIU own ®O®m©O®U\ [\mwm mm own OM OO wnIuowwe N: LDMCDJNNO‘IJ' H333 33 (ID-:1 (1): CD: N m m \0 3 M 3 3 N _. ‘- ’\ \ ,-\ 339.1310 “To. 0.93.9999“. 03093.0. :1“. '1“? “3°C. ‘9“. WUIUOUILUE (\Lfl LnMCOLOMOkDm \O\OC\JL{'\ r—‘|\D O3 (\J\O H3 m b— (\J H \0 (\1 M m 00 L0 M H apraowo ‘9“? 5“L‘°?“3“303°C2“.‘ "WT“? “T“? “3“) 0.“? OT“? mnruomme 33 L03\O3MH003 \03H3 O3 \03 \03 H3 0.! LI‘\ (\J (\J m (‘0 :T r-1 (*1 M SQ‘BUOQJBOIQ \OO OONOLROMC) \OOMO 00O L{'\O LnO O mntuomme o o I o o o a o o o I u o o o a o o o 0 o r—1t\ Mr~3t\Lr\t\Hr~ Ht\\0t\ 3N H3 COIN \C[\ at 3 3 3 3 Lf\ 3 m M an 3 3 II (\J a) >>3IIZ 332323212331 33233; 3 3:1: 32:: III 3H0. HQHQHQHQ HQHCLH 310.300. O. U) a a 3 (0 U) s) U) m L .C (U r—4 :3 :3 W1 :5 SC. :30. '0 H HU) H *1 LOLNLQCDLLQ Mm: H03 (11:3 Lu’\£. mumOWlOLmOOm MSHHQJCU Lu (Ur-4m 3L17r-4"r—* OH Er-iuj Huci’fl-HO (UMID ID Ob .' ml 63 I (00!» [01‘ l 1.03 £:>MoH(Y‘)-H me-Hm-HOM-HNMWCUMLL c0:> #360 C) 0 L57. DODQJOD-HQ-H'UFZW >r—i Cir-4| l mm: l HI .Cl :3 I <1) (CIHO cUUO °|U -|O t10 U 0 q <1 6‘ (<0. ‘4 Ad > r0 trogen content based on 0.“? g per liter, the amount in one g of urea. O H was taken at 1“ day ‘ all p 4 qua, 3-135 ** V- V. vclv mutant V-63-1? 125 Cantharellus clavatus (C-63-8) was able to uti- lize only ammonium bicarbonate, and produced a higher dry mycelial weight than with urea. The former had a dry weight of “1.6 mg, the latter was 31.“. At 1“ days, the pH with ammonium bicarbonate was 7.0, and 6.“ with urea, other nitrogen source media remained close to the original pH or lower. Few mycelial pellets formed; hyphae remained single stranded in submerged culture. Dry mycelial weight for g. cibarius (C-63-15) was higher in cultures of ammonium citrate, ammonium sul- fate or ammonium tartrate than with urea; the respective 1“ day pH readings were 3.5, 3.0 and 3.0. Other terminal pH readings were generally “.5 or higher. The other am- monium compounds produced moderate submerged hyphal yields, while nitrates were not utilized. Urea in Humfeld's me- dium gave greater hyphal growth than any of the inorganic nitrogen source media for g. cibarius (C-63-86).‘ Hyphal pellets develOped only in the urea medium, all other cul- tures produced hyphal strands. ‘An Opposite effect was registered with g. cibarius (C-63-96) compared to the other Cantharellus strains. All the nitrogen sources, except ammonium bicarbonate and ura— nium nitrate, were utilized by the mycelium producing dry mycelial weights around 300 mg and above at 1“ days incu- bation. Terminal pH readings were quite varied. Except 126 for ammonium bicarbonate, ammonium nitrate and uranium nitrate, mycelium of g. cibarius (C-63-96) developed dense pellet formation in submerged culture and grew thick flask rings at splash levels. Urea, ammonium citrate, calcium nitrate, and am— monium tartrate each produced good mycelial dry weights at 1“ days with Agaricus campestris (A-63-l36), while other inorganic nitrogens were not utilized. Urea had a termi- nal pH of 6.8, while the other three cultures were 3.5 to “.5. The pH values for the other media remained near “.5 except for pH 1.9 with uranium nitrate and pH 7.0 with am- monium bicarbonate, a consistency with the two latter in- organic nitrogens found in all 12 fungal cultures with one exception. Hyphal pellets formed in the cultures which produced good mycelial dry weights. A. bisporus (A-63-137) had greatest hyphal growth in urea in Humfeld's medium while all the other nitrogen sources had less dry mycelial weights. Both mutant strains of A. bisporus had a reduced mycelial dry weight in the urea medium compared to the parent culture. A. bisporus pleomorph (A-63-l38) utilized most of the ammonium com- pounds at about the same rate as urea, all producing simi- lar dry weights. Magnesium nitrate and sodium nitrate media both produced over twice the amount of hypha as did urea, other nitrate sources were poor sources. With A. bisporus rhizomorph (A-63—139), only the ammonium tartrate 127 flasks had hyphal growth equal to urea cultures, all other inorganic nitrogen sources were not good sources. The pH values for media supporting good growth with A. bisporus and its mutants remained around “.5 to 5.0 except for the Humfeld's medium with ammonium bicarbonate. Both mutants had poor pellet formations in media containing an inorganic nitrogen source. Lepista nuda (L-63-l33) grown in urea or ammonium bicarbonate had dry mycelial weights around 290 mg at the end of 1“ days incubation. Terminal pH was 6.0 for the urea medium, and “.0 for ammonium bicarbonate. Very poor growth occurred in all other inorganic nitrogen sources. Pleurotus ostreatus (P-63-5l) grew well in urea and ammo- nium citrate, and had terminal pH readings of 6.“ and 6.8 respectively. All other ammonium and nitrate nitrogen sources produced minimal growth results and the pH readings remained at “.5 except for the usual low pH of uranium ni- trate and ammonium bicarbonate. Inorganic nitrogens sup— porting good growth developed moderate pellet formation. Volvariella volvacea (V-63—l35) was able to uti- lize ammonium bicarbonate, ammonium citrate, ammonium ni— trate, ammonium tartrate, calcium nitrate, potassium nitrate, and sodium nitrate and produced similar mycelial dry weights as urea did at 1“ days incubation. Except for pH 6.“ in the urea medium and pH 7 in ammonium bicarbonate medium, all other good hyphal producing media remained at 128 pH “.5. Ammonium sulfate, and magnesium nitrate media were moderately good growth promoters for V—63-l35. v. volvacea mutant (V-63-l3“) grew best in ammonium bicarbonate, and well in urea, ammonium chloride, ammonium citrate, ammonium tartrate, calcium nitrate, cobaltous ammonium sulfate, cu- pric nitrate, magnesium nitrate, potassium nitrate, and sodium nitrate. Terminal pH readings at the end of 1“ days for inorganic nitrogen source media with the Volvariella mutant remained at the initial “.5 except for ammonium bi- carbonate. 1. volvacea (V-63-135) grew in submerged cul— ture as a mass of interwoven hyphae. y, volvacea (V-63- 13“) grew only as isolated hyphal strands. Vitamin Requirements In Table 12, ten vitamin sources are listed that were each added in concentrations of 0.5 mg per liter to glucose casein hydrolysate with purified agar. Four fungi, Volvariella volvacea (V—63-l35), Lepista nuda (L-63-l33), Cantharellus cibarius (C-63-86) and Pleurotus ostreatus (P—63-5l), were selected for this study. Hyphal growth measurements in mm at two day intervals after inoculation were taken for each of the four fungi growing on each of the 10 vitamin media and control, and averaged from repli— cates of 3 cultures. Y: volvacea (V—63-l35) had growth differences during the first two days of incubation but then growth OO HO OO NO OO OO HO HO OO OO OO NH HN OO ON ON ON NN HO HO NO OO NO OH NH ON NN OH OH OH NN ON NN ON ON O O OH NH HH HH OH NH OH OH OH OH O HOumOum O O O O O O O O O O O O mspmmppmo O.H O.H O.H O.H O.H N O.H N O.N N O.H N mspopsmHO O NN OH OH OH O OH ON OH OH OH NH 0.0 OH O OH OH O OH OH OH O OH OH O OH O O O O HH OH NH O OH O N O 0.0 O O O O OH O O O O OOIOmuo H O O N H H O O O N O O OOHOOOHO 0.0 H 0.0 0.0 0.0 0.0 0.0 H 0.0 0.0 H N OOHHOOOOOOOO OH OH OH OH OH OH OH ON OH OH ON NH NH OH OH OH OH OH OH OH OH NH OH OH O NH O O H OH NH NH OH O NH O O O O O O O O O O O 0.0 O o, O O O O O O O O O O O O OOHuOOuO m O O O.N O.N O N O.N O.N N N O.N N OOOO mOmHOOH OO OO NH OO OO OO OH HO O» OO OO NH OO NO HO OO OO OO OO OO HO OO NO OH OO OO OO OO NO OO HO HO NO OO OO O ON ON ON ON ON ON ON ON ON ON ON O OOHuOOI> NH HH NH OH OH OH OH OH OH OH OH O mm0m>Ho> H O.N O H H O O O O O O N OHHOHOO>H0> O 1. J J qu mu H 1.100 0. 99 Paid WOAMQ o H n I UAR 7:? I Use I as be. u I. 1 0. IPJ p? B GUT. O I.0 Tum 3, B I. o oat. so 0 ufio 3 0.0 Dz J m u J J00. I. I. ROI. I. J CI. 0 I. T. I._O u u 1_m u 0. In TL u B D. X E 8 T: 00 e A a I. _ O . I u u a .mcHEmpH> wchkucoo OHOOE pmwm co CQHOOHSOOCH 909mm mambnmucH mac 03» pm mHoumEHHHHE :H cpZOHw Hmnmzmla.mH mHnt 130 rates became equalized. P—amino benzoic acid, ascorbic acid, biotin, calcium pantothenate, niacin and niacinamide media had initial hyphal growth, during the first two days after inoculation, of about 5 to 8 times greater than con- trol. Pyridoxine hydrochloride, riboflavin, rutin or thia- mine media had hyphal growth rates equal to hyphae grown in the medium without a vitamin source. Calcium pantothe— nate and niacinamide had a slightly reduced growth rate compared with other vitamins, they equaled growth in the control medium. Hyphal growth of p. nug§_(L—63-133) and g. gs: treatus (P—63-51) showed no significant growth rate dif- ferences on any of the 10 vitamin agar media when compared to the control culture. 9. cibarius (C-63-86) had approx- imately similar hyphal growth for all vitamin media except control and niacinamide. Mycelium grown on the latter two agar media grew only half or one fourth as fast as mycelium on the other nine vitamin media. Cantharellus cibarius (C-63-86) appears to be the only one of the four fungi selected for vitamin study that showed a dependency upon vitamins for mycelial growth. Growth Hormones Five growth hormones; gibberellic acid, kinetin, 2,“-dichlor0phenoxy acetic acid, indoleacetic acid, and indolebutyric acid, were each added in concentrations of 0.1%, 131 0.01% and 0.001% to 2% malt Oxoid agar no. 3 and then inoc— ulated with v. volvacea (C-63-l35), Q. nuda (L-63—133), g. cibarius (C-63-86) or g. ostreatus (P-63-5l). Hyphal meas— urements in mm were taken seven times at two day intervals. Growth hormones at 0.1% concentration killed the inoculum due to difficulty in controlling the pH, concentrations of 0.01% limited growth also due to the low pH factor compared with control media, with or without acetone. No growth differences were noted in the two control media; acetone was used as Solvent for the hormones. At 0.001% concen- tration, no hyphal growth differences were noted with Eg- pista, Cantharellus or Pleurotus in any of the growth hormone media as compared to the two controls for each species. y. volvacea (V-63-l35) had an increased growth with four of the hormones especially gibberellic acid, and a retardation with another. Growth readings in mm for Volvariella were as follows at 0.001% hormone concentration at the second and fourth day of incubation respectively: gibberellic acid, 22, 3“; 2,“-dichlor0phenoxy acetic acid, 15, 22; indoleacetic acid, 19, 25; indolebutyric acid, 18, 23; and kinetin, 1.“, 9; compared to control with acetone, 9, 26; and without acetone, 9, 2?. After four days incu- bation, hyphal growth on all hormone cultures simulated deve10pment of the control cultures and in eight days covered the entire agar surface of the petri plates. 132 Spawn Media Table 13 indicates the suitability of spawn media for growth of X. volvacea (V—63-l35), Q. nuda (L—63-l33), g. cibarius (C-63—86) and B. ostreatus (P—63-51), incubated 2 weeks at 2“0 C unless otherwise indicated. X. volvacea (V-63-135) had no hyphal growth on seeds of rape, vetch, rye at 120 C or spruce needles; poor growth with flax, milo, rye at 180 C or sunflower; moderate growth with orange cane, spelt, sudan grass or rye with glucose; and heavy hyphal growth on lupine, millet, sorghum, wheat, rye in Humfeld's medium, rye at 2“0 C, 30° C, 36° C and ground corn cobs. p. nuda (L-63-l33) had no growth with spawn media of rape, vetch, rye at 360 C or spruce needles; poor growth with flax, lupine, millet, rye at 12° C or sunflower seeds; moderate growth with milo, sorghum, rye with glucose, rye in Humfeld's medium,rye at 180 C, 2“° C, 30° C or ground corn cobs; heavy mycelial growth with orange cane, spelt, sudan grass or wheat. g. cibarius (C-63-86) had no growth on vetch, rye at 360 C, or ground corn cobs; poor growth on rape, sorghum, rye at 12° C or spruce needles; moderate growth on lupine, orange cane, wheat or sunflower; heavy growth with flax, millet, milo, spelt, sudan grass, rye with glucose, rye in Humfeld's medium, rye at 180 0, 2M0 0 or 300 c. g. ostreatus (P-63-51) had hyphal growth on spawn media of vetch; poor growth with rape, rye at 12° C, 360 C, 133 Table l3.--Suitability of spawn media for growth of four selected fungal cultures.* Volvariella Lepista Cantharellus Pleurotus ** volvacea nuda cibarius ostreatus Substrate 7:63:13? L:63:l33 6:33:36” P-63-51 flax l l 3 3 lupine 3 1 2 3 millet 3 l 3 3 milo l 2 3 3 orange cane 2 3 2 3 rape 0 0 l l sorghum 3 2 l 3 spelt 2 3 3 3 sudan grass 2 3 3 3 vetch 0 0 0 0 wheat 3 3 2 3 corn cobs, ground 3 2 0 3 spruce needles 0 0 l l sunflower seeds 1 l 2 3 rye 3 2 3 3 rye, with glucose 2 2 3 3 rye, with Humfeld's medium minus carbohydrate 3 2 3 3 rye, 12°C 0 1 1 1 rye, 18°C 1 2 3 3 rye, 300C 3 2 3 3 rye, 360C 3 0 0 l u Cultures grown for 2 weeks. *‘H‘ O 2“ C unless indicated. Key: no growth poor growth moderate growth heavy growth LUMP-‘0 13“ or spruce needles; and heavy growth with all other sub- strates listed. Examination of y, volvacea (V-63-l35) inoculum on spruce needles, vetch or rye at 120 C spawn media showed that hyphae were dead, showing coagulated protoplasm. The Volvariella inoculum on rape produced an extremely dense mycelial growth over the inoculum plug and 2 mm of the adjacent rape seeds indicating nutrients were supplied by the agar plug. Mycelium growing on spawn media supporting poor growth was not dense, the growth was light and wispy over the grain surface and throughout the spawn medium. Occasional aerial or submerged dense tan hyphal areas appeared; these were composed of chlamydospore masses. The spawn cultures of moderate growth had a greater mycelial density, more aerial growth, some hyphal tufts and larger chlamydospore masses. Some flasks, with heavy growth produced dense tan mycelial mats over the surface and throughout the spawn, growth density was equal to basidiocarp tissue in some areas. Aerial hyphae in flasks of heavy growth filled the entire flask volume. Rye and wheat spawn developed the best growth for Volvariel- la. g. nuda (L—63-l33) grown on spawn media support— ing poor growth had white cottony mycelial development over the surface with no aerial growth. There was little growth into the grain from the culture surface. Moderate growth cultures produced thin mycelial growth throughout all the 135 spawn media, surface growth became more dense and slight aerial hyphal deve10pment occurred. Heavy growth yielded a mycelial mass over and throughout all the spawn media, but no tissue formation. Some aerial cottony growth de- veloped over a portion of the surface areas. Best growth was supported on spelt, sudan grass or orange cane spawn. g. cibarius (C-63-86) hyphal inoculum was killed on vetch spawn, ground corn cobs or rye at 36° C. Media supporting poor growth had cottony, thin growth over and through all spawn, at times wispy tufts appeared on the spawn surface. Moderate growth spawn had dense hyphal de- ve10pment over and throughout the media, some solidation, little aerial hyphal production, and was tan to cream in color. Dark brown exudate dr0plets appeared on the culture surface of several spawn media. Heavy growth cultures had very dense hyphal growth; tissue formation was common throughout the media. The surface again did not support aerial growth, only a dense mycelial mat was present, exu— date dr0plets formed small surface pools of dark tan li- quid (Figure 15). Rye and Humfeld's medium, and spelt spawn developed a mass of fructifications after 12 weeks incubation (Figure 16). There was little differentiation in the round dense bodies; no lammae formed. 3. ostreatus (P-63-51) hyphal inoculum was killed on vetch spawn. Flasks with poor growth showed thin white mycelial development over and throughout the media and had 15. 16. Figure 15.--Cantharellus cibarius (C-63-86) exudate formation with three month old culture, growing on millet grain. Figure l6.--Cantharellus cibarius (C-63-86) fructifications on rye grain with Humfeld's medium minus carbohydrate, three month old culture. 137 a somewhat cottony surface. Only rye at 36° C, rye at 120 C, spruce needles or rape spawn had poor growth, all other media supported heavy mycelial growth. Extremely dense hyphal growth developed, solidation was common on surface and throughout the media, rhizomorphs were abun- dant. Aerial growth at times filled the entire flask volume and grew out of the cotton stoppered flasks. Fructifications formed within 2 to 6 weeks on sudan, orange cane, millet, rye, rye with 2 g glucose per flask, rye with Humfeld's medium without glucose, or ground corn cob spawn media. Basidiocarps grown on corn cobs had large stipes and small caps (Figure 1?) while other fructifications were more equally proportioned such as those grown on 2% malt agar slants (Figure 18). An interesting observation concerning Pleurotus fructifications, when inoculations were made on 2% malt agar in petri plates, is that only rudimentary basidiocarp initials appeared and were composed of undifferentiated mycelium (Figure 19). At times only rhizomorphs formed on the 2% malt agar petri plates (Fig— ure 20). A microsc0pic view of early rhizomorph formation can be seen in A. bisporus rhizomorph (A-63-139) material (Figure 21). The cultures not forming fructifications de- veloped small droplets of yellow exudate on the colony surface. High temperatures of 36° C or above killed myce— lium of Cantharellus, Lepista and Pleurotus but Volvariella showed the greatest hyphal growth at 360 C. Volvariella 138 l7. 18. Figure l?.--Pleurotus ostreatus (P-63-51) basidiocarp formation on ground corn cobs, six weeks. Figure 18.--P1eurotus ostreatus (P-63-51) fructification on a 2% malt agar slant, six weeks old. ' Figure 19.--Pleurotus ostreatus (P-63-51) basidiocarp initials on 2% malt agar, five weeks old. Figure 20.--Pleurotus ostreatus (P-63-51) development of rhizomorphson 2% malt agar in a six week old culture. 1“O hyphae was extremely dense, large tissue areas formed through- out the rye spawn medium, and chlamydospore production was at its greatest. Cantharellus formed fructifications on rye spawn at 18° c, and did not develop at 12°, 2“°, 30° or 360 c. Pleurotus fructifications on rye spawn formed between 180 and 2“° C only. 9. cibarius (C-63-86) also produced immature fructifications on “% and 6% malt agar petri plates at “ weeks incubation. The structures were columnar projections 2 cm in length and 2 mm in diameter growing from the agar surface and were composed of psuedoparenchyma and compact hyphae. The fructifications produced in culture were exa- mined cytologically. Pleurotus ostreatus (P-63-51) basidio- carp grown on a 2% malt agar slant formed mature hymenia complete with basidiospores (Figure 22). Tissue removed from a Pleurotus fructification revealed a typical basidiomycete dolipore septa with parenthesomes (Figures 23 and 2“). A simple pore between two adjacent cells was found in tissue from Cantharellus cibarius (C-63-86) (Figure 25). g. ostreatus (P—63-51) was inoculated on 2% malt agar plates and incubated in inverted or upright positions, with or without 10% KOH present in the petri dish cover, in light or in dark. Fructifications were produced in cultures placed in continual light, inverted, without KOH; continual light, inverted, with KOH; and in natural light, alternating periods of light and dark, no KOH, inverted plates. Pleuro— tus cultures developed aerial tufts and a cottony surface in l“l Figure 21.--Agaricus bisporus rhizomorph (A-63-139) showing early formation of rhizomorphs (r, rhizo- morphs). x 3,“00 Figure 22.--Hymenium and basidiospores of Pleurotus ostreatus (P-63—51) grown on 2% malt agar slant. x 2, 00 l“2 Figure 23.--P1eurotus ostreatus (P-63—51) showing one dolipore septum in cross section from a fruiting body (8, septal swelling; p, pore in septal pore ap- paratus; scm, subcap matrix; cd, discontinuous pore cap; m, mitochondrion). x 33,000 l“3 Figure 2“.--Pleurotus ostreatus (P—63-51) cell with dolipore septa on adjacent walls from a fructifica- tion grown on ground corn cob spawn medium. x 23,375 1““ Figure 25.--Cantharellus cibarius (C-63-86) adjacent cells isolated from a fruiting body showing a simple pore septum (sp, simple pore; er, endoplasmic reti— culum; w, walls of adjacent cells). x 56,500 l“5 continual light, right side up, no KOH; continual dark, in- verted plates or right side up, with or without KOH; in natural light, alternating periods of light and dark, in- verted, with KOH; and natural light, alternating periods of light and dark, plates right side up, no KOH. Hyphal growth in petri plates containing 10% KOH was slightly less than plates without KOH. Potassium hydroxide has the capacity of absorbing CO2 but no relation to fructification was noted when the level of C02 was changed. 3. ostreatus was inoculated into autoclaved beech logs by filling 2" holes, 5" apart, 1 1/2" deep with rye spawn then covering the inoculum with drill shavings and taping to hold in place. After 2 weeks at 3“° C incubation, the wood chips were covered with a dense hyphal growth. Mycelium slowly grew over the log and in 3 months, basidio- carp initials appeared. The fructifications grew to one inch in height, but did not differentiate from the cylindri- cal hyphal mass. 01d decomposed beech logs were also inoc- ulated, mycelial growth was greater and developed faster but the same abnormal fructifications appeared. Logs con- taining rhizomorphs were transported from the collection site near Rose City and placed in a protected area near a building in East Lansing over winter. The following spring and fall, a number of Pleurotus sporophores developed. y_. volvacea (v-63-135), .L_- nuda (L-63-l33), g. cibarius (C-63-86) and 3. ostreatus (P—63-51) were selected for mushroom house studies and were grown under conditions 1“6 suited for Agaricus bisporus production, using rye spawn. Volvariella had some mycelial growth over the compost, but with the addition of the casing soil, no further growth oc- curred. Lepista produced a dense hyphal growth over the compost surface having a purple tint typical of Lepista basidiocarps. A mycelial mat also covered the casing soil but no fructification initials formed. The Cantharellus inoculum did not grow on the compost. After Pleurotus mycelium grew at a fast rate within and on the compost sur- face, tissue formation developed. When casing soil was added, mycelial growth on the soil surface was quite good but basidiocarp button stages failed to appear. Colonnyorphology on 20 Selected Media Tables 1“ through 25 briefly describe morphological char— acteristics and colony measurements of the twelve fungi when grown on twenty selected agar media (appendix). The basis for use of these 20 selected agar media was to pick out colony characteristics that could be used to recognize a specific strain or possible variants in these strains. The cultures were incubated four weeks before descriptions were made. Following the descriptive tables, Cantharellus cibarius (C-63-86), Egpista nuda (L-63-l33), Pleurotus gs: treatus (P-63-51) and Volvariella volvacea (V-63-135) are each pictured growing on four selected media (Figures 26- 29). 1“? Table 1“.—-Colony morphology of Cantharellus clavatus (C-63— grown on twenty agar media for one month. Blood agar base: no growth. Brewer anaerobic agar: diameter of colony 1.8 cm, tan col- or, dense growth, slightly course texture, irreg- ular margin. Chlamydospore agar: diameter of colony 7.2 cm, light growth, smooth surface with some aerial tufts, irregular margin, cream color, agar color change from blue to pink adjacent to colony. Corn meal agar: diameter of colony 7.“ cm, dense growth, white color, numerous aerial tufts, cottony tex- ture, smooth margin. Liver spleen glucose agar: no growth. Micro assay culture agar: diameter of colony 6.8 cm, waxy texture, moderate growth, aerial tufts at colony margin, cream color. Mycological agar: diameter of colony 7.“ cm, dense growth, matted texture, many aerial tufts, cream color, smooth margin. Mycosel agar: no growth. Nutrient agar: entire petri plate covered with moderate hy- phal growth, smooth surface, white color. 0. Modess agar: entire petri dish covered, dense growth, granular texture, white color. 1“8 Table l“.--Continued. Potato dextrose agar: entire petri dish surface covered, white color, aerial tufts, cottony surface, dense growth. Prune agar: entire plate covered, tufted, slightly aerial, white color, granular to cottony texture. Rice extract agar: poor hyphal growth over entire surface, cottony texture, white color, some small dense aerial tufts, large amount of subsurface growth. Russian medium: diameter of colony 6 cm, tan color, margin smooth, dense surface growth with tufted clumps, slightly matted. Russian medium minus casein and tryptophan: moderate sur- face growth, diameter of colony “ cm, smooth sur- face, no aerial development. Russian medium minus vitamins: diameter of colony 5 cm, tan color, smooth margin, dense growth, slightly matted. Thioglycollate medium: diameter of colony 7.2 cm, brown to black color, dense growth, matted cottony texture. Tomato juice agar special: entire petri plate covered, dense growth, matted cottony texture, light cream color. Tryptic soy agar: no growth. l“9 Table l“.——Continued. Violet-red bile agar: diameter of colony 3.5 cm, agar color change from purple to pink, waxy colony surface, irregular margin, white to yellow color, white aerial hyphae, moderate growth. 150 Table l5.—-Colony morphology of Cantharellus cibarius (C-63- 15) grown on twenty agar media for one month. Blood agar base: diameter of colony “.5 cm, dense hyphal growth, cream color, smooth margin and surface growth. Brewer anaerobic agar: diameter of colony 6.8 cm, cream color, moderate growth, smooth margin and surface. Chlamydospore agar: diameter of colony 5.3 cm, smooth mar- gin and surface, dense growth, cream to white color. 2 Corn meal agar: entire petri plate covered, moderate growth, rough texture, white color, no aerial development. Liver spleen glucose agar: diameter of colony 2 cm, smooth surface and margin, cream color, slightly waxy texture. Micro assay culture agar: entire plate covered, waxy sur- face, some colony folds radiating from point of inoculation, cream color. Mycological agar: entire petri plate covered, dense growth, waxy surface texture, cream color, smooth margin. Mycosel agar: no growth. Nutrient agar: diameter of colony “.5 cm, dense growth, cream color, smooth texture and margin. 0. Modess agar: entire petri dish covered, growth dense, light cream color, smooth surface. 151 Table 15.--Continued. Potato dextrose agar: entire petri dish covered, moderate growth, cream color, smooth texture, no aerial hyphae. Prune agar: entire petri dish covered, moderate growth, cream color, surface slightly rough with some hy- phal tufts. Rice extract agar: entire petri dish covered, thin surface growth, smooth texture, cream color, no aerial growth. Russian medium: diameter of colony 6.5 cm, moderate sur— face growth with some subsurface growth, cream to yellow color, smooth texture and margin. Russian medium minus casein and tryptOphan: diameter of colony “.2 cm, poor growth, cream color, smooth margin. Russian medium minus vitamins: diameter of colony 5.0 cm, moderate surface growth, cream color, smooth tex- ture and margin. Thioglycollate medium: entire petri plate covered, dense growth, cream color, waxy texture, smooth margin. Tomato juice agar special: entire petri plate covered, dense growth, cream to white color, matted cottony 'surface, slightly aerial. 152 Table 15.--Continued. Tryptic soy agar: diameter of colony 3.5 cm, waxy surface, moderate subsurface growth, some colony folds, white color, smooth margin. Violet-red bile agar: diameter of colony “.5 cm, waxy tex- ture, dense growth, folded and heaped surface, cream color, smooth margin. 153 Table l6.——Colony morphology of Cantharellus cibarius (C- 63-86) grown on twenty agar media for one month. Blood agar base: diameter of colony “.5 cm, smooth texture and margin, surface folds, white color, moderate growth. Brewer anaerobic agar: diameter of colony 2.5 cm, tan color, smooth texture and margin, poor growth density. Chlamydospore agar: diameter of colony 6.8 cm, blue color of agar changed to pink, many aerial hyphal tufts, rough texture, white color, moderate growth, smooth margin. Corn meal agar: diameter of colony 7.5 cm, many hyphal tufts, slightly aerial cottony texture, moderate growth, white color. Liver spleen glucose agar: diameter of colony 2.2 cm, dense growth, white color, matted surface growth, mar- gin smooth. Micro assay culture agar: diameter of colony “.9 cm, many aerial tufts of hyphae, dense growth, cream color, red to brown color for reverse surface of colony, smooth margin. Mycological agar: diameter of colony ?.3 cm, numerous small -hyphal tufts, very dense growth, cream to yellow color, smooth surface, irregular margin. 15“ Table l6.--Continued. Mycosel agar: no growth. Nutrient agar: diameter of colony 6 cm, dense hyphal growth, granular texture, white color, smooth margin, no aerial growth. 0. Modess agar: diameter of colony 5 cm, smooth surface and margin, tan color, dense hyphal growth. Potato dextrose agar: entire petri dish covered, numerous aerial tufts, very dense growth, white color, mat- ted cottony texture. Prune agar: entire petri dish covered, very dense growth, white color, numerous aerial hyphal tufts, fine cottony texture. Rice extract agar: entire petri dish covered, thin growth, smooth surface, no aerial growth, moderate sub— surface hyphal growth, white color. Russian medium: diameter of colony ? cm, smooth to granular texture, small aerial hyphal wisps radiating from colony center, white colon,smooth margin. Russian medium minus casein and tryptOphan: diameter of colony 5 cm, granular texture, dense growth, smooth margin, surface growth only, white color. Russian medium minus vitamins: diameter of colony 6 cm, granular texture, moderate growth, white color, smooth margin. 155 Table 16.--Continued. Thioglycollate medium: diameter of colony 5.3 cm, tan color, moderate growth, smooth margin and surface, no aerial growth. Tomato juice agar special: entire petri dish covered, light cream color, very dense growth, rough texture, many small aerial tufts, reverse surface dark brown. Tryptic soy agar: diameter of colony 6 cm, white color, dense growth, smooth surface with some folds, no aerial deve10pment. Violet-red bile agar: diameter of colony 3.5 cm, gray col- or, reverse surface black, matted cottony texture, some aerial growth, smooth margin, surface folds. 156 Table l?.--Colony morphology of Cantharellus cibarius (C- 63-96) grown on twenty agar media for one month. Blood agar base: diameter of colony “ cm, smooth texture, and margin, folds and breaks, cream to white in color, thin growth. Brewer anerobic agar: diameter of colony 5.“ cm, smooth texture and margin, dense growth, folded surface, cream color. Chlamydospore agar: diameter of colony 2.0 cm, white color, dense cottony growth, blue color of agar cleared around colony to pink, reverse colony surface black, irregular margin. Corn meal agar: diameter of colony 7.2 cm, slight waxy texture, some aerial hyphal tufts, cream to white color, smooth margin. Liver spleen glucose agar: diameter of colony 7.“ cm, smooth surface and margin, moderate growth, white color, extremely folded surface. Micro assay culture agar: diameter of colony 6.8 cm, dense growth, white color, smooth texture with some folds. Mycological agar: diameter of colony 7.6 cm, slightly folded, rough surface, heaped center, white col- or, smooth margin. 157 Table l7.-—Continued. Mycosel agar: diameter of colony 6.8 cm, dense surface growth, slightly folded, smooth to waxy at cir- cumference, center of colony white and periphery cream. Nutrient agar: diameter of colony 5 cm, white color, dense O. Modess growth, some subsurface growth, fairly smooth margin. agar: diameter of colony “ cm, white color, slightly cottony surface, moderately dense growth, smooth margin. Potato dextrose agar: entire petri plate covered, smooth surface, light cream color, no aerial growth. Prune agar: entire petri dish covered, white to cream col- or, dense growth, waxy texture, no aerial growth. Rice extract agar: entire petri plate covered, cream col- or, thin growth, some aerial tufts. Russian medium: diameter of colony “.5 cm, light tan col-' or, circumference of colony darker brown, dense growth, rough texture. Russian medium minus casein and tryptOphan: diameter of colony 3 cm, tan color, moderate growth, rough texture, smooth margin. 158 Table l?.--Continued. Russian medium minus vitamins: diameter of colony “ cm, tan color, smooth margin, moderate growth, rough tex- ture. Thioglycollate medium: diameter of colony 6.“ cm, dense growth, smooth surface and margin, some folds, cream color. Tomato juice agar special: diameter of colony 7.2 cm, dense growth, cream to white color, matted cottony tex- ture, smooth margin. Tryptic soy agar: diameter of colony 6 cm, smooth paper- like texture, surface folds, margin smooth, densely compact growth. Violet-red bile agar: diameter of colony “.8 cm, waxy tex- ture, heaved center, some folds, tan color. 159 Table 18.--Colony morphology of Agaricus campestris (A-63- 136) grown on twenty agar media for one month. Blood agar base: no growth. Brewer anaerobic agar: diameter of colony 1.2 cm, cream color, waxy texture, smooth margin. Chlamydospore agar: diameter of colony 2 cm, white color, cottony texture, aerial hyphae, blue color of agar around colony cleared to pink, smooth mar- gin, reverse surface of culture black. Corn meal agar: diameter of colony 7 cm, white color, mod— erately dense, some aerial hyphal strands, smooth margin. Liver spleen glucose agar: diameter of colony 0.5 cm, cream color, waxy texture, dense growth, heaped. Micro assay culture agar: diameter of colony 0.9 cm, tan color, waxy surface, heaped, dense hyphal growth. Mycological agar: diameter of colony 6.5 cm, dense growth, smooth surface and margin, brown color. Mycosel agar: no growth. Nutrient agar: diameter of colony 2 cm, white color, dense growth, some aerial hyphae, smooth margin, granu- lar texture. 0. Modess agar: diameter of colony 3 cm, waxy texture, smooth surface and margin, white color. 160 Table 18.—-Continued. Potato dextrose agar: diameter of colony 7.“ cm, cream color, smooth surface and margin, moderately dense growth. Prune agar: diameter of colony “.2 cm, white color, dense growth, some aerial tufts, rough texture. Rice extract agar: entire petri dish covered, white color, rhizomorphs present, moderate growth, some aerial deve10pment. Russian medium: diameter of colony 3 cm, buff color to dark brown at circumference, dense growth, smooth margin, granular texture. Russian medium minus casein and tryptOphan: diameter of colony 1.5 cm, buff color, granular texture, smooth margin, moderate growth. Russian medium minus vitamins: diameter of colony 0.5 cm, tan color, some aerial tufts, dense surface growth, much subsurface hyphal growth. Thioglycollate medium: no growth. Tomato juice agar special: diameter of culture “ cm, dense growth, heaped center, some surface folds, matted cottony texture, white to gray color. Tryptic soy agar: no growth. Violet—red bile agar: diameter of colony 0.7 cm, waxy tex- ture, heaped surface, cream to tan color. 161 Table 19.--Colony morphology of Agaricus bisporus (A-63-l3?) grown on twenty agar media for one month. Blood agar base: diameter of colony 0.2 cm, cottony sur- face, slightly elevated, tan color, reverse sur- face white, smooth margin. Brewer anaerobic agar: diameter of colony 2.9 cm, cream to white color, cottony texture, smooth margin, tan color pigment diffused in agar. Chlamydospore agar: diameter of colony 2.“ cm, cottony surface, moderate growth, blue color of agar changed to pink around colony, smooth margin, cream color. Corn meal agar: diameter of colony 6.6 cm, dense growth, matted cottony texture, white color, some aerial hyphal tufts, smooth margin. Liver spleen glucose agar: diameter of colony 2.5 cm, mat- ted cottony surface, dense growth, cream color, smooth margin. Micro assay culture agar: diameter of colony “.8 cm, waxy surface, cream color, reverse surface of colony brown, smooth margin. Mycological agar: diameter of colony 6.5 cm, cream to white color, diffused tan pigment in agar, rhizomorph formation, tufted aerial hyphae. Mycosel agar: no growth. 162 Table l9.--Continued. Nutrient agar: diameter of colony 5 cm, white color, sparce growth, smooth surface and margin. 0. Modess agar: entire plate covered, thin growth, numerous aerial hyphal tufts, white color. Potato dextrose agar: diameter of colony 5.8 cm, many aerial tufts, moderately dense growth, cottony texture, smooth margin, white color. Prune agar: diameter of colony 6.9 cm, white color, matted texture, smooth margin, some aerial hyphae. Rice extract agar: entire petri plate covered, white color, rhizomorph formation, moderate growth, some aerial growth. Russian medium: entire plate covered, cream to white color, some rhizomorph formation, few aerial hyphal tufts. Russian medium minus casein and tryptOphan: diameter of col- ony 6 cm, white color, dense growth, cottony tex- ture, smooth margin. Russian medium minus vitamins: diameter of colony “ cm, large amount of subsurface growth, irregular mar- gin, cottony surface, white color. Thioglycollate medium: no growth. Tomato juice agar special: diameter of colony 5.6 cm, 163 Table l9.——Continued. matted cottony texture, cream to white color, dense growth, reverse surface of colony dark brown, smooth margin. Tryptic soy agar: diameter of colony 2 cm, brown color, granular texture, reverse surface of colony dark brown, smooth margin. Violet-red bile agar: diameter of colony 2.5 cm, smooth surface and margin, cream color, slightly folded, moderate growth. 16“ Table 20.--Colony morphology of Agaricus bisporus pleomorph (A—63-l38) grown on twenty agar media for one month. Blood agar base: diameter of colony 5 cm, dense growth, tan color, some aerial growth, smooth margin, granular texture. Brewer anaerobic agar: diameter of colony “.5 cm, cottony texture surface, cream color, reverse surface of colony brown, dense growth, smooth margin. Chlamydospore agar: diameter of colony 6.2 cm, few aerial hyphal tufts, white color, blue agar color change to pink adjacent to colony, irregular margin. Corn meal agar: entire petri plate covered, aerial hyphae fills petri plate volume, white color, dense growth. Liver spleen glucose agar: diameter of colony 5.“ cm, tan color, matted cottony texture, dense growth, smooth margin. Micro assay culture agar: diameter of colony 5.5 cm, tan color, waxy surface, heaped center, reverse side of colony brown color, smooth margin. Mycological agar: entire petri plate surface covered, dense cottony growth, cream to white color. Mycosel agar: no growth. Nutrient agar: diameter of colony “.5 cm, white color, dense growth, some aerial hyphae, rough surface, smooth margin. 165 Table 20.-—Continued. O. Modess agar: entire petri plate covered, moderate aerial deve10pment, white color, rough surface. Potato dextrose agar: entire petri plate covered, white color, moderate growth, some aerial tufts, rough texture. Prune agar: entire petri plate covered, densely aerial hy- phae,petri volume filled with hyphae, white color. Rice extract agar: entire petri plate covered, several rhizomorphs developed, some aerial cottony growth, not dense, white color. ‘ Russian medium: entire petri plate covered, cottony tex- ture, a few rhizomorphs, white color, moderate growth. Russian medium minus casein and tryptOphan: entire petri plate covered, white color, moderate growth, cot- tony texture, no rhizomorphs. Russian medium minus vitamins: diameter of colony 5 cm, smooth margin, matted cottony texture, white color. Thioglycollate medium: no growth. Tomato juice agar special: diameter of culture 7.2 cm, cream color, dense cottony growth, aerial hyphae. Tryptic soy agar: diameter of colony 1 cm, tan color, re- verse colony surface brown color, dense growth, smooth margin, cottony texture. 166 Table 20.-—Continued. Violet-red bile agar: diameter of colony “.1 cm, colony perimeter waxy, cottony textured center, white color, irregular margin. 167 Table 21.--Colony morphology of Agaricus bisporus rhizomorph (A—63-l39) grown on twenty agar media for one month. Blood agar base: diameter of colony 1 cm, dense growth, tan color, smooth margin, granular texture. Brewer anaerobic agar: diameter of colony 3.? cm, tan col- or, brown pigment diffused into agar, dense growth, irregular margin, matted texture. Chlamydospore agar: diameter of colony “.9 cm, blue color of agar changed to pink adjacent to colony, thin surface growth, irregular margin, cream color. Corn meal agar: diameter of colony 6 cm, cream to white color, moderate growth, numerous aerial tufts of hyphae,irregu1ar margin. Liver spleen glucose agar: diameter of colony 3.7 cm, tan color, dense surface growth, some rhizomorph for- mation, irregular margin. Micro assay culture agar: diameter of colony 3.2 cm, dense growth, waxy texture, tan color, smooth margin. Mycological agar: entire petri dish surface covered, nu- merous rhizomorphs, some basidiocarp initials, very dense growth, tan color. Mycosel agar: no growth. Nutrient agar: diameter of colony 7 cm, white color, moder- ately dense growth, matted cottony texture, some rhizomorphs, smooth margin. 168 Table 21.--Continued. O. Modess agar: entire petri dish covered, white color, some aerial hyphal tufts, moderate growth, cot- tony texture. Potato dextrose agar: diameter of colony 6.“ cm, dense growth, smooth to waxy texture, cream color, smooth margin. Prune agar:l entire petri dish covered, numerous rhizomorphs, good growth, white color, some aerial hyphae, rough texture. Rice extract agar: entire petri plate covered, moderate growth, good rhizomorph formation, some aerial rhizomorphs, irregular margin, some subsurface growth, cream color. Russian medium: entire petri plate covered, matted cottony texture, some rhizomorphs, some submerged hyphal patches in agar. Russian medium minus casein and tryptOphan: diameter of colony 5 cm, moderate growth, rough texture, cream color, irregular margin. Russian medium minus vitamins: diameter of colony 6 cm, rough surface growth, irregular margin, subsur- face hyphal growth. Thioglycollate medium: no growth. 169 Table 21.--Continued. Tomato juice agar special: diameter of colony 6.2 cm, brown color, reverse side of colony dark brown, dense growth, somewhat aerial, rough texture. Tryptic soy agar: diameter of colony 2 cm, waxy surface with folds, smooth margin, some subsurface growth. Violet-red bile agar: diameter of colony 3.1 cm, smooth texture and margin, no aerial growth, dense deve1- 0pment, tan color. 170 Table 22.-~Colony morphology of Lepista nuda (L-63-133) grown on twenty agar media for one month. Blood agar base: entire surface of petri dish covered with thick growth, some aerial hyphae, tan color, re- verse colony surface dark brown, rough texture. Brewer anaerobic agar: entire petri plate covered, buff color, matted cottony texture, moderate growth. Chlamydospore agar: diameter of colony 5.2 cm, some rhizo- morphs, moderate growth, aerial cottony hyphae, blue color of agar adjacent to colony changed to pink, irregular margin. Corn meal agar: diameter of culture 7.“ cm, cream to white color, extremely aerial, not dense, little sur- face growth. Liver spleen glucose agar: diameter of colony 5.7 cm, tan to cream color, extremely dense growth, matted cottony texture, smooth margin. Micro assay culture agar: entire petri dish surface cov- ered, waxy surface, violet to black color, pig- ment diffused into agar, smooth margin. Mycological agar: entire petri dish covered, dense growth, tan to yellow color, matted to rough texture, some aerial hyphae. Mycosel agar: no growth. Nutrient agar: growth over entire petri plate, white color, abundant aerial growth, cottony texture. 171 Table 22.-—Continued. O. Modess agar: entire petri dish covered, thin growth, cottony texture, no aerial hyphae, white color. Potato dextrose agar: entire petri plate covered, white color, moderate growth, slightly aerial, some- what cottony. Prune agar: entire surface of petri dish covered, moderate growth, tan to white color, some aerial hyphal tufts. Rice extract agar: entire surface of petri plate covered, thin growth, white color, abundant aerial hyphae, some subsurface growth. Russian medium: entire petri dish surface covered, white to tan color, thin growth, cottony texture, aerial deve10pment, no subsurface hyphae. Russian medium minus casein and tryptOphan: entire petri plate covered, white color, moderate growth, cot- tony texture, aerial hyphae. Russian medium minus vitamins: entire petri dish covered, white color, moderate growth, cottony texture, aerial hyphal deve10pment. Thioglycollate medium: entire petri plate covered, dense growth, matted cottony texture, cream to white color. 172 Table 22.--Continued. Tomato juice agar special: diameter of colony 6.2 cm, cream color, brown pigment diffused in agar, cot— tony texture, reverse surface of colony dark brown, densely aerial, irregular margin. Tryptic soy agar: ”entire petri dish covered, dense growth, white color, small cottony tufts, dark brown pig— ment diffused into agar, reverse surface of col- ony brown to black. Violet-red bile agar: diameter of colony 3.3 cm, waxy sur- face, smooth margin, some folds, central area violet black, circumference cream color. 173 Table 23.--Colony morphology of Pleurotus ostreatus (P-63-51) grown on twenty agar media for one month. Blood agar base: diameter of colony 3 cm, thin growth, sub- surface mycelium present, tan color, irregular margin. Brewer anaerobic agar: diameter of colony 6.9 cm, small aerial hyphal tufts, dense growth, white color, rough texture. Chlamydospore agar: diameter of colony “.5 cm, white color, aerial hyphal tufts, blue color of agar changed to pink adjacent to colony, moderately dense growth, irregular margin. Corn meal agar: entire petri plate covered, aerial hyphal tufts, white color, thin surface growth. Liver spleen glucose agar: entire petri plate covered, yel- low to white color, dense surface growth, slightly aerial, rough texture. Micro assay culture agar: entire petri plate covered, cream color, matted cottony texture, numerous aerial tufts, rough texture, growth over petri dish sides, yellow exudate drops on colony surface. Mycological agar: entire petri dish covered, very dense growth, growth over sides of plate, cottony texture, yellow exudate drops on surface, white to yellow color. 17“ Table 23.—-Continued. Mycosel agar: no growth. Nutrient agar: entire petri plate covered, white color, dense surface growth, rough texture. O. Modess agar: entire petri plate covered, tan to white. color, moderate growth, rough texture, not aerial. Potato dextrose agar: entire surface of petri dish covered, dense growth, white color, rough texture, some aerial hyphae. Prune agar: entire petri dish surface covered, moderate growth, aerial hyphae, white color, rough texture, some mycelium growing over petri dish side. Rice extract agar: entire petri dish covered, thin growth, several aerial hyphal tufts, white color, rough texture. Russian medium: entire petri plate covered, dense growth, white color, tufts of aerial hyphae. Russian medium minus casein and tryptophan: entire petri plate covered, dense growth, white color, tufted aerial growth. Russian medium minus vitamins: entire petri plate covered, 1 no aerial hyphae, subsurface growth, moderate growth, rough texture. Thioglycollate medium: diameter of colony 6.8 cm, white color, cottony growth, aerial hyphal tufts. 175 Table 23.--Continued. Tomato juice agar special: entire petri plate covered, very dense growth, cottony, yellow to white color, hy- phal growth out of plate, yellow droplets of exu- date on surface. Tryptic soy agar: diameter of colony 5 cm, white color, dense growth, rough texture, smooth margin, aerial hyphal tufts. Violet-red bile agar: diameter of colony “.8 cm, buff c01- or, waxy to smooth texture, no aerial growth, ir- regular margin. 176 Table 2“.-—Colony morphology of Volvariella volvacea (V—63— 135) grown on twenty agar media for one month. Blood agar base: entire petri dish covered, cottony tex- ture, white color, moderate aerial growth, poor surface growth. Brewer anaerobic agar: entire petri dish covered, tan col- or, moderate growth, some aerial growth. Chlamydospore agar: diameter of colony 3.2 cm, wispy aerial growth, poor surface growth, white to tan color, irregular margin, purple color of agar adjacent to colony changed to pink. Corn meal agar: entire petri plate surface covered, many aerial hyphal tufts white to tan color, moderate growth. Liver spleen glucose agar: entire petri plate surface cov- ered, dense aerial growth filling plate, tan col- or, grew out of dish. Micro assay culture agar: entire petri dish covered, dense surface growth, very dense aerial cottony deve10p- ment, tan color, grew out of dish. Mycological agar: entire petri plate covered, dense aerial growth, tan color, cottony texture, grew out of plate. Mycosel agar: very thin growth on agar surface. Nutrient agar: entire petri plate covered, light tan col- or, moderately dense aerial growth. 177 Table 2“.-—Continued. O. Modess agar: poor surface growth covering petri plate. Potato dextrose agar: entire petri plate covered, moderate growth, some aerial hyphae, cream color, colony radiating from inoculation point. Prune agar: entire petri dish covered, moderate growth over surface, fair deve10pment of aerial hyphae, tan color. Rice extract agar: entire petri dish covered, moderate surface growth, several dense aerial areas, tan to white. Russian medium: sparce surface growth over entire petri plate, aerial hyphal tufts, tan color, some sub- surface hyphal growth. Russian medium minus casein and tryptophan: sparce surface growth, some aerial tufts, tan to white color, some subsurface growth. Russian medium minus vitamins: light growth over petri dish agar surface, greater submerged growth than sur- face development, some aerial hyphal strands. Thioglycollate medium: entire petri dish surface covered, dense aerial hyphal growth, tan color, plate vol- ume filled with hyphae. 178 Table 2“.--Continued. Tomato juice agar special: entire plate covered, very dense aerial deve10pment, tan color, hyphal growth out of plate. Tryptic soy agar: entire petri dish covered, sparce growth, moderate aerial hyphae, tan color. Violet-red bile agar: diameter of colony 6.1 cm, very dense surface growth, loose cottony texture, tan color, irregular margin. 179 Table 25.--Colony morphology 0f Volvariella volvacea mutant (V-63-13“) grown on twenty agar media for one month. Blood agar base: entire petri dish surface covered, white color, dense growth, matted cottony texture, some- what aerial. Brewer anaerobic agar: entire petri plate surface covered, moderately aerial growth, white to tan, matted cottony texture. Chlamydospore agar: very thin mycelial growth over petri plate, white color, Some aerial hyphae. Corn meal agar: entire surface of petri plate covered, rough texture, aerial hyphal tufts, white to tan color. Liver spleen glucose agar: entire petri plate covered, cream to white color, dense growth, matted cot? tony texture. Micro assay culture agar: entire petri dish surface cov- ered, very dense growth, aerial hyphae present, cottony texture, some fructification initials. Mycological agar: entire petri dish surface covered, rough texture, white color, dense growth. Mycosel agar: no growth. Nutrient agar: thin hyphal growth over entire petri dish surface, white color, not aerial. 180 Table 25.—-Continued. O. Modess agar: sparce surface growth over entire plate, no aerial hyphae, tan color. Potato dextrose agar: entire petri plate covered, moderate growth, some aerial hyphae, cream color. Prune agar: diameter of colony 7.2 cm, moderate surface growth, granular texture, smooth margin, slightly cottony, cream color. Rice extract agar: moderate growth over entire petri plate surface, some subsurface mycelium, some aerial tufts of hyphae, white color. Russian medium: entire petri plate surface covered, white color, thin growth, some dense aerial hyphal growths. Russian medium minus casein and tryptOphan: entire petri dish surface covered, white color, thin growth, some aerial hyphal masses. Russian medium minus vitamins: very thin hyphal growth over petri plate surface, white to cream color, no aerial hyphae. Thioglycollate medium: entire petri dish surface covered, dense growth, tan color, matted cottony texture, some aerial hyphae. 181 Table 25.—-Continued. Tomato juice agar special: entire petri plate surface cov- ered, matted cottony texture, cream color, some fructification initials. Tryptic soy agar: sparce hyphal growth over plate surface, white color, some aerial hyphal strands. Violet-red bile agar: diameter of colony 6 cm, extremely dense growth, aerial mycelium, several fruiting initials, white color. 182 c. d. Figure 26.--Cantharellus cibarius (C-63—86) growing on the following media: (a) Potato dextrose agar; (b) Mycological agar; (0) Micro assay culture agar; (d) Violet-red bile agar. c. d. Figure 2?.--Lepista nuda (L—63—133) growing on the following media: (a) Chlamydospore agar; (b) Tomato juice agar special; (c) Violet-red bile agar; (d) Thioglycollate medium. c. d. Figure 28.-~Pleurotus ostreatus (P-63-51) growing on the fol- lowing media: (a) Tomato juice agar special; (b) Brewer anaerobic agar; (c) Potato dextrose agar; (d) Chlamydospore agar. c. d. Figure 29.--Volvariella volvacea (V-63—135) growing on the fol- lowing media: (a) Mycological agar; (b) Violet-red bile agar; (0) Micro assay culture agar; (d) Prune agar. 186 No growth of g. clavatus (C-63-8) occurred on blood agar base, liver spleen glucose agar, or tryptic soy agar while the three strains of g. cibarius (C-63-15, C-63-86, and C—63-96) had growth. The C-63-96 strain was the only one of the four Cantharellus cultures to grow on mycosel agar. Cantharellus clavatus (C-63-8) did not grow on tryptic soy agar while all three 9. cibarius strains did. Cantharellus clavatus (C-63-8) had the ability to cause color change from purple to pink in violet-red bile agar, the three 9. cibarius strains did not. Only 9. cibarius (Cé63-15) did not produce an agar color change from blue to pink using chlamydospore agar, the three other Cantharellus strains did. All four strains had colony color differences when grown on violet-red bile agar but the morphology was similar. The three 9. cibarius strains grew as dense cream to tan colored colonies with a smooth surface or margin on thioglycollate medium while 9. clavatus (C-63-8) produced a dense cottony colony, brown to black in color. Agaricus campestris (A-63-l36), the wild strain, did not grow on blood agar base and tryptic soy agar while A. bisporus and its mutants had some growth. The colony of A-63-l36 was waxy and smaller on brewer anaerobic agar, liver spleen glucose agar, O. Modess agar, and violet-red bile agar, compared to the larger cottony colonies of the three A. bisporus strains on the same media. The pleomorphic strain of A. bisporus, A—63-l38 , grew more vigorously and 187 more densely on most all agar media compared to the parent strain, rhizomorphic strain, and A. campestris. The pleo- morphic strain was less pigmented than the other strains. Agaricus campestris (A-63-l36) had a brown colony circum- ference not found with the three A. bisporus strains on my- cological agar. Less growth occurred with all four strains when casein and tryptOphan or vitamins were eliminated from the Russian medium. Lepista nuda (L-63—l33) produced rhizomorphs on chlamydospore agar. Waxy colonies formed on micro assay culture agar and violet-red bile agar instead of the usual cottony textured colonies. Colony colors were violet to black on micro assay culture agar, white aerial growth oc- curred and a brown pigment diffused into the tryptic soy agar givingzareverse black colony surface. Tan or cream surface growth occurred and the reverse surface was brown on blood agar base and tomato juice agar special, while growth on most other agars was white. Pleurotus ostreatus (P-63-51) changed the blue color of chlamydospore agar to pink adjacent to the colony. Numerous yellow exudate dr0ps formed on the colony surface when grown on micro assay culture agar, mycological agar, and tomato juice agar special. Colony color was white to yellow on most of the 20 agars, colony texture also had little variation. 188 Volvariella volvacea (v-63-135) grew quite well on most of the agar media and produced dense aerial hyphae while growth of the mutant strain, V-63—13“, was very much reduced. Mycosel agar supported no growth for the mutant but some growth for the parent. The parent was able to produce a color change from purple to pink with chlamydo- spore agar while the mutant did not. The tan color of aerial hyphae, indicating chlamydospores, was present with the parent strain on chlamydospore agar, liver spleen glu- cose agar, mycological agar, nutrient agar, prune agar, rice extract agar, Russian medium, tomato juice agar special, tryptic soy agar, and violet-red bile agar while the 1. volvacea mutant (V-63-13“) strain had a cream to white color and an absence of chlamydospores. Fructification initials occurred with the mutant strain on tomato juice agar Special and violet-red bile agar while initials were absent with the parent strain. DISCUSSION The purpose of this work was to determine first the carbohydrate and nitrogen requirements of three wild mushrooms Lepista nuda, Cantharellus clavatus, and g. g;- barius which are considered desirable for adaptation to commercial mushroom production. Volvariella volvacea, a mushroom grown commercially on rice straw in the Philippines, with little or no information on nutritional requirements, was also used in this study. A wild strain of Agaricus campestris and A. bisporus, the commercial mushroom, were also compared. Additional investigations were made on the effect of other factors on the growth and fructification of these organisms including: growth hormones, vitamins, types of spawn media, temperature, and environmental conditions in a mushroom house. Based on reports in the literature, a pH of “.5 was selected for all growth studies in submerged culture. Urea was the nitrogen source in all liquid culture eXperi- ments except for studies in the utilization of nitrogen sources. Humfeld and Sugihara (109) found that the utili- zation of urea does not change the pH of the medium suf- ficiently to have an unfavorable effect on growth. In 189 190 media studies involving fungi, a high pH affected solubili— ties while acidity affects the entry into the cell of meta— bolites (“1). Lilly and Barnett (1“3) kept the initial pH of media low to avoid isomeriZation of closely related monosaccharides such as glucose, fructose and mannose. Bohus (26) believes the hydrogen ion concentration on the medium determines what carbon and nitrogen sources will be at the disposal of the species, effects changes of colloids within the fungal cell and determines cell membrane perme- ability to the components of nitrogen source mixtures. Volvariella and Cantharellus species grew more favorably in acid media than alkaline, while Agaricus and Pleurotus species grew in only mildly acidic media. Lepista produced better mycelial growth in acid conditions in those experi- ments which lasted for shorter periods of time. The pH changes in media during the course of the experiment were caused by fungal metabolites (1“2). Fungi are able to produce acids, or bases such as ammonia from deamination of amino acids and proteins, thus changing the environmental pH. The utilization of cations such as the ammonium ion tends to drive the pH down resulting in a more acid medium; utilization of nitrate ion or other anions in— creases the hydroxyl ion concentration to create a more basic medium. Investigating the most useful carbon sources for mycelial growth of Cantharellus clavatus (C—63-8) showed that fructose-1,6- diphosphate produced the highest yields 191 followed by pectinol A, and pectin. The organic acids galac- turonic and tannic acid were best suited for a 10 day period of hyphal growth, while succinic acid was best for 20 days incubation for g. clavatus. All three strains of g. cibarius utilized fructose- l,6—diph0sphate as the best carbon source. However, strains of Q, cibarius had variation in selectivity of the remain- ing best carbon sources. Raffinose, a trisaccharide with one sucrose linkage to melibiose, is usually utilized readily by the majority of the fungi (Cochrane, “1); however, only C-63-86 strain readily utilized this carbon source. Strain C-63—96 had remarkable growth rates compared to the other 9. cibarius strains, and also produced a water soluble yellow-green pigment. Extensive dry mycelial weights re- sulted from utilization of D-mannitol, and D-sorbitol. Many other carbon sources were readily utilized by the strain, indicating greater adaptability to growth under natural con- ditions. The wild strain of Agaricus campestris (A-63-l36) had greatest hyphal growth with D-fructose, followed by D-glucose, fructose-1,6-diphosphate and raffinose. Similarly, for the cultivated A. bisporus (A-63-137) parent and pleo- morphic (A-63-l38) strains, fructose-l,6-diphosphate, pecti- nol A, pectin, xylan, and D-cellobiose were excellent carbon sources. The utilization of D-cellobiose by the cultivated strain A—63-l37 as well as the pleomorphic form A-63—138 192 indicates that both produced the required enzymes to separ- ate the 8 glucosidic linkage in cellobiose. The rhizomorphic strain of A. bisporus, A-63-139, differed from the other two strains in that it could not utilize xylan, and also in that growth was much less with D—cellobiose and pectinol A. The utilization of pectin as a carbon source was previously reported by Treschow (273). He also reported that glucose, xylose, L-arabinose, and oxalate were good carbon sources. The rhizomorphic strain was similar to the parent and dif— fered from the pleomorphic form by not utilizing well the monosaccharides D—glucose and D-mannose. The pleomorphic mutant, A-63-l38, was similar to the parent A. bisporus (A—63-l37) in the degree of utilization of organic acids, the rhizomorphic mutant, A-63-139, was more readily able to utilize oxalic and less capable of utilizing tannic acid. Results obtained with the A. bisporus strain and and mutants agreed with those reported by Cochrane (“1) in that glucose, fructose, galactose, xylose, L-arabinose, some mannitol, maltose, and sucrose were good carbon sources. Differences in carbon source usefulness reported by Garibova (81) indicated that race a utilized glucose, starch and mal— tose best, race A used glucose and starch, while race 3 used starch, glucose and xylose. Styler (266, 267) found with his A. bisporus strains that xylose, dextrose and maltose were the good carbon sources. Bohus (26) found that only those polysaccharides which give the proper monosaccharides 193 by hydrolysis were suitable carbon sources for the culti- vated mushroom. He obtained best growth with his A. Elg- pgrgg strains by using D-xylose, D-glucose, D-fructose, and hemicellulose, while organic acids and other carbon sources were poor. All sugars, starch, hemicellulose, cellulose and lignin were utilized by the A. bisporus strain selected by Lambert (133). As can be seen from the reports in the literature on carbon utilization by strains of A. bisporus, there is some variation in the results probably due to variations in strains and environmental conditions. This was evident in the variations in carbon utilization of the three strains studied. Waksman (281) and Klioushnikova (12“) found that A. campestris was able to utilize hemicellulose, but found differences in the usability of lignin. In an early work by Duggar (51), all carbon sources were poorly used due to the high osmotic concentrations reached in his experiments. Humfeld and Sugihara (107, 109) selected several carbon sources and obtained similar results with their A. campestris strain as did others. However, in these experiments, hexoses pentoses, disacchardes, soluble starch and dextrin were utilized by A. campestris. In the wild strain of A. cam— pestris (A—63-l36) similar results were found except that monosaccharides were the best carbon source. Lepista nuda (L-63-l33) (=Tricholoma personatum) had exceptionally good growth in media containing D—mannose, D—glucose, D—fructose, or galacturonic acid. Several 19“ Tricholoma species selected by Melin (175) and Norkrans (201) also grew best with the same three sugars. Tricholoma fumosum (175) was reported as a strong cellulose decomposer while T. nudum (227) was not able to use cellulose. Other carbon sources such as starch, raffinose, and inulin varied greatly in their usefulness for different Tricholoma species. Tricholoma flavobrunneum (201) failed to utilize cellobiose but other species of the same genus grew well with it as the sole carbon source. In order of decreasing yield of dry mycelial weights for T.ggdgm, Reusser et al. (227) selected the following carbon sources: pyruvate, xylan, sucrose, maltose, D-mannose, raffinose, D—arabitol, L-rham- nose, D—galactose, D-fructose, D—xylose, dextrin, glucose, D—ribose, cellobiose, sorbitol, L—arabinose, starch, dul- citol, mannitol, inulin, salicin, lactose, L-sorbose, and cellulose. Pleurotus ostreatus (P-63—51) developed approxi— mately three times greater mycelial growth with fructose- l,6-diphosphate than with any other carbon source. D—fruc- tose, D-sorbose, D—cellobiose and D-maltose were of some use to 3. ostreatus while all other mono—, di-, and tri- saccharides were of little importance. In comparison, Block, Tsao and Han (2“) found corn steep liquor best, molasses moderate, glucose fair, and corn starch poor for mycelial growth of E. ostreatus. It is interesting that D—sorbose was utilized by E. ostreatus (P-63-51) as it is 195 not utilized by most fungi and is definitely toxic to some (Cochrane, “1). The mono-, di-, and trisaccharides were of minor importance to both Volvariella volvacea (V-63-l35) and the mutant strain V-63-l3“. Fructose-1,6-diphosphate was readily utilized by both strains. Glycogen and pectinol A were excellent carbon sources for the parent strain, while the mutant strain assimilated these poorly. A change in the ability to assimilate glycogen by the mutant strain in— dicates that this organism can not utilize starch readily. Fungi unable to use glycogen would not be able to utilize starch as insofar as is known the same enzymes attack both polysaccharides (Cochrane, “1). The parent produced better hyphal growth in media containing galacturonic, oxalic or succinic acids while the mutant strain could not utilize these particular acids. Succinic acid was not utilized by 1. volvacea mutant (V-63-l3“). The twelve test organisms I mentioned under "Materials and Methods" were grown in eight different car- bon sources at various concentrations to determine if the carbon source in the basal medium had been a limiting factor. The Cantharellus species grew increasingly better with greater amounts of available carbon except for g. cibarius (C-63—15) in the presence of D-fructose. Agaricus bisporus pleomorph (A-63—l38) was the only Agaricus strain able to grow best with all selected sugars at the highest 196 concentrations indicating the carbon source may still be a limiting factor in this case. The other three strains all were varied in utilization of the different sugars. Pleuro- tus ostreatus (P-63-51) grew at higher carbon levels slightly better than Lepista nuda (L—63-l33). Concerning carbon utilization, Perlman (213) listed three spheres of influence affecting the utilization of energy sources by fungi. They include the physical avail- ability of carbon source to fungal cells; cultural conditions such as the presence of other nutrients in the media, aer- ation, incubation temperature, concentration of nutrients; and adaptation of the strain to the substrate. Fungal cells utilize disaccharides in one of two ways (“1, 2“6): by sac- charases Splitting the compound molecule on the outer sur- face of the cell to monosaccharides which are then transported into the cell by carrier systems for monosaccharides, or by direct movement of disaccharides into the cell by specific membrane transport systems. Lilly and Barnett (1“2) found the availability of carbohydrates to depend upon the produc— tion of the necessary extracellular hydrolytic enzymes. Intermediate metabolic products should also serve as a source of carbon for the fungus. The composition, structure and configuration of organic compounds affect utilization, but the effeCts of these factors are different for different fungi (1“2). The metabolic pathways of carbohydrates and their components differ, depending upon the environmental 197 conditions and the fungus involved. The growth promoting effect of organic acids, added to the buffer capacity of media, act as chelating agents, and compensate for a defi- ciency in the cyclOphorase system (78). Variation in car— bon utilization by the twelve test organisms may be explained by some of these conditions just mentioned. Growth curve studies of the twelve fungal strains and mutants at various concentrations of glucose produced interesting results. Cantharellus clavatus (C-63-8) at 25 g per liter had little mycelial growth indicating carbon was a limiting factor. Maximum growth occurred at 30 days with 75 g per liter, then autolysis followed. At 75 g per liter growth was the greatest for g. cibarius (C—63-15), the peak occurredchuflxm;the first 10 days of growth, then autolysis occurred. In contrast, Cantharellus cibarius (C-63-86) de- veloped the typical lag, log, peak and decline sigmoid phases at 25 g glucose per liter, but had little or no noticable lag phase at 50 or 75 g. No typical growth curves formed with the wild strain of Agaricus campestris (A-63-l36). At 25 g glucose per liter, an extended peak phase occurred before autolysis. A rapid growth increase develOped during the first few days in 50 or 75 g glucose per liter, both concentrations had a slight increase in hyphal growth following a sharp decrease, probably due to autolysis. In contrast, extended sigmoid curves developed for A. bisporus (A-63—l37) in 25 and 75 g 198 glucose per liter. At 25 g per liter, the glucose concen- tration was not sufficient for growth of the rhizomorphic mutant, but greatly increased hyphal growth for the pleo- morphic form. A similar growth delay occurred for A. gig- pgrgg rhizomorph (A-63-139) at 50 g glucose per liter as was formed with the parent strain, but the extended lag phase was absent with the pleomorph mutant at this glucose concentration. A sigmoid curve was produced at 75 g glucose per liter for Lepista nuda (L-63-l33), but only the growth rate increase phase occurred between 10 and “0 days at 25 or 50 g glucose per liter. Pleurotus ostreatus (P—63-51) did not have enough carbonaceous material present at 25 g glucose per liter for sufficient growth, but at 75 8 glucose per liter produced a sigmoid curve. For Volvariella volvacea at 75 g, the parent strain had autolysis occur at 20 days while the mutant began a steady growth increase at 20 days after a lag phase. Several nitrogen sources were selected from am- monium and nitrate compounds, and amino acids to determine which is best suited for each of the twelve fungal strains and mutants. Peptone was by far the best source of nitrogen for hyphal growth of Cantharellus clavatus (C-63-8). Nitrates were not utilized and ammonium only in the form of ammonium bicarbonate was usable. Amino acids were good nitrogen sources, especially L-proline. 199 The three 9. cibarius strains varied in their nitrogen utilization requirements as they did for carbon sources. Urea was better utilized by g. cibarius than 9. clavatus. Inorganic nitrogen sources produced growth better than urea for the three 9. cibarius strains. Pep- tone was a good nitrogen source and a number of the amino acids equaled or produced greater mycelial growth than urea. As with carbon sources, mycelial growth with various nitrogen sources for C-63—96 was several times greater com- pared to growth of the other 9. cibarius strains. 0n the basis of experimental condition, Agaricus campestris (A-63-136) readily utilized urea as a nitrogen source as well as ammonium citrate, ammonium tartrate, pep- tone and a number of amino acids. Less mycelial production occurred when arginine was used although Iwanoff and Tos- chewikowa (111) reported a marked mycelial growth increase occurred. Similarly, Humfeld and Sugihara (107, 109) found ammonium nitrogens, urea, peptone, amino acids and mono- sodium glutamate good nitrogen sources for A. campestris. In an early work by Duggar (51), A. campestris grew fairly well with asparagine, urea, peptone, casein and albumen. Koch (125) found that Psalliota (=Agaricus) campestris could utilize urea, arginine, asparagine, aspartic acid, ammonium tartrate and alanine which were some of the best nitrogen sources in these studies, however arginine was not as readily utilized by the strains studied. 200 Agaricus bisporus and the two mutants produced greatest mycelial dry weights when grown in peptone as the nitrogen source. Urea was a better nitrogen source for the parent strain than for either the pleomorphic or rhizomor- phic mutant, and had greater growth than with any inorganic nitrogen source. The pleomorph form (A-63—138) excelled in hyphal growth with ammonium compounds, while ammonium tar- trate was the only ammonium nitrogen source to equal urea for mycelial production with the rhizomorphic strain. Some of the amino acids were better than urea in mycelial pro— duction for the two mutant strains, A-63—138 (pleomorph), and A-63-l39 (rhizomorph). Similar results reported by Styler (266, 26?) indicated A. bisporus utilized ammonium salts, glycine, asparagine, peptone, proteins, and urea as good nitrogen sources. According to Garibova (82), A. bisporus races can be separated according to preference for individual amino acid nitrogen sources: race A utilizing glycine, a alanine for race A, and asparagine for race 9. Jennison and Perritt (11?) found Psalliota (=Agaricus) campestris utilized the L-isomer (natural) of amino acids better or exclusively, while the D-isomer (unnatural) may actually be inhibitory. However, both isomers of proline, alanine and leucine can be utilized. In these studies, D-histidine was not usable as a nitrogen source by A. campestris and the three strains of A. bisporus. The A. bisporus strain studied by Fraser 201 (63) developed greater growth responses when amino acids were used in different combinations, but growth was pro- moted to some degree when phenylalanine, methionine, pro- line, tryptophane, and tyrosine were used singly. Fungi utilizing nitrate nitrogen must be able to reduce the nitrogen to the oxidation level of ammonia (1“2). Ammonium salts or nitrates are suitable sources of nitrogen although with nitrates there may be a lag in growth before the necessary enzymes concerned are produced (192). Bohus (26) found that the source of nitrogen for A. bisporus is important for carbon utilization and the ammonium nitrogen utilization is modified by pH. The cation (NH“+) is the same in every ammonium compound; utilization depends on the anion. Media can become exceedingly acid if ammonium ions are greater than anion uptake. The rate of amino acid utilization is a specific character of strains. Treschow (273) reported Psalliota (=Agaricus) bispora f. avelanea was unable to use nitrate nitrogen, grew moderately in ammonium nitrogen, and grew best in amino acids. This is similar to the growth responses for the A. bisporus strains. Both urea and peptone were good as nitrogen sources for Lepista nuda (L-63-133) (=Tricholoma personatum). Ammonium bicarbonate and glycine were excellent sources; other useful amino acids include DL-alanine, L-glutamic acid, and DL-methionine. Melin (175) found nitrate nitro- gen was poor for eight Trichomoma species, but inorganic 202 and organic ammonium salts were easily used. Twenty-one amino acids, plus potassium nitrate, ammonium sulfate, am— monium nitrate, ammonium tartrate, ammonium phosphate, am— monium chloride, and urea were useful for T. 22322 (Reusser, et al., 227). Falanghe et al. (57) found ammonium acetate, ammonium tartrate and ammonium sulfate in decreasing order of usefulness for T. Agggm. Falanghe (56) increased growth of A. campestris, T. nudum, but not Cantharellus cibarius with Vinasse, a fermentation product of beet sugar high in ammonium salts. Glutamic and aspartic acid along with their corresponding keto acids accelerated growth of seven Tricholoma species; T. gambosum and T. imbricatum were greatly stimulated by glutamine and asparagine (Norkrans, 202). Ammonium and organic nitrogen are assimilated by all Tricholoma species, nitrate nitrogen by T. nudum only, and various amino acids stimulate growth of different species (Norkrans, 201). Urea was the best nitrogen source for T. fumosum and T. vaccinum. The utilization of nitrogen sources by T. Egggm is in general agreement with the results reported in the literature for T. ggdgm_and other species of Lepista as urea was usually the best nitrogen source, fol— lowed by amino acids, then ammonium salts, and with the ni- trate form a poor source. Peptone and urea were the best nitrogen sources for Pleurotus ostreatus (P—63—51), closely followed by am- monium citrate, and DL-alanine. The other compounds including 203 amino acids were of little value as nitrogen sources. Tsao (27“) and Block et al. (2“) noted nitrate and ammonium salts produced poor growth with T. ostreatus while peptone and casamino acids were quite good for hyphal growth, which is similar to the results for nitrates and peptone for strain P—63-51. Koch (125) selected asparagine, glutamic acid, glycocoll, and alanine as nitrogen sources for a strain of the same species. Leonian and Lilly (138) found the follow- ing amino acids useful for T. ostreatus: D-arginine, L- aspartic acid, D-glutamic acid, glycine, DL-alanine, DL- isoleucine, B alanine, L-histidine, Lehydroxyproline, L-proline, L-tryosine, and DL-Serine, while those not well utilized included L-trypt0phane, DL—valine, and DL—leucine. Hacskaylo et a1. (92) found asparagine best, ammonium nitro- gen good, ammonium sulfate poor and nitrate nitrogen poor for growth of a strain of T. ostreatus. Pleurotus corticatus had better growth with several amino acids but could not utilize ammonium nitrate (138, 139, 262), a pattern that is followed by some of the T. ostreatus strains. Several inorganic nitrogen sources better for hyphal growth than urea for Volvariella volvacea (V-63—135) and the mutant V-63-l3“ included: ammonium bicarbonate, ammonium citrate, ammonium tartrate, calcium nitrate, po- tassium nitrate and sodium nitrate. The mutant strain could utilize cobaltous ammonium sulfate as well as urea while the parent strain could not. Similarly, the parent 20“ strain of T. volvacea could utilize ammonium salts better than the mutant. Peptone produced the greatest hyphal growth for both parent and mutant of any nitrogen source tested. Amino acids equivalent to urea for growth for the V—63-l35 strain include: DL-alanine, L-asparagine, L- proline, and DL—tryptOphane. The utilization of inorganic and organic nitrogen sources by this organism would account for the adaptability to the method of cultivation in the Philippines and other areas. Compounds utilized such as nitrates and amino acids are usually in plant material used in the compost for cultivation of these mushrooms. The shorter carbon chain length amino acids generally were more readily utilized by the strains studied. Jennison, Newcomb and Henderson (116) concluded from their studies with amino acids with A. campestris that the shorter 3-“ carbon chain length--g1ycine, valine, a alanine--averaged more growth than the 5 or 6 carbon acids--1eucine, isoleu- cine, and norleucine. The shorter chain acids containing an OH group--serine and threonine--were considerably poorer; those containing sulfur-—cystine, cysteine, methionine-- were even more poorly assimilated. The heterocyclic trypto- phane, histidine, proline, and hydroxyproline followed no pattern. Jennison et al.(1l6,117) listed the reasons for poor growth to be pH concentration, buffer action, redox potential, osmotic concentration, lack of minerals or vitamins, inadequate kinds and amounts of carbon or nitrogen, unfavorable 205 balance of constitutents, incubation time and synergistic action of organic acids. Changes in amino acid require- ments may be induced by mutation in fungi but are uncommon in nature, when they occur naturally they are more often partial rather than absolute requirements (192). The vari- ations in amino acid requirements between the parent and mutants may be due to mutation for A. bisporus and due to strain variation that occurred in nature for Pleurotus ostreatus. The many nutrilites present in malt or yeast extract (see Appendix) serve as good constituents of basal or stock culture media. No vitamin heterotr0phy was noted for Volvariella volvacea, Lepista nuda, Cantharellus cibarius or Pleurotus ostreatus under cultural conditions selected for the experi- ments. Hyphal length in mm was greater the first two days of growth for T. volvacea with 6 of the 10 vitamins added singly to the agar media, but then all growth thereafter equaled control. The other three fungal strains had no growth effects with vitamins. The vitamins act as co- enzymes in fungal metabolism and may be synthesized or exo- genously supplied by the organism (192). Absolute vitamin deficiencies in fungi are not known to be influenced by the environment, while conditioned deficiencies may be affected either by nutritional or physical environment factors such as temperature, composition and pH of the medium (1“2). Tsao (27“), Kurancowa (129), and Block, et a1. (2“) found 206 thiamine increased hyphal growth of T. ostreatus, and Koch (125) added the vitamin to a medium for fructification of that species. No observed difference was noted with the addition of thiamine for T. ostreatus, however, the period of time was not sufficient to determine if fructification was affected. Lilly and Barnett (1“0) believed the vitamins to be autotrophic for T. ostreatus but it is entirely pos— sible under other environmental conditions that a deficiency would evolve. Thiamine was found necessary for T. cortica- EEE (138). Melin and Norkrans (178) used thiamine and pyrimidine to increase hyphal growth of Tricholoma albo- brunneum. Melin et al. (177, 178) believed thiamine, pyri- midine or pyrimidine-thiazole heterotr0phy existed for several Tricholoma species. Tricholoma imbricatum is par- tially heterotrophic for pantothenic acid, T. fumosum is partially deficient for nicotinic acid, T. fumosum grew equally well in thiazole and pyrimidine or thiamine while “ other Tricholoma Species grew better in thiazole + pyri- midine, pyrimidine, or thiamine (176). Thiamine, biotin, and inositol greatly increased growth of T. nudum (126). Psalliota hortensis f. albida increased vegetative growth in media containing thiamine and biotin (Fraser, 62). Treschow (273) increased hyphal growth of Psalliota bispora f. avellanea also with thiamine and biotin. Fraser (63) found only thiamine increased hyphal growth of A. bisporus. Robbins and Kavanagh (2““) saw no growth effect upon Agaricus 207 campestris with these vitamins. On the contrary, Hoffman and Hunt (103) increased mushroom yields of Agaricus bis- porg§_with nicotinic acid, pantothenic acid, biotin, thia— mine or pyridoxine added singly to the medium or with riboflavin + nicotionic acid + pantothenic acid, or thia- mine + nicotinic acid in combination. There was no growth effect on the wild strain of Agaricus campestris, A. bis— pgrgg or the two mutants with vitamin B1 in the eXperiments, corresponding to the results reported by Robbins and Kavanagh (2““). Growth hormones were of no significance to the 12 selected fungal strains at low concentrations while at high concentrations growth was inhibited apparently due to pH changes in the medium. Fraser (62) noted that Psalliota hortensis f. albida had a marked growth increase with 1AA in the presence of thiamine and biotin. Heteroauxin is a growth inhibiting substance for Pleurotus corticatus (13?), and 2,“-D increased germination and lengthened spore viability of T. ostreatus (129). In selecting various seeds and other material as spawn media for growth of T. volvacea, A. nuda, Q. cibarius, and T. ostreatus, different temperatures were selected to determine the best suited range for hyphal growth on rye grain. Optimal temperature for hyphal growth of the organ- isms were as follows: T. volvacea, 30-36°C; A. nuda, 18-30°C; Q. cibarius, 18—30°C; g. ostreatus, 18-30°C. Except for 208 Volvariella at 36° C, growth was inhibited. Many plant seeds furnish essential nutrients for good spawn production of the four genera. When 9. cibarius was grown on rye or spelt spawn media, immature fructifications occurred after three months. Immature basidiocarps formed for T. ostreatus within two to Six weeks on several spawn media. Changes in the environment might trigger the maturation of the basidio- carps. Mushroom house environmental conditions suited for production of Agaricus bisporus were unfavorable for basi- diocarp development of the selected strains of Volvariella, Pleurotus, Cantharellus or Lepista. Only hyphal growth occurred. Additional variations in the conditions in the mushroom house, such as changes in temperature, modifications in compost, type of casing soil, light, and moisture are just some of the important factors that need further investigation with respect to the fructification problem. SUMMARY The ability of Cantharellus clavatus Fries, three strains of Q. cibarius Fries, Lepista nuda (Bull. ex. Fries) Cooke, Pleurotus ostreatus (Jacq. ex. Fries) Kummer, Agari- cus campestris L. ex. Fries, A. bisporus (Lange) Singer and pleomorphic and rhizomorphic mutants of A. bisporus, Volva— viella volvacea (Bull. ex. Fries) Singer and one mutant of V. volvacea to utilize various nutrilites was studied. Agaricus bisporus and Volvariella volvacea have been grown on a commercial basis. The others are considered as de- sirable species for cultivation by the mushroom industry. A pH of “.5 was selected for the growth studies in submerged culture since several of the 12 species and mutants produced optimum growth at that pH level, at a temperature range of l8-30° C. Volvariella was able to grow up to 36° C. For the carbon and nitrogen studies, fructose-1,6- diphosphate and peptone, respectively, were generally the best sources for mycelial growth of Cantharellus clavatus and three strains of g. cibarius. g. clavatus (C-63-8) pro- duced Optimum mycelial growth with pectinol A as the carbon source and L-proline for the nitrogen source. The sugar alcohols and amino acids were utilized best for g. clavatus. g. cibarius (C-63-15) grew well with pectinol A and L-proline. 209 210 Cantharellus cibarius (C-63-86) grew best with pectinol A as the carbon source and with urea as the nitrogen source. The third strain, C-63-96, was the only one to produce a water soluble yellow-green pigment in submerged culture. It also had much greater mycelial growth than other 9AA- tharellus strains, and grew best with fructose—1,6-diphos- phate and peptone. Agaricus campestris (A-63-l36) grew best with D-fructose and L—glutamic acid, while sugar alcohols and amino acids were good for mycelial growth. A. bisporus (A-63-137) best utilized xylan as the carbon source with peptone, urea, and L-proline as the best nitrogen sources. The polysaccharides and inorganic nitrogen sources were slightly better for mycelial production of strain A-63-l37. The pleomorphic mutant, A-63-l38, grew best with D-cello- biose and creatine. The rhizomorphic strain, A-63-l39, utilized all carbon and nitrogen sources about equally well. Lepista nuda (L-63-133) produced greatest mycelial growth when D-mannose was used as the carbon source and gly— cine was the nitrogen source. A number of amino acids were good nitrogen sources. Pleurotus ostreatus (P-63—51) grew best with fruc- tose-1,6-diphosphate and urea as the carbon and nitrogen sources. The polysaccharides were good carbon sources while a number of nitrogen sources were readily utilized. 211 Volvariella volvacea (V—63—l35) produced maximum growth with fructose-1,6-diphosphate as the carbon source, and peptone as the nitrogen source. Other good carbohydrate sources were polysaccharides while amino acids and inorganic nitrogen compounds were equally good nitrogen sources. The mutant, V-63-13“, of T. volvacea (V-63-l35) grew best with the same carbon and nitrogen groups as did its parent, but less vigorously. Growth curves were made for each organism grown over a period of “0 days in three different concentrations of glucose. In most strains used, 75 g of the carbohydrate source per liter was sufficient for maximum growth of the organism. P-aminobenzoic acid, ascorbic acid, biotin, cal- cium pantothenate, niacin, and niacinamide produced an ac— celerated hyphal growth rate for T. volvacea for the first “ days after inoculation; then growth leveled off to the control rate. The other organisms were not affected by the vitamins. The growth hormones selected were of no Signifi- cance to the test organisms. Several agar media were selected for growth of the 12 fungi, and morphology of these colonies was described. Several spawn media were selected and utilized by T. volvacea (V—63—l35), A. 229% (L-63-133), g. cibarius (C-63—86), and T. ostreatus (P-63—51). Immature fructifi- cations occurred with g. cibarius (C-63-86) when grown on 212 rye or spelt spawn. No tissue differentiation develOped within the fruiting-body. T. ostreatus (P-63-51) produced basidiocarps on several spawn media. The basidiocarps dif- ferentiated into modified stipes, and pilei with lamellae and spores. Light and electron microsc0py identified doli- pore septa in mycelium of Pleurotus, a simple pore in Cantharellus, crystals in both genera, and other cellular organelles in Volvariella and Lepista. The same four fungal strains selected for spawn studies were subjected to growth conditions suitable for A. bisporus mushroom production on horse manure compost. No fructifications occurred.‘ The mycelial mat of Lepista 229% which developed over the compost had a light blue coloration similar to normal basidiocarps. Pleurotus and Volvariella develOped some hyphal growth but Cantharellus did not grow. l....|1 iiiiillll APPENDIX The following stock culture media are those men- tioned in the text but not identified as to contents. The accompanying number in parentheses refers to the bibliography. Medium A (263): glucose 15.00 g sucrose 15.00 g Bacto peptone 5.00 g Bacto yeast extract 5.00 g magnesium sulfate 0.50 g potassium phosphate, di—H 1.00 g potassium chloride 0.50 g ferrous sulfate 0.01 g agar 15.00 g distilled water 1,000.00 ml 2% Malt agar (“): malt extract 25.00 g agar 15.00 g distilled water 1,000.00 ml 213 21“ The twelve strains were grown on the following agar media for culture identification. Blood agar base (“9): infusion from heart muscle tryptose sodium chloride agar distilled water Brewer anaerobic agar (“9): Bacto yeast extract Bacto tryptone proteose peptone no. 3 sodium chloride sodium thioglycollate sodium formaldehyde sulfoxylate resazurin agar Chlamydospore agar (“9): Corn meal ammonium sulfate monopotassium phosphate biotin trypan blue purified polysaccharide agar distilled water agar (“9): corn meal infusion agar distilled water 375. 10. 09090909 15. 1,000. U‘l 00000 H OOOOOO 090909090909 .002 .0 g OOI—‘NU‘IOU'IU'I (\D OOU'IF-‘H OOOHOOO 09 1,000. 50.0 g 15.0 g 1,000.0 g 215 Liver spleen glucose agar (“9): beef liver infusion beef spleen infusion proteose peptone dextrose agar distilled water Micro assay culture agar (“9): Difco proteose peptone no. 3 Bacto yeast extract Bacto dextrose monopotassium phosphate sorbitan monooleate complex agar distilled water Mycological agar (“9): Bacto soytone Bacto dextrose agar distilled water Mycosel agar (“9): phytone Bacto dextrose actidione chloromycetin agar distilled water 250.0 250.0 10.0 10.0 20.0 1,000.0 5.0 20.0 10.0 2.0 0.1 10.0 1,000.0 10.0 10.0 15.0 1,000.0 10.0 10.0 0.“ 0909090909 09 ml g g g 0.05 g 15.5 8 1,000.0 ml 216 Nutrient agar (“9): Bacto beef extract Bacto peptone agar distilled water 0. Modess agar (18“): potassium phosphate magnesium sulfate ammonium chloride ferric chloride (1% sol) glucose malt extract agar distilled water Potato dextrose agar (“9): infusion from potatoes Bacto dextrose agar distilled water Prune agar (“9): infusion from prunes agar distilled water Rice extract agar (“9): extract from white rice agar distilled water 3.0 5.0 15.0 0.5 0.5 0.5 g g g 1,000.0 ml 8 g 8 10 dr0ps 5.0 5.0 20.0 1,000.0 200.0 20.0 15.0 1,000.0 36.0 15.0 1,000.0 20.0 20.0 1,000.0 8 g 8 ml ml ml 217 Russian medium (121): ammonium nitrate g potassium phosphate g magnesium sulfate g calcium chloride g sodium chloride g casein hydrolysate with 0.025 g tryptophan 5.0 m1 ferrous chloride 0.6 mg cupric sulfate 0.23 mg sodium borate 0.0“5 mg sodium molybdate 0.0“3 mg manganese chloride 0.0“6 mg zinc sulfide “.3 mg nicotinic acid — 1.0 mg para-amino-benzoic acid 1.0 mg riboflavin 1.0 mg ca-pantothenate 1.0 mg biotin 1.0 mg thiamine 1.0 mg glucose 15.0 g agar 20.0 g distilled water 1,000.0 ml Russian medium minus casein and tryptOphan. Russina medium minus vitamins. Thioglycollate medium (“9): Bacto yeast extract 5.0 g Bacto casitone 15.0 g Bacto dextrose 5.0 g L-cystine 0.25 g 218 sodium chloride 2.5 g thioglycollic acid 0.3 mg agar 0.75 g distilled water 1,000.0 m1 Tomato juice agar special (“9): tomato juice (“00 ml) 20.0 g Bacto peptone 10.0 g Bacto peptonized milk 10.0 g agar 20.0 g distilled water 1,000.0 ml Tryptic soy agar (“9): Bacto tryptose 17.0 g Bacto soytone 3.0 g Bacto dextrose 2.5 g sodium chloride 5.0 g potassium phosphate 2.5 g agar 15.0 ml distilled water 1,000.0 m1 Violet-red bile agar (“9): Bacto peptone 10.0 g Bacto lactose 10.0 g Bacto bile salts 1.0 g Bacto yeast extract 5.0 g agar 15.0 g neutral red 0.05 g crystal violet 0.00“ g distilled water 1,000.0 ml 219 Components of Difco malt extract: A dehydrated barley infusion containing diastase, dextrin, dextrose, protein and salts from barley. Analysis of Bacto yeast extract, furnished by Difco Laboratories, Detroit, Michigan: Percent ash 10.10 total N 9.18 chloride 0.19 total sulphur 1.39 PPM lead 16.00 arsenic 0.11 manganese 7.80 zinc 88.00 COpper 19.00 Percent phosphorus 0.89 iron _ 0.028 silicon dioxide . 0.052 potassium 0.0“2 sodium 0.32 magnesium 0.03 calcium 0.0“06 arginine ~ 0.78 aspartic acid 5.1 glutamic acid 6.5 220 Percent glycine 2.“ histidine 0.92 isoleucine 2.9 leucine 3.6 lysine “.0 methionine 0.79 phenylalanine 2.2 threonine 3.“ tryptOphane 0.88 tryosine 0.60 valine 3.“ Micrograms per gram pyridoxine 20.0 biotin ' 1.“ thiamine 3.2 nicotinic acid 279.0 riboflavine 19.0 folic acid 0.3 Analysis of Bacto peptone, furnished by Difco Laboratories, Detroit, Michigan: total nitrogen 16.16% primary proteose N 0.06% secondary proteose N 0.68% peptone N 15.38% ammonia N 0.0“% free amino N 3.20% amide N 0.“9% mono-amino N 9.“2% di-amino N “.07% tryptophane 0.29% tyrosine 0.98% 221 cystine organic sulphur inorganic sulphur phosphorus chlorine sodium potassium calcium magnesium manganese iron ash lead arsenic zinc c0pper silicon dioxide arginine aspartic acid glutamic acid glycine histidine isoleucine leucine lysine methionine phenylalanine threonine valine pyridoxine biotin thiamine nicotinic acid riboflavine 0.22% 0.33% 0.29% 0.22% 0.27% 1.08% 0.22% 0.05% 0.056% nil 0.0033% 3.53% 15.00 ppm 0.09 ppm 18.00 ppm 17.00 ppm 0.0“2% 8.00% 5.90% 11.00% 23.00% 0.96% 2.00% 3.50% “.30% 0.83% 2.30% 1.60% 3.20% 2.50 0.32 0.50 35.00 “.00 per per per per 1:121:11: per 0909090909 10. 11. 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