Physiology of Fungi Physiology of Fungi C a VINCENT W. COCHRANE Professor of Biology Wesleyan University Physiology of Fungi New York • John Wiley & Sons, Inc. london • chapman & hall, limited COPYRIGHT © 1958 BY JOHN WILEY & SONS, INC. All rights reserved. This book or any part thereof must not be reproduced in any form with- out the written permission of the publisher. LIBRARY OF CONGRESS CATALOG CARD NUMBER: 58-13456 PRINTED IN THE UNITED STATES OF AMERICA Preface A lthough study of the physiology of the fungi has not kept pace with that of, for example, bacterial metabolism, it has in recent decades had much the same development. It seems fair to say that there have been two disparate types of study. Research on some problems — reproduction, parasitism, development — has been, with sig- nificant but not very numerous exceptions, descriptive. At the other extreme, it has been possible to develop basic biochemical knowledge on the implicit assumption that the microbial cell is a small bag of enzymes which only require to be extracted and enumerated. Our need now is to extend the valuable results of the abstract analytical study to the type of problem that has remained in the descriptive, natural history, stage. This, however, is for the future. We work with what we have, and it is the purpose of this book to summarize and organize our knowledge of the physiology of the fungi, indicating whenever possible some of the problems that lie immediately ahead. If there is one guiding prin- ciple, it is that the fungi, however unusual or unique they may be as functioning intact organisms, can be studied at the cellular and molecu- lar levels only by taking account of the findings of other disciplines, particularly bacterial physiology. This is the point of view of com- parative biochemistry. No doubt, in a decade or two, some more useful principle of organization will emerge, but for the present an Vi PREFACE ordering of knowledge around this central principle seems valid. To stress the fundamental biochemical similarities of all organisms is to assert in a concrete instance the general proposition of the unity of all living things, the unity of the science of biology. The separa- tion of the various fields of biology has caused incalculable damage to all, possibly most to the botanical sciences. This is not to deny the need for specialization within biology, but when this specialization reaches the point where a student can, for example, be considered well trained in botany without having had any contact with animal sciences or with bacteriology, it seems time to reconsider. Some way must be found, both at the teaching and at the research level, to ensure that biologists of all sorts will be conversant with each other's problems and will be able to draw on each other's experience in the prosecution of their own specialties. The literature on the physiology of fungi is very large and is grow- ing rapidly. Much of it is of necessity repetitious and limited in scope to particular organisms or to practical applications. Some of it fails to meet criteria of accuracy or logic. I have attempted to exercise the author's prerogative of selection so as to exclude work which, in my judgment, falls into either category. However, I have preferred to err on the side of citing too much literature rather than too little. All-inclusiveness is, of course, out of the question; the references are designed to give immediately most of the work of the last three decades and to open up the entire literature for the student or re- search worker. In this connection, it is to be regretted that the "old- fashioned" review of the literature has been largely squeezed out of modern journal articles. No doubt it was often overdone and wasted valuable space, but the current practice of publishing the past month's work in the form of a letter to the editor has even less to recom- mend it. Professor Frank Dickens once spoke in a seminar to the effect that for the purposes of biochemistry the yeasts are considered to be ani- mals. With something of the sort in mind, I have omitted much of the physiological work on yeasts, feeling that it has been well treated elsewhere and that its importance to the filamentous fungi is of a back- ground nature only. On the other hand, I have included the aerobic actinomycetes on the ground that ecologically and physiologically they have close affinities to the true fungi. Both groups are filamen- tous, both require oxygen for growth, both form exogenous spores, and both occur in similar habitats. The scope of the book is then the filamentous fungi and actinomycetes and that work on the yeasts which has a direct bearing on the physiology of the true fungi. PREFACE VH In the naming of organisms I have followed the usage of the orig- inal author unless confusion is thereby introduced. A few excep- tions to this rule have been made, e.g., in the use of Streptomyces for the aerobic actinomycetes forming conidia in chains. Two important and interesting topics have been almost entirely omitted: the genetics of fungi and the interactions of fungi with other organisms in nature. Both of these subjects deserve more space than could here be given to them, and the second — the ecology of fungi — probably requires a type of conceptual approach different from that of this book. Individual chapters have been read in manuscript by S. H. Hutner, Frederick Kavanagh, Albert Kelner, Leonard Machlis, S. E. A. McCallan, W. C. Snyder, and C. E. Yarwood. It is a pleasure to acknowledge their advice and help. I am grateful, too, to the many authors and publishers who have allowed me to use published mate- rial, and to the staffs of the Olin Library of Wesleyan University, the Mann Library of Cornell University, and the New York Agricultural Experiment Station Library. A considerable part of the work embodied here was done during a sabbatical leave spent as guest of the Department of Plant Pathology of the University of California at Berkeley, to the members of which I am greatly indebted. The Board of Trustees of Wesleyan University generously provided support for this period. Jean Conn Cochrane has given invaluable assistance in all phases of preparation of the manuscript, which without her help would have been poorer in quality and later in appearance. Vincent W. Cochrane October 1958 Contents Page Chapter 1 Cultivation and Growth 1 1. Methods of Cultivation 4 2. Preservation 4 3. The Dynamics of Growth 5 4. Dimorphism 10 5. The Measurement of Growth 11 6. Temperature and Growth 13 7. pH and Growth 19 8. Oxygen and Growth 23 9. Carbon Dioxide and Growth 24 10. Water and Growth 24 11. Bioluminescence 26 Bibliography 27 2 The Composition of Fungus Cells 35 1. The Major Constituents of the Cell 36 2. Mineral Constituents of the Cell 37 3. The Cell Wall 39 4. Cellular Carbohydrates 42 5. Nitrogenous Constituents 43 6. Cell Lipids 45 Bibliography 49 IX 75412 X CONTENTS Page Chapter 5 Carbon Nutrition 55 1. Problems of Method 55 2. Monosaccharides and Their Derivatives 60 3. Oligosaccharides 66 4. Polysaccharides 75 5. Organic Acids 80 6. The Lipids 82 7. Other Carbon Sources 83 8. The Concentration Factor in Carbon Nutrition 85 9. Enzyme Induction: Adaptive Growth and the Utilization of Mixed Carbon Sources 86 10. Efficiency of Growth 89 11. Conclusion 90 Bibliography 92 4 Carbon Metabolism 99 PART I INTRODUCTION Bibliography 103 5 Carbon Metabolism 104 PART II THE METABOLISM OF CARBOHYDRATES AND RELATED COMPOUNDS 1. The Metabolism of Monosaccharides 104 2. The Metabolism of Oligosaccharides 105 3. The Metabolism of Other Glycosides 109 4. The Metabolism of Polysaccharides 110 5. The Metabolism of Amino Sugars 121 6. The Metabolism of Polyhydric Alcohols 122 Bibliography 124 6 Carbon Metabolism 131 PART III ORGANIC ACIDS, LIPIDS, AND OTHER COMPOUNDS 1. The Metabolism of Aliphatic Organic Acids 131 2. The Metabolism of Lipids 148 3. The Metabolism of Aromatic Compounds 164 4. Derivatives of Pyrone 178 5. Derivatives of Furan 180 6. Biological Methylation 181 7. Biological Chlorination 183 8. Penicillin 184 Bibliography 187 CONTENTS XI Page Chapter 7 Respiration 202 1. Problems of Method 203 2. Factors in Respiration 206 3. Endogenous Respiration 210 4. Oxidative Assimilation 212 5. The Embden-Meyerhof Pathway 212 6. The Phosphogluconate Oxidation Pathway 217 7. The Non-phosphorylative Oxidation of Glucose 220 8. Pentose Fermentation and Oxidation 221 9. The Citric Acid Cycle 222 10. Dicarboxylic Acids in Respiration 226 11. Carbon Dioxide Fixation 228 12. Terminal Respiration 229 Bibliography 232 S Nitrogen Nutrition and Metabolism 241 1. Inorganic Sources of Nitrogen 242 2. Proteins and Peptides 250 3. Utilization of Amino Acids and Amides 256 4. Biosynthesis of Amino Acids 265 5. Amines 273 6. Metabolism of Nucleic Acids and Their Derivatives 274 7. Urea 277 8. Ergot Alkaloids 279 9. Miscellaneous Nitrogen Compounds 280 Bibliography 283 9 Inorganic Nutrition and Metabolism 300 1. Phosphorus 301 2. Potassium 303 3. Sulfur 304 4. Magnesium 306 5. Iron 307 6. Zinc 308 7. Copper 309 8. Manganese 310 9. Molybdenum 310 10. Calcium 311 11. Cobalt 312 12. Other Inorganic Nutrients 313 13. Bioassay of Minerals 313 14. Problems in Mineral Nutrition 314 Bibliography 317 x[[ CONTENTS Page Chapter 10 Vitamin Requirements of Fungi 323 1. Problems of Method 324 2. The Nature of Vitamin Requirements 325 3. The Metabolism of Vitamins 328 4. Fungi in the Bioassay of Vitamins 328 5. Thiamine 329 6. Biotin 333 7. Pyridoxine 335 8. Riboflavin 336 9. Nicotinic Acid 337 10. Inositol 339 11. Para-aminobenzoic Acid 340 12. Pantothenic Acid 341 13. Pteroylglutamic Acid 342 14. The Vitamins B^ 343 15. Vitamins K 344 16. Other Growth Factors 344 17. The Occurrence of Vitamin Deficiencies in the Fungi 345 18. Vitamins, Taxonomy, and Ecology 346 Bibliography 347 // Reproduction 356 1. Light 357 2. Temperature 362 3. Water Relations 364 4. Oxygen, Carbon Dioxide, and Volatile Substances 365 5. Acidity 366 6. Carbon Nutrition 367 7. Nitrogen Nutrition 369 8. Mineral Nutrition 370 9. Vitamins and Unidentified Factors 371 10. Specific Reproductive Hormones 373 11. Metabolism and Reproduction 375 12. The Sporulation of Plant Pathogens 377 13. Conclusions 378 Bibliography 380 12 Spore Germination 387 1. Measurement 388 2. The Germination Process 389 3. Spore Metabolism 392 4. Maturation and Dormancy 395 5. Spore Longevity 398 CONTENTS Xlll Page 6. Nutrition and Germination 400 7. pH and Germination 403 8. Water and Germination 404 9. Temperature and Germination 407 10. Oxygen and Germination 412 11. Carbon Dioxide and Germination 413 12. Biological Effects on Spore Germination 413 Bibliography 415 Chapter 13 The Action of Physical Agents 423 1. High Temperature 423 2. Low Temperature 425 3. Visible Light 427 4. Ultraviolet Radiation 429 5. Ionizing Radiations 433 6. Electromagnetic Radiation of Long Wavelength 435 Bibliography 435 14 The Action of Chemical Agents 440 1. The Response to Toxicants and Its Measurement 441 2. Tolerance and Resistance 448 3. Selective Accumulation 450 4. Surface Action 453 5. Hydrogen-Ion Concentration 454 6. Structure and Activity 457 7. The Action of Heavy Metals 465 8. The Action of Sulfur 472 9. Quinones and Other Unsaturated Compounds 475 10. Chelation and Fungistatic Activity 478 Bibliography 485 Organism Index 499 Subject Index 508 v mass. SS^j 1 . Cultivation and Growth Xhe precise definition of growth in fungi depends on the method of measurement used. Most methods determine, directly or indirectly, the increase in mass of an inoculum after a known in- cubation in a complete medium. Increase in mass or in number of cells may therefore be used as an operational definition of growth. In considering cultivation and growth we shall first concern ourselves with methods of cultivation and preservation of fungi, the dynamics of growth, dimorphism, and the measurement of growth. Then we turn to external factors — temperature, hydrogen-ion concentration, oxygen, carbon dioxide, and water — as they affect growth. The same external conditions also influence spore germination (Chapter 12); radiant energy effects are taken up in Chapter 13. 1. METHODS OF CULTIVATION Cultures of fungi are usually maintained on conventional agar slants, and the agar petri dish has been employed in experimental work. Most quantitative studies, however, require a liquid medium which may be either surface (still) or submerged (aerated) culture. The general technique of surface culture is too well known to require description here. It is not always realized that normal aerobic growth can be obtained only if the inoculum floats on the surface of the liquid. Many spores float naturally; for those that do not, use of a bran-cultivated inoculum (206) or inclusion of a small amount of agar, 1 Z CULTIVATION AND GROWTH gum arable, or polyvinyl alcohol in the medium (334) may be neces- sary. Two major objections attach to the use of surface culture for fungi. The cells of the developed mat are not only visibly of different types — aerial and submerged growth — but also are exposed to different en- vironmental conditions. Aerial hyphae are remote from the nutrient supply and are bathed in the atmosphere; the lower cells, by contrast, are in close contact with nutrients and with soluble metabolic products but suffer from a partially anaerobic environment (167). Examination of submerged hyphae in such a culture shows them to be highly vacuolated. The culture as a whole is probably always deficient in oxygen. This deficiency is shown by the favorable effect on sugar utilization of an increase in surface : volume ratio (208) and by the effect, mentioned below, of aeration on growth rate of most fungi. The commonest and most useful laboratory-scale aerated culture apparatus is the shaker, first thoroughly studied by Kluyver and Per- quin (161). Flasks are mounted on a platform which is either rotated rapidly or moved horizontally with a reciprocating motion. Problems of design are considered by Shu and Johnson (270), Paladino (230), and Chain and Gualandi (54). Representatives of all the major groups of fungi and of the genus Streptomyces have been grown successfully in stirred or shake culture. The growth rate is usually at least double that of the same organism in surface culture (1, 61, 162, 286, 319, 335), although some fungi do not show this effect (14, 268) and some grow so poorly that the method cannot be used for them. One of the chief advantages, apart from more rapid growth, is that shaker-grown mycelium is usually homo- geneous enough to be used directly in respirometric and other meta- bolic studies. In shake culture each colony is exposed to a uniform environment in all spatial directions; hence the typical colony is a globose structure. Colonial morphology varies, however, with the species — even within a genus — and with the medium (48). Sporulation is usually, although not always, suppressed — another factor of homogeneity. Microscopic characteristics of the mycelium are also affected by aeration and agita- tion (76, 83). Although a shaken culture has a measurably higher content of dis- solved oxygen and rate of oxygen diffusion than a still culture (283), for certain purposes a still higher rate of oxygen supply may be de- sirable (249, 252). This usually arises when a high concentration of a readily utilizable carbon source is employed; the peak oxygen de- mand under specified conditions is 16 mM oxygen per liter per hour METHODS OF CULTIVATION O for Ustilago zeae and 56 mM per liter per hour for Aspergillus niger (97). Rates of aeration of this magnitude require an apparatus in which a sterile medium can be agitated and aerated. Various labora- tory-scale devices have been described (10, 55, 142, 168, 252), and some are commercially available. Theoretical and practical problems of de- sign have been studied in several laboratories (9, 49, 68, 97). Growth on solid media other than agar is useful in special problems; such materials include soil (224, 260), sawdust (6), bran (206), and cotton (25). The use of plant materials sterilized by treatment with propylene oxide is particularly valuable in the induction of sporula- tion (Chapter 11) and may have wider application in the study of physiology. The inoculum used for cultures is usually a population of spores from an agar slant. A common occurrence in the fungi is the replace- ment of the normal type by a variant arising in culture; this develop- ment has been analyzed especially with regard to sporulation. Other examples include the loss of biochemical capacities (63), change in virulence to a host plant (202), and change in assimilatory capacity (246). The ultimate recourse is to preservation of cultures in a dormant state (p. 4). Methods of obtaining single-spore cultures of fungi are reviewed by Hildebrand (136); such cultures may be neces- sary in order to recover or purify the desired type. It must, however, be remembered that some spores are multinucleate (Chapter 12) and may contain genetically different nuclei. Three physiological aspects of the inoculation process may be men- tioned. First, growth is more rapid with a heavy than with a light inoculum; in addition, a heavy inoculation of a submerged culture usually results in the development of small colonies, with less danger that oxygen diffusion into the colony will limit growth or respiration. Second, the use of old spores of low viability is to be avoided; both the age and the conditions of growth of the inoculum, especially medium and temperature, should be standardized. For certain fungi the "chem- ostat" provides an easily standardized inoculum (221). Finally, a large inoculum may be required in order to initiate growth on an un- favorable medium (235). When rapid growth is a desideratum, the inoculum may be preger- minated spores or mycelium (207). Mechanically macerated mycelium can be used as inoculum (233, 259). This is often the method of choice for fungi which do not sporulate (3). The treatment, to minimize injury, should be as brief as possible. Special methods of cultivating particular fungi are too numerous to mention here. Continuous-flow apparatus may have some general 4 CULTIVATION AND GROWTH interest; these include devices in which only the solution is renewed (325) and those in which both nutrients and cell population are con- trolled (138). Culture media for fungi are described by Fred and Waksman (101), Levine (176), and Lilly and Barnett (180). In general, it has been too readily assumed that fungi need and tolerate high nutrient levels and high acidity; this is true of many fungi but by no means of all. For certain purposes, concentrated media are of course useful, but mor- phology, physiology, and reproduction are best studied in more dilute media than those often used. 2. PRESERVATION Interest in the preservation of fungi springs from the desire to avoid excessive labor in maintaining stock cultures and, more important, the wish to maintain cultures with a minimum of genetic change. The problem is to maintain a viable inoculum in a non-growing condition. Cultures and herbarium specimens often survive for surprisingly long periods after drying, probably by virtue of the formation of long- lived spores. Zobl (344) reviews early records of up to 21 -year survival; in his own work he found that survival of dried cultures is common in ascomycetes and basidiomycetes, but the Mucorales are relatively short- lived. The sclerotia of several fungi, which may live for as long as 13 years (336), are less viable if stored dry (294). A few of the more leathery basidiomycete sporophores survive dry for a period of years; more succulent types are less durable (41, 43). In the soil tube method (5, 121), a fungus is transferred to moist sterile soil, preferably brought to pH 6-7 with calcium carbonate, and grows and sporulates until the soil dries out. The limited data avail- able indicate that although some fungi survive at least 5 years in soil tubes, too many do not live that long, and the method therefore is to be regarded as a special one not adapted for general use. Preservation of cultures under sterile mineral oil is relatively sim- ple and has the advantage that sporulation is not essential (39, 267). Limited tests indicate, however, that although many fungi survive at least 2 years, others die out earlier (146, 331, 333). Lyophilization of spores suspended in serum, gelatin, dextran, or other colloidal materials appears to be a usable method, although again not successful with some fungi, e.g. the Entomophthorales and the dermatophytes (199, 239, 265, 337). Simple vacuum drying without freezing is suitable for many fungi (244). The structure of the fungus is important; thus, oogonia but not sporangia or mycelium, of Pythium THE DYNAMICS OF GROWTH 0 spp. can be successfully lyophilized (281). Haskins and Anastasiou (126) report successful use of a modification of the lyophil technique. Too rapid rehydration of vacuum-dried uredospores is apparently injurious (265a). For sporulating fungi, lyophilization appears to be the best single method; until more information is available, however, preservation of many fungi will have to be on an experimental basis. Lyophiliza- tion is comparatively easy and apparatus is commercially available. Preliminary studies (197) indicate that spray drying of fungus spores may also be feasible. 3. THE DYNAMICS OF GROWTH Growth cannot be precisely defined; a provisional operational defini- tion is increase in either mass of cells or number of cells. Mass — dry weight, usually— in the filamentous fungi may be deceptive inasmuch as it can represent in part the accumulation of polysaccharides, lipids, or wall materials without any increase in living protoplasm. Con- versely, in Neurospora crassa protein synthesis appears to continue for a period of time after weight increase has ceased (7). The time course of growth of filamentous fungi in liquid shaken cultures is exemplified by the data of Figure 1. Similar curves are ob- tained with surface cultures (2, 105, 248). Typical curves are charac- terized by three major phases, with transitions between them: 1. A phase of no apparent growth. 2. A phase of rapid and approximately linear growth. 3. A phase of no net growth or of autolysis and decline in dry weight. It should not, however, be assumed that all data fit this scheme. In particular, a two-phase growth curve, in which an initial growth period is succeeded by a second, with a leveling off of growth inter- vening, has been reported (30). The second phase may represent polysaccharide synthesis only, without increase in other cell com- ponents, or it may depend on a mobilization of nitrogen from older hyphae and use of it for new growth. The observations of Morton and Broadbent (205) on Scopulariopsis brevicaulis support the second ex- planation; at least in a medium relatively high in carbon, it appears that nitrogen is reutilized after exhaustion of the available exogenous nitrogen. Probably by suitable adjustment of cultural factors, especially nu- trient concentrations, a variety of growth curves very different from those of Figure 1 can be obtained. Provision of calcium, for example, CULTIVATION AND GROWTH Figure 1. The growth of Fusarium solani in an aerated medium. Open circles, dry weight; closed circles, optical density. to Streptomyces fradiae delays the onset of the autolytic phase (132). The growth rate varies not only between species, but between differ- ent isolates or clones of the same species; this has been found to be true in both liquid and agar cultures (71, 88, 89, 130). The first phase, that of no apparent growth, presumably has two components: a genuine lag phase before spore germination, and a phase in which growth is occurring but is undetectable by the methods used. The details of this growth period have not been studied in the fungi. In bacteria it appears that growth, as measured by protein and nucleic acid synthesis, begins immediately, with no true lag period (298). The second phase is that of rapid growth. As Emerson (86) has shown, the cube root of the dry weight increases linearly with time over a major portion of the period (Figure 2). It should be stressed, how- ever, that this relation holds only if no external factor, e.g., oxygen or nutrient concentration, is limiting to growth. Conversely, a curve con- forming to that of Figure 2 indicates that the organism is growing un- restrictedly. The morphological basis of mycelial development is that growth THE DYNAMICS OF GROWTH ' occurs only at hyphal tips (272, 273, 280). Interior cells of the myce- lium do not normally contribute to the new growth directly, although they supply nutrients to peripheral cells, especially to aerial structures in still culture. Growth in any one time period is therefore a function, not of the total number of cells present, as in bacteria and yeasts, but of the number of hyphal tips and of the rate at which these tips are supplied nutrients. Some of the chemical events during the phase of rapid growth are illustrated in Figure 3; these include especially utilization of carbo- hydrate, nitrogen, and phosphate. Metabolic products, e.g., acids, may or may not appear in the medium at this time. Characteristically, respiratory activity is at a maximum (Chapter 7). Few studies have considered critically the relation of reproduction to growth phases; casual observation and the data of Robinson (248) on Sporodinia grandis indicate that in still culture spores usually de- velop at the end of the phase of rapid growth; spores may, however, appear earlier (209). The third and last phase is, as shown in Figure 3, characterized usu- ally by a decline in mycelial weight and in the appearance of nitrogen and phosphate in the medium. The loss in weight varies from neg- ligible to extreme; a common pattern is a rapid loss of weight for a 40 Time, hours Figure 2. The growth of Neurospora crassa as a function of time. Curve 1, loga- rithmic plot; curve 2, cube root plot; curve 3, linear plot. Redrawn from Emerson (86), by permission of the Williams and Wilkins Company. CULTIVATION AND GROWTH Time Figure 3. Growth of and changes in the medium by Aspergillus oryzae. Redrawn from Crewther and Lennox (62), by permission of the Australian Journal of Bio- logical Sciences. short time with no further change thereafter (151). Autolyzing myce- lium of Penicillium griseojulvum undergoes an extensive breakdown of chitin, carbohydrates, and protein, catalyzed by enzymes of the fungus (275). Other autolytic products include ammonia (74, 80, 160), free amino acids (71, 108), organic phosphorus compounds (200), and sul- fur compounds (137, 245). Cells at the end of the growth phase appear vacuolate; younger cells have a denser and more homogeneous protoplasm (49). Such old cells, of course, are found even during the growth period in regions remote from the growing hyphal tips. From the rather limited data available, the cessation of active growth appears to be determined by either of two factors. In concen- trated media, toxic metabolites can be shown to accumulate and these materials, earlier called "staling substances," demonstrably retard growth (27, 37, 236). Probably organic acids, in high-carbohydrate THE DYNAMICS OF GROWTH 9 media, and ammonia, in high-nitrogen media, are the compounds most frequently involved. In better balanced and more dilute media the principal factor in cessation of growth is exhaustion of the carbohydrate supply (15, 74, 79), as shown by the data of Figure 3. The extent of autolytic breakdown, once growth has ceased, varies, as mentioned above, with species and cultural conditions. Autolytic breakdown of Aspergillus niger mycelium is much more extensive in nitrate than in ammonium salt media, possibly because of the alkalin- ity of the nitrate medium (15). However, autolysis of a thermophilic Streptomyces sp. is more rapid at acid than at neutral pH (158). So much for growth in liquid media. A fungus colony on an agar surface follows a different course. Typically, a period of little or no growth is succeeded by the establishment of a constant growth rate which is maintained unless toxic metabolites accumulate (36, 93, 308). The growth rate tube of Ryan et al. (253) may be used to show this linear relation of extension to time; the data of Figure 4 are typical. Growth on soil should follow the same course if measured by horizontal spread only; specialized rhizomorphs of Armillaria mellea grow several times as rapidly as unorganized mycelium (111). Neurospora crassa and other fungi respond to certain toxicants, e.g., 1 1 1 1 ! 2 1 I 200 1/ /3 E E - — o 100 — /4 — 5^ n -i — ~V I i 6 . i I 3 6 Time, days Figure 4. The linear growth of several fungi in growth rate tubes. Curve 1, Neurospora crassa; curve 2, Rhizopus oryzae; curve 3, R. nigricans; curve 4, Sordaria fimicola; curve 5, Fusarium oxysporum lycopersici; curve 6, Monilinia fructicofa. 10 CULTIVATION AND GROWTH sorbose, by a more colonial and less rapid growth on agar (293). The growing hyphal tips are killed and new branches then form and grow laterally (181). 4. DIMORPHISM Several fungi pathogenic to man are found in infected tissues in a unicellular yeastlike form, but when cultivated at room temperature grow out in a mycelial form. The term dimorphism strictly applies to these forms only, but related phenomena in the normally filamentous fungi may provisionally be considered as in the same category. A dimorphic fungus is one, therefore, in which a reversible transforma- tion from a mycelial to a non-mycelial and unicellular growth type occurs. Dimorphism is common in the yeasts (262), and unicellular bacteria may be caused experimentally to form filaments by, for example, magnesium deficiency (216, 326). Some normally filamentous fungi, e.g., smut fungi, grow in culture as unicellular budding cells. The fungi listed in Table 1 include both the pathogenic dimorphic fungi Table 1. Factors Affecting Non-mycelial Growth in Fungi Organism Candida albicans, C. tropicahs Blastomyces dermatitidis Histoplasma capsulatum Histoplasma farciminosum Paracoccidioides brasiliensis Sporotrichum schencki Trichosporon capitatum Ceratostomella ulmi Dematium pullulans Fusarium oxysporumf. nicotianae Mucor guillermondii Ophiostoma multiannulatum Romano a terricola Non-mycelial Phase Favored by Cysteine, NH4C1, glucose carbon source (152, 211, 214, 217) High temperature (35-37°C) cultivation (177, 256) Cysteine, high temperature (255) High C02 (40) High temperature (37°C) cultivation (82, 256) HighC02 (81, 254) Cysteine (217) Aeration (75), low pH (26) Low aeration (11), low carbohydrate (263) Aeration (339) Low aeration (186) High (12 mg per liter) inositol (107) Aeration (299) and those filamentous fungi in which production of a unicellular growth phase can be induced reversibly. It should be stressed that in this second group the morphology of the unicellular phase may not be the same for all forms listed; i.e., the available information does not discriminate between, say, conidia and hyphal fragments. The diver- THE MEASUREMENT OF GROWTH 11 sity of non-mycelial growth phases is rendered more likely by the vari- ety of stimuli which induce the phase in different fungi (Table 1). Several theories, reviewed elsewhere (152, 262), have been proposed to explain dimorphism in the fungi pathogenic to animals. The most attractive concept is that synthesis of new protoplasm and cell division are separate processes. Filaments form when growth occurs under conditions not permitting rapid cell division. The studies of Nickerson and co-workers (152, 211, 212, 214, 215, 217) indicate that a supply of reduced organic sulfhydryl compounds is essential for rapid cell divi- sion and hence for maintenance of the unicellular condition. At least in principle, all the stimuli known to affect dimorphism in the patho- genic fungi could act through their effect on sulfhydryl content of the cells. Whether this will apply to the non-pathogenic fungi listed in Table 1 is not known. The yeast phase of Blastomyces dermatitidis and Paracoccidioides {Blastomyces) brasiliensis is multinucleate, that of some other di- morphic fungi uninucleate (82). The yeast and mycelial forms of Blastomyces spp. differ in their respiratory capacities (213). Dimorphism in yeastlike fungi suggests, of course, that the true yeasts may have arisen by a more or less permanent acquisition of the uni- cellular habit. It is also perhaps legitimate to ask whether the fila- mentous habit in the true fungi and the algae is an expression of the same metabolic idiosyncrasy as the filamentous habit of Blastomyces and Histoplasma. 5. THE MEASUREMENT OF GROWTH As mentioned earlier, the meaning of growth is determined by the method of measurement chosen. Some methods are useful only for particular organisms or for special problems; no method is so general that it can be recommended for all. The most widely used and within limits the most satisfactory meas- urement of growth is by determination of dry weight of the mycelium. The principal limitation is that weight may reflect accumulation of polysaccharides or other reserve materials rather than synthesis of new protoplasm. Oxidative assimilation (Chapter 7) is often very active in the fungi under conditions which restrict protein synthesis. Various procedures may be used for the determination of dry weight. Often a coherent mycelium can be removed from a liquid medium, washed, and dried in a tared container. More commonly filtration is required; tared filter paper, protected from atmospheric moisture, or porous discs (ceramic or sintered glass) held in a demountable suction 12 CULTIVATION AND GROWTH assembly are practical filtration devices. Gelatinous growth which filters slowly may be separated by centrifugation. The use of solvents (16) to hasten drying is practicable, of course, only if the extracted lipids are negligible. Drying is usually effected by high temperature, 70-80°C. The dry weight of agar colonies, although of dubious significance in most contexts, has been determined by scraping or peeling off of the surface growth (91). Removal of agar with hot water results in a significant loss in dry weight (67). Turbidimetric measurements of growth are suitable for those fungi which grow as single cells or very short dispersed hyphae. Ustilago xnolacea is of the first type, and turbidity is correlated with dry weight (22). Methods have been evolved primarily for studies on bacteria (305, 306); although most workers use a colorimeter, in principle the nephelometer is better adapted to turbidity measurements. A turbidi- metric method should, of course, always be calibrated against dry weight or other standard measure; Figure 1 illustrates the correlation. Determination of growth by measurement of the total cellular nitrogen has been used only rarely in work with fungi (222). Although the chitin nitrogen complicates interpretation, this is the best method for determination of growth defined as synthesis of protoplasm, and deserves wider use. Linear growth on agar is the least laborious method of estimating growth and has been correspondingly popular. The radial spread of a colony in a petri dish or linear advance in a growth rate tube (93, 253) are most frequently determined. Growth is commonly expressed as the constant rate established after the initial period of slow growth. Naturally, the impurities present in agar rule out this method for certain problems. There are, however, more fundamental objections. Chaudhuri (56) found no response of colony diameter to even a four- fold dilution of the nutrient medium. Nor does the rate of advance on agar always respond to necessary vitamins (106, 179) or essential ions (238); strain differences in growth on agar are not always reflected in dry weight measurements (133). Chaudhuri (56) found, on the other hand, excellent correlation of radial growth on agar with dry weight and germ tube growth in tem- perature studies on Verticillium albo-atrum; more recently, Domsch (77) has confirmed the correlation in temperature response. Wood- destroying fungi usually respond to temperature in agar culture about as they do in wood blocks (317). One crucial detail is often over- looked: the depth of the medium influences growth and must be standardized (28, 36, 56). TEMPERATURE AND GROWTH 13 We may conclude that the agar method is adequate for studies of some environmental factors, e.g., temperature and (probably) com- position of the atmosphere, although even in these experiments the results are not always correlated with dry weight (94, 134, 145, 250). It is decidedly not adequate for nutritional studies. Whether it should be used for essentially chemical problems like the effct of pH or toxic agents is still uncertain. The burden of proof that spread on agar is a fair index of growth rests on the investigator who chooses to use the method. Its value cannot be assumed. 6. TEMPERATURE AND GROWTH Temperature affects growth, spore germination, reproduction, and indeed all activities of the organism. Typical curves of growth as a function of temperature are shown in Figure 5. There is characteristi- cally a linear portion in which growth increases directly with tem- perature, an optimum range which may be narrow or rather broad, and a descending limb as the temperature becomes too high for growth. The curve as a whole is usually skewed to the right; an empirical method for converting this skewed curve to a straight line is described by Cohen and Yarwood (58). The temperature-growth curve tends to become more nearly sym- metrical as the optimum becomes lower: fungi with optima of 22-24°C or less often have a much less skewed curve (53, 98, 143, 171, 311). Not all low-temperature fungi, however, follow this pattern (113, 164). Fungi with high temperature optima generally have the typical skewed curve even more pronouncedly; in a few cases, the high opti- mum is associated with a broader range of good growth, but this is not the rule. It should be noted that the concept of an optimum, usually taken for granted, is not in fact very clear. The response of Phycomyces blakesleeanus to temperature (247) illustrates the principal ambiguities in the concept of optimum temperature. First, growth is most rapid at 20-25 °C, but the maximum total amount of growth at limiting levels of thiamine is greatest at 10°. Second, the effect of temperature on final dry weight can be almost wiped out if the nitrogen supply is made limiting. Another example of the dependence of temperature characteristics on other factors has been elucidated by L. Fries (103). Coprinas fimetarius grows poorly at 44°C because of the failure of methionine biosynthesis to keep pace with other processes; if exogenous methionine is supplied, growth at the elevated temperature is normal. The ap- 20 Temperature, CC (a) 40 Figure 5 A. Temperature and growth (dry weight) of fungi. Curve 1, Cephalospo- rium costantii, from data of Treschow (311); curve 2, Pullularia pullulans, from data of Rennerfelt (243); curve 3, Coprinus sp., from data of Johnson and Jones (154). 0 20 Temperature, °C (b) Figure 5B. Temperature and growth (colony diameter on agar) of fungi. Curve 1, Verticillium tricorpus, redrawn from Isaac (144) by permission of the Cambridge University Press; curve 2, Polyporus vaporarius, from data of Gaumann (112); curve 3, Pythium debaryanum, from data of Roth (250). 14 TEMPERATURE AND GROWTH 15 parent optimum temperature for radial growth of Sclerotinia fructicola is affected by pH (301). It is clear that temperature optima and ranges reported are valid only under specified conditions of time, medium, and method of meas- urement. There is no single temperature optimum of growth. Never- theless, we shall continue to employ the concept of an optimum tem- perature, on the grounds that it is empirically useful and that gross differences in apparent optima provide a basis for ecological studies. A given metabolic process — respiration, antibiotic production, or vitamin synthesis — does not, of course, necessarily respond to tem- perature in the same way as does growth. Studies on penicillin produc- tion in particular support this generalization (49, 227), as do studies of bacterial growth and metabolism (78). Over the range in which growth is proportional to temperature, the relation may be expressed by the value of Q10, defined as the ratio of the growth rate at a given temperature to that at a temperature 10°C lower. The values found by Fawcett (92) in studies on four fungi are comparable to those reported for bacterial growth (298). The shape of the temperature-growth curve is somewhat affected by the time of incubation. Growth at suboptimal temperatures is often so slow that it is not apparent at all at a time when cultures at more favorable temperatures are growing vigorously. Growth at supra- optimal temperature may begin rapidly but slow down or cease en- tirely after a period of time (34, 223, 278, 286). Both of these factors, of course, affect the curve, so that use of only one incubation period may introduce error. Most fungi make at least some growth over a 25- or 30-degree range of temperatures, but narrower ranges are known, e.g., in Merulius lacrymans (53) and Capnodium salicinum (98). Narrow ranges are often associated with low optima, but the reverse is not true — many fungi with low optima grow over a normally wide range of tempera- ture. Temperature optima for plant pathogenic fungi have been collected by Togashi (307) and are depicted graphically in Figure 6. Not all of these reports can be accepted, and details of the distribution are unduly affected by popular incubator temperatures, but it can be seen that most members of this ecological group have optima in the region 20-30°C and about half have their optima between 26 and 30°C. The relation between temperature and habitat is not yet entirely clear. Many fungi are probably world-wide in distribution, i.e., their growth is limited by factors other than temperature. Species of Allo- myces, on the other hand, are clearly confined naturally to the tropical 16 CULTIVATION AND GROWTH 20 25 30 Optimum temperature, °C 35 40 Figure 6. The optimum temperatures for growth of plant pathogenic fungi. Com- piled by Dr. C. E. Yarwood from Togashi (307). and warmer temperate regions of the earth (84). Subtropical strains of a species may have a higher temperature optimum than strains from temperate regions (38). The distribution of Phymatotrichum omni- vorum is determined by winter soil temperature (90). The few marine fungi studied so far have temperature optima for growth that are some- what higher than the temperature of the sea (8). The occurrence and dominance of fungi in decomposing plant materials are largely deter- mined by temperature (150, 223). Dermatophytic fungi grow best in culture at 25-35°C, i.e., below the host body temperature (284); fungi causing systemic mycoses grow well at 37° but optima have not been reported. It is significant that Asper- gillus fumigatus, a thermotolerant species, can cause disease in warm- blooded animals. Some examples of high and low temperature optima are collected in TEMPERATURE AND GROWTH Table 2. Low and High Temperature Optima in the Fungi Fungus Criterion of Growth Optimum, ' Botrytis cinerea (115) Dry weight 15-18 Gnomonia spp. (135) Linear extension 17-19 Hendersoniella sp. (98) Linear extension 18 Herpotrichia nigra (113) Linear extension 15 Mastigosporium album (23) Linear extension 15 Phacidium infestans (232) Dry weight 15 Phoma apiicola (17) Linear extension 16-20 Phycopsis sp. (98) Linear extension 15 Phytophthora primulae (309) Linear extension 15-20 Actinomyces (Streptomyces) sp. (123) Linear extension 35-37 Aspergillus fumigatus (243) Dry weight 37 Aspergillus nidulans (223) Linear extension 40 Coprinus fimetarius ( 1 04) Dry weight 35-40 Lenzites trabea (53) Linear extension 35 Mucor pusillus (218) Linear extension 40-46 Rhizopus chinensis (330) Linear extension 37.5-40.5 Sclerotium (Rhizoctonia) bataticola (224, 310) Linear extension 31-35 Sepedonium sp. (223) Linear extension 45 17 Table 2. It should be mentioned that not only may species within a genus differ widely (300, 330), but strains or geographical isolates of the same species may respond differently (83a, 143). Surprisingly many fungi are able to grow, although usually only slowly, at 0° or slightly less. Plants under snow may be infected by the parasitic "snow molds" — Ty pint la spp., Fusarium nivale, and an un- identified basidiomycete (32, 65, 242). Phacidium infestans, cause of a disease of pines, is able to grow in culture at —3°, although its optimum temperature is about 15° (232). A second ecological group of fungi tolerant of low temperature is commonly associated with the spoilage of refrigerated foods. Fungi of many different genera fall into this group and some, especially strains of Cladosporium and Sporotrichum, grow at temperatures well below zero, e.g., —5 to —8°. The literature on these forms is reviewed by Berry and Magoon (19) and, more briefly, by Pehrson (232). Again, although these fungi tolerate extremely low temperatures, their optima are higher (122). Survival of spores and mycelium at temperatures too low for growth is discussed briefly in Chapter 13. Most fungi are unable to grow at 35-40°C. There are, however, two general types of exceptions to this rule. The first group is a 18 CULTIVATION AND GROWTH miscellaneous one, examples of which appear in Table 2; it includes saprophytes — especially coprophilic forms — and several wood-destroy- ing fungi. The second group, the true thermophiles, is defined by the ability to grow at 50°C or higher and the inability to grow at tempera- tures below about 30°. Relatively few such fungi are known; ex- amples include Thermomyces lanuginosus (Monotospora lanuginosa) (135a, 313), Thermoascus aurantiacus (201), Penicillium duponti (240), and Chactomium thermophile (169). No study of the funda- mental basis of this behavior in fungi has appeared. Truly thermophilic forms also are found among the actinomycetes, reviewed by Bernstein and Morton (18). The obligately thermophilic Micromonospora vulgaris is distinguished by the high resistance of its conidia to heat damage (87). Both fungi and actinomycetes are active in decomposition of compost at 50°C (320). Killing of fungi by high temperature is considered elsewhere (Chap- ter 13); spores are the usual experimental material. It need only be noted here that mycelium is easily killed by elevated temperatures and that many fungi die slowly when held in culture at a temperature just above the maximum for growth. Fluctuations in temperature induce two types of zonation, the more common of which results from the presence of zones of reproductive structures (Chapter 11). In a second type of temperature-induced zonation the zones are entirely mycelial; this phenomenon occurs in Cercospora beticola (60) and in other fungi (20, 124). The data compiled by Togashi (307) indicate that the optimum temperature for spore germination is generally very close to that for growth in culture. There are instances, however, in which spore germination is less inhibited than growth by supraoptimal tempera- tures (185, 330). Spore germination at temperatures too low for growth has been reported in Rhizopus spp. (330), and is presumably common. The relation between a fungus-incited plant disease and temper- ature is rather more complex, inasmuch as at least two organisms are necessarily involved. In general, storage and transit rot diseases are most severe at the temperature which favors mycelial growth of the pathogen; examples include the decay of sweet potato by Rhizopus spp. (172, 330) and storage rots caused by Rhizoctonia sola?ii (170, 332). Typically, however, the pathogen will grow in culture at tem- peratures below the minimum for infection (33). The vascular wilts of plants caused by Fusarium spp. often have the same pattern, i.e., the disease optimum is close to that of the pH AND GROWTH 19 pathogen (155, 156, 302, 322). However, it is not certain that the relation is as simple as it appears, and in some wilt diseases tempera- ture affects the host (100, 174) and, probably, other soil organisms which influence the disease (264). For a few plant diseases it has been definitefy shown that differences between temperature optima for disease and for growth of the patho- gen occur and reflect the effect of temperature on the host. This has been established particularly for the seedling blight of wheat and corn caused by Gibberella saubinetii (72, 73) and a root rot of sweet clover caused by Plenodomus meliloti (258), and has been suggested for other diseases (114). Finally, there are some plant diseases in which the observed action of temperature appears to be exerted on the pathogen, but on spore germination or sporulation rather than on mycelial growth (95, 155, 156). It is conceivable, of course, that pathogenicity, as distinct from growth, is affected by temperature, but so far this has not been demon- strated. 7. pH AND GROWTH Under given conditions, a fungus will grow maximally over a certain range of initial pH values of the medium, and will fail to grow at high and low extremes. However, it must be borne in mind that pH is not a unitary factor; the mechanism of its action differs at different concentrations of hydrogen ion. For example, one part of the pH-growth curve may reflect the effect of a low pH on enzyme systems, another the effect of a high pH on metal solubilities. Acidity may also affect the entry of essential vitamins (341), surface metabolic reactions, entry into the cell of organic acids, or the uptake of min- erals (226). It is not surprising, therefore, to find that almost any factor in the environment may change the shape of the pH-growth curve. Such factors include temperature (232, 327), time of harvest (175, 210), gross changes in the medium (187, 340), growth factor supply (178), calcium and magnesium levels (314), and nitrogen source (140). Furthermore, pH is affected during growth by metabolic activities — raised by absorption of anions or production of ammonia from nitrogenous compounds, lowered by formation of organic acids or absorption of cations. These effects of growth on pH complicate results, particularly in the poorly buffered media commonly em- ployed. It seems probable that many of the broad optima recorded 20 CULTIVATION AND GROWTH Table 3. pH Optima of Some Fungi and Actinomycetes* Organism Aphanomyces euteiches {21 A) Aspergillus flavus (28) A. niger (28) Boletus variegatus (198) Botryosporium (?) sp. (149) Cercospora kikuchii (70) Chalara quercina (14) Chytridium sp. (61) Colletotrichum hibisci (314) Coprinus sp. (154) Fomes annosus (89) F.fraxineus (203) Fusarium aquaeductum (229) F. aurantiacum (260) Geotrichum sp. (229) Marasmius graminum (182) Merulius confluens (219) Mycogone perniciosa (311) Neurospora crassa (253) Ophiobolus graminis (327) Penicillium expansum (28) P. glaucum (2) Phlyctorhiza variabilis (251) Phycomyces blakesleeanus (47) Pythiogeton sp. (52) Sepedonium sp. (230) Septoria pepli (266) Streptomyces coelicolor (57) iS". scabies (318) Tricholoma nudum (222) Trichophyton persicolor (285) Trichosporon cutaneum (229) Verticillium malthousii (311) V.psalliotae (311) * For reasons discussed in the text, these figures must be considered as approxi- mations and as limited to the conditions of the particular experiment. f Defined for this table as the pH range tested in which the growth was within 10 per cent of the maximum growth. % With added calcium and magnesium in the medium. riterion of Growth Optimum f Linear extension 4.5-6.5 Linear extension 5.5-8.4 Linear extension 4.4-7.5 Dry weight 5.0 Dry weight 6.6-7.4 Dry weight 4.1 Dry weight 4.5 Dry weight 5.2-7.5 Linear extension 3.5-8.0J Dry weight 4.8-6.9 Linear extension 4.6-4.9 Linear extension 6.0-7.0 Dry weight 4.0-9.0 Linear extension 6.3-7.0 Dry weight 3.0 Dry weight 5.7-6.4 Dry weight 4.0 Dry weight 6.7 Linear extension 4.3-6.5 Dry weight 4.9-7.4 Linear extension 4.4-7.5 Dry weight 5.0-6.5 Dry weight 7.2-7.6 Dry weight 3.5-4.6 Dry weight 6.5 Dry weight 7.0-8.5 Linear extension 4.8-6.8 Dry weight 6.9-7.7 Dry weight 6.9 Dry weight 5.0-6.0 Dry weight 6.5-7.0 Dry weight 4.0-9.0 Dry weight 5.3 Dry weight 6.7-7.0 pH AND GROWTH 21 in Table 3 reflect the ability of the fungus to raise or lower the pH of an initially unfavorable medium; data on Colletotrichum linde- muthianum (173) illustrate this phenomenon. The values of Table 3 should, therefore, be viewed with some cau- tion; at best they offer a rough guide to choice of media and a possi- ble indication of ecological preferences. They are not to be regarded as absolute figures in any sense. A double pH optimum is frequently reported, a minimum appear- ing between two maxima in the curve. This type of response has been exhaustively analyzed in studies on Coprinus spp. (102, 104). It appears that the minimum merely reflects pH-dependent unavail- ability of one or more inorganic elements; provision of iron, zinc, and calcium in available forms eliminates the double optimum and re- places it with a broad single optimum zone. An organism may also be able to grow, even quite well, at a pH which does not permit the initiation of growth (189). The metabolic basis of this phenomenon is not clear; again, the artificialitv of the simple pH-growth curve is emphasized. In contrast to the bacteria and the actinomycetes, fungi are rela- tively more able to invade acid environments, although we should avoid the common assumption that they are restricted to such environ- ments. In culture, the larger basidiomycetes are often unable to grow at an initial pH above 7.0; examples include most species of Ma- rasmius (182), and Tricholoma (222). Correspondingly, limited ecological studies of these fungi indicate that they are in general restricted to acid environments (338). However, Marasmius rotula grows in culture and is found in nature on substrates of widely dif- ferent pH (182, 338), and several other fleshy basidiomycetes, notably species of Coprinus, actually require alkaline conditions for best growth (104, 120, 312). Biological types of fungi may only roughly be characterized as to pH preference. Most plant pathogens — exceptions are evident in Table 3 — grow best in media with an initial pH of 5.0 to 6.5. Species of Streptomyces are marked by a relative intolerance of acidity; very few can grow at a pH lower than 5.0. The same intolerance of acidity appears also in other groups, e.g., the lower phycomycetes (61, 85). The dermatophytic fungi, perhaps because they are usually grown in complex media, seem rather indifferent to pH over the range 4.0 to 10.0 (284, 285). Other biological activities of the fungi respond to pH, but not necessarily in the same way as growth. As a rule, the pH range for sporulation is narrower than that for vegetative growth (Chapter 22 CULTIVATION AND GROWTH 11). The range for spore germination is similarly more narrow (185, 291); this is to be expected, inasmuch as the design of the spore germination experiment does not permit sufficient time for metabolic activities of the organism to modify the pH of the medium. Soil-borne plant diseases may or may not show the same pH curve as do the fungi which cause them (96, 260); here, of course, the effect of pH on the plant presumably exerts an important influence, and the com- plex effect of pH on the soil itself may be even more decisive. In most physiological studies it is necessary that the pH be con- trolled, at least within limits. Periodic addition of alkali (51) and the use of solid calcium carbonate in the medium (99) are satisfactory for certain problems, but in general it is necessary to employ a buffer system. Of these, the most common and generally useful is the phosphate buffer. The usefulness of phosphate is limited, however, by the tolerance of the fungus to the ion; this may be as low as 0.003 M (61) or as high as 0.20 M (66, 151), and must be determined for each organism. Other buffer systems must be employed with caution: acetate is often toxic (64, 183, 291), and citrate may be utilized by the fungus as a source of carbon, with consequent displacement of the apparent pH optimum (64). Phthalate, borate, and citrate buf- fers are satisfactory for Neurospora crassa (253); a-hydroxybutyrate has been proposed as a nonutilizable buffer for studies on fungi (303). Equipment for automatic pH control has been described (50). Physiological buffer systems can be devised for particular problems, e.g., acetate as carbon source and ammonium ion as nitrogen source (188), or mixtures of ammonium and nitrate ions (141). In both these systems, utilization of an anion is balanced by utilization of a cation and the pH is thereby held relatively constant. In conclusion, the pH-growth curve has, to say the least, a limited usefulness. In designing experiments of this type, the first question to ask is whether the information to be gained is worth the effort required. Once such an experiment is decided upon, two rather ele- mentary precautions should be observed. First, the final pH of the medium, after growth, must be reported. Second, the results will be more meaningful if buffer systems are used to control pH; in the alkaline range, addition of sodium hydroxide to control pH is followed by absorption of carbon dioxide from the air, with a con- sequent false impression of the upper pH limit of growth. The internal pH of the fungus cell — and the fungus cell is usually large enough so that the concept of pH may legitimately be applied — is not known with any precision. Indicator methods, which have obvious uncertainties, yield in most hands a value of about 6.0 (4, OXYGEN AND GROWTH 23 193, 219), close to the value for resting cells of yeast (59). Bunning (44) reports a lower internal pH, below 5.0, for Aspergillus niger. Whether or not the internal pH responds to changes in the acidity of the external medium is uncertain: there may be some effect (44, 228), but it is to be presumed that any large change would result in injury and death. The principal effects of external pH are probably on permeability and other surface phenomena. 8. OXYGEN AND GROWTH Fungi are commonly thought of as strictly aerobic, and this opinion is basically correct. However, the quantitative relations of growth and oxygen supply vary considerably among different forms. Visually observable growth of fungi occasionally occurs under ni- trogen or in a liquid-filled container (61, 191, 220). This limited growth probably depends on oxygen previously dissolved in the medium. Blastocladia pringsheimii grows well in tank nitrogen or carbon dioxide but fails to grow if oxygen is chemically absorbed by pyrogallol; the fungus has, therefore, a very low but real oxygen de- mand (85). Achlya prolifera has been reported to grow in the presence of pyro- gallol (134). However, this report cannot be accepted without con- firmation, in view of the overwhelming evidence that other fungi and species of Streptomyces fail to grow at very low or zero oxygen pressures (69, 94, 119, 127, 166, 192, 195, 204, 234, 237, 257, 292, 342, 343). When growth on agar is the criterion, it appears that most fungi grow as well at 20-40 mm oxygen pressure as at atmospheric (160 mm) (69, 116, 118, 261, 295). However, the dry weight is usually affected at somewhat higher oxygen pressures; growth of Ophiobolus graminis is reduced at 105 mm oxygen (94), Aspergillus oryzae grows best at oxygen pressures higher than atmospheric (292). Contradictory data may be cited (12, 166), but it seems that mass increase is more sensi- tive to oxygen deficit than is the more diffuse and thinner growth of a colony on agar. In keeping with the relatively low oxygen requirement for growth, fungi successfully colonize environments in which oxygen is limited, e.g., relatively stagnant aquatic milieu. It is doubtful whether invasion of woody tissues is ever prevented by lack of oxygen (295), and the same is probably true of invasion by vascular pathogens and decay fungi. The number of fungi usually falls off at lower soil depths (31, 45, 147), but this may be caused more by high carbon dioxide 24 CULTIVATION AND GROWTH than by low oxygen (46). The adverse effect of flooding the soil on the growth and survival of fungi is, however, believed to reflect a deficit in oxygen (287, 288, 289). Very high oxygen pressure may somewhat reduce growth or change the mycelial habit, but does not usually have a lethal effect (159, 328). Many discussions of aerobiosis in the fungi have confused growth and fermentation. Numerous fungi ferment sugars anaerobically (Chapter 7); this capacity has no essential bearing on growth, and these forms should not be referred to as anaerobes. The term an- aerobe, in fact, cannot be applied to any fungus; all require at least some oxygen and none has as yet been reported to grow better at low than at atmospheric oxygen pressure, although reducing substances favor growth of Armillaria mellea (148). The vigorous anaerobic fermentation of some aquatic phycomycetes (Chapter 7) is coupled with a low oxygen demand and this relation is presumably a causal one. 9. CARBON DIOXIDE AND GROWTH Growth stimulation by carbon dioxide is considered later, with carbon dioxide fixation (Chapter 7). High carbon dioxide pres- sures generally inhibit the growth of fungi but the level at which inhibiton appears is quite variable. Thus, Alternaria solani is mark- edly inhibited by 38 mm of carbon dioxide (159) and Coniophora cerebella by 23 mm (345), but many other fungi are only slightly retarded in growth by carbon dioxide pressures of 150 mm or more (12, 69, 117, 295). An isolate of Zygorhynchus vuilleminii found only below the soil surface is retarded about 35 per cent by 152 mm of carbon dioxide, while the surface soil inhabitant Penicillium nigri- cans is completely inhibited under the same conditions (46). Finally, we may mention Blastocladia pringsheimii, which grows well in an atmosphere of tank carbon dioxide and which requires carbon dioxide for the formation of resistant sporangia (85). 10. WATER AND GROWTH The association of moisture and fungal growth is well known in relation to the deterioration of natural and manufactured products. Fungi require relatively high moisture levels, but most of the higher fungi can grow in the absence of liquid water. WATER AND GROWTH 25 Two related types of study concern us here: the effect of relative humidity on growth and the effect of osmotic pressure on growth. Both presumably measure the ability of the organism to draw water from its environment. Spore germination per se is considered else- where (Chapter 12); but it should be noted that experimental designs used in growth studies do not fully discriminate between spore germi- nation and mycelial development. Relative humidity effects must, of course, be determined by grow- ing the organism on a substrate which is in equilibrium with the atmosphere. Agar and other solid substrates — bran, wood blocks, fabrics, etc. — have been used. Unfortunately, quantitative determina- tion of growth on such materials is difficult, and the data are rather imprecise. This imprecision makes it hard to decide whether rela- tive humidity itself or the moisture content of the equilibrated sub- strate is the more important factor in fungus growth. Data can be cited for each view (21, 277). Probably the amount of water in the substrate is the more important: thus if cotton and wool are incu- bated at 92 per cent relative humidity only the wool, which absorbs more water at this humidity than does cotton, supports growth of fungi (21). The influence of moisture on fungal deterioration of economically important materials has been studied intensively and reviewed often (13, 184, 271, 277). Fungi tolerant of low humidity (85 to 90 per cent) include Steream frustulosum (13), Schizophyllum commune (131), and Aspergillus spp. (163, 276). A few fungi attack substrates at as low as 65 per cent relative humidity (276), but most are limited to much higher humidities, 95 per cent or more. Tolerance to low relative humidity appears, as expected, to be associated with tolerance to high osmotic pressure (131, 157); this relation is especially clear in spore germination experiments (Chap- ter 12). Species in which spore germination occurs at low humidity also tolerate a low humidity during growth (321, 323), and it is reasonable to infer that spore germination is more affected by marginal water supply than is mycelial growth (329). Studies on the role of osmotic pressure are always complicated by possible non-osmotic effects of the solute. Sodium chloride is dis- tinctly inhibitory to some fungi (207); other complications are dis- cussed by Brancato and Golding (29) and Stuart (290). Tolerance of high osmotic pressure is specific. Thus, a mutant of Neurospora crassa is inhibited at 0.17 M glucose (139); at the other extreme, species of Aspergillus can grow in media with an osmotic pressure of as high 26 CULTIVATION AND GROWTH as 100 atmospheres (28, 282). Most fungi cease growth or are markedly inhibited when the concentration of soluble sugar exceeds about 2.0 M (109, 129, 159). Obligate halophily — an absolute salt requirement — has been re- ported for several fungi (194, 225, 315, 316), although at least some marine fungi do not require salt (8). Further quantitative studies on halophily in marine fungi would be of great interest. Protoplasm is presumably always more concentrated than the ex- ternal solution, and limited data support this assumption for the fungi (35). The indications are that the internal concentration, as measured by freezing point depression or by plasmolysis methods, increases as the external concentration is raised (195, 225, 231). In Aspergillus niger the relative amount of bound water is greater when the organism is grown in concentrated glucose (304). Ohtsuki (225), relying on the cryoscopic method, reports an osmotic concentration in Aspergillus glaucus var. tonophilus equivalent to approximately 250 atmospheres, i.e., the freezing point of the expressed cell sap was -21°C. Thatcher (296, 297), using plasmolytic methods, found that the osmotic pressure of plant pathogenic fungi exceeds that of their hosts and may be a factor in parasitism. Osmotic pressure of hyphae was found in this study to be 15.5-41.3 atmospheres, that of haustoria of rust fungi to be 18.6-21.9 atmospheres. Fungi are more active at moderate than at high soil moisture (287); this is presumably to be ascribed to the effect of excessive soil water on aeration. 11. BIOLUMINESCENCE The phenomenon of bioluminescence in organisms generally is re- viewed by Harvey (125); Wassink (324) critically reviews its occur- rence in fungi, and the biochemistry of the process is summarized by Johnson et al. (153). Bioluminescence occurs in at least 17 species of the basidiomycetes, possibly in one ascomycete, and has not been reported from phycomycetes (125, 324). Within a species, both lu- minescent and non-luminescent strains occur in nature (24, 42); in Panus stipticus luminescence is governed by a single gene (190). Lumi- nescence occurs in sporophores, rhizomorphs, sclerotia, and vegetative hyphae (42, 125, 241). As Robert Boyle discovered almost three cen- turies ago, oxygen is necessary; modern work shows that the oxygen re- quirement for fungal luminescence is much higher than that for bac- terial (128). The reaction is temperature sensitive and is reversibly BIBLIOGRAPHY 27 Figure 7. Emission spectra of bio- luminescence in Photobacterium fisch- (left) and Armillaria mellea en (right). Redrawn from Spruit-van der Burg (279), by permission of El- sevier Publishing Company, Amster- dam. 500 Wavelength, m/x 600 inhibited by narcotics (42). Active extracts have not as yet been pre- pared from fungi; by analogy with other luminescent organisms, we may safely assume that the process is enzymatic. 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II, 97: 222-244. 2. The Composition of Fungus Cells u T nder this head are considered those constituents which as a class if not individually are common to most or all fungi. Materials which are found only in some species are discussed, if sig- nificant, in the section on metabolism; thus, sugar alcohols and carote- noids do not occur in the mycelium of all fungi and so are not included here, but are considered in, respectively, Chapters 5 and 6. Known vitamins produced by fungi are reviewed in Chapter 10. The theoretical significance of chemical composition is somewhat limited. It will be seen that, within bounds set by the metabolic capacities of the species, the composition varies widely with the en- vironment. The quantities of fat, carbohydrate, ash, wall material, and total nitrogen are all more or less responsive to the culture me- dium. In composition, the principal difference between fungi and bacteria is in the high chitin content of most fungi. Chitin is present in the bacteria (176); the green algae do not have chitin walls. Absence of chitin in the Oomycetes, discussed below, in turn suggests a polyphy- letic origin of the true fungi. A second difference between fungi and bacteria is also quantitative rather than absolute: lipid accumulation is more common and attains higher levels in the fungi than in most bacteria. Mycobacterium and related forms, however, are exceptions to this rule and may have a rather high lipid content (130). 35 36 THE COMPOSITION OF FUNGUS CELLS 1. THE MAJOR CONSTITUENTS OF THE CELL Growing fungus cells are, of course, largely water; early measure- ments indicate that 85-90 per cent of the fresh weight of mycelium and fleshy sporophores is water. Presumably the more leathery sporo- phores and sclerotia have less water (25, 197). The conidia of most fungi appear to have a relatively low water content, e.g., 17.4 per cent in Aspergillus oryzae (171), 25 per cent in Monilinia jructicola, and 6 per cent in Penicillium digitatum (195). It is probable, as suggested by Yarwood (195), that most of the water in these spores is hygroscopic. Their water content is largely, therefore, a function of the environ- mental humidity; this may explain some of the rather high values re- ported in the early literature. Species of Erysiphe are unusual in that the conidia have a high water content (195, 196). With this is associated the ability to germinate at low relative humidity (Chapter 12). The bound water of Aspergillus niger mycelium, defined as water which is removed by heat but not by in vacuo drying over a desiccant at moderate temperatures, amounts to 7.1-8.4 per cent of the dry weight when the fungus is cultivated on 5 per cent glucose; at higher glucose concentrations the bound water rises to as much as 30 per cent of the dry matter (182). The elementary composition of fungi has been studied chiefly with respect to carbon and nitrogen. The carbon content of a number of fungi studied by Heck (78) was 40-44 per cent of the dry weight; Pinck and Allison (128) report a somewhat wider range — 43 to 53 per cent. Aspergillus oryzae mycelium is about 49 per cent carbon (173), and A. niger mycelium about 46 per cent (129), but other species of Aspergillus may be as high as 55 per cent (194). Most fungi are 40-50 per cent carbon, and the value is relatively little affected by cultural conditions (173, 194) or age (126). Bacteria and viruses in general have carbon contents of the same order as fungi (130). The mycelium of Streptomyces griseus has slightly less carbon — about 38 per cent of the dry weight (58). In contrast to the relative uniformity of carbon content, the total nitrogen of fungal mycelium is quite variable; both the kind and the concentration of the nitrogen source influence the composition. The major constituents of the mycelium of several fungi are listed in Table 1. These data cannot be considered typical of all fungi, since it is known that at least four of the fractions — carbohydrate, protein, lipid, and ash — are strongly affected by cultural conditions ydrate Chitin Protein Lipid Ash 9.00 — 22.75f 2.20 5.33 34.4 8.4 21.2$ 22.4 — — — 38. 8f 9.72 5.52 60.0 10.6 16.4J 1.4 — 45.0 5.5 12.0§ 34.5 — 32.42 — 23.94f 4.49 6.24 MINERAL CONSTITUENTS 37 Table 1. Major Constituents of Cultivated Fungi and Actinomycetes* Carbo- Organism Aspergillus oryzae (spores) (171) Gliocladium roseum (157) Rhizopus japonicus (99) Penicillium chrysogenum (157) Penicillium griseojulvum (165) Phymatotrichum omnivorum (42) * As per cent of dry weight. t Estimated from total nitrogen only. \ Estimated from total nitrogen after subtraction of chitin nitrogen. § Determined in a chitin-free fraction. and differ in amount from one species to another. As discussed below, those protein values of Table 1 which are based only on the total nitrogen are almost certainly high. 2. MINERAL CONSTITUENTS OF THE CELL The ash content of fungi varies with the medium used and, pre- sumably, with species. Some figures reported for total ash as per cent of dry weight are: Rhizopus japonicus, 5.52 (99); Aspergillus niger, 2.39 (129); A. oryzae, 7.19 (78); Coprinus radians, 4.97 (78); Penicil- lium notatum, 28.3-34.0 (50). Fruiting bodies collected in the field may be as low as 1.08 or as high as 29.8 per cent ash; most have 5-10 percent (197). The composition of the ash of a few fungi is summarized in Table 2. Although no figures should be considered typical, it is clear that the most abundant minerals are phosphorus and potassium. Probably none of the analyses of Table 2 is complete, and inspection raises some obvious problems, e.g., the accumulation of silicon in Rhizopus japoni- cus and aluminum in Penicillium notatum. The total ash content and the concentration of individual elements are influenced by the environment and by the stage of development. In Aspergillus niger the total ash of conidia is about three times that of mycelium; the increase is confined, however, to a few of the ele- ments, particularly phosphorus, potassium, and magnesium (5, 139). Autolysis is accompanied by loss of phosphorus (Gl) and by a decline in total ash (100). Among the environmental effects, the simplest is the increase in the internal concentration of an element in the my- celium as its external concentration is raised (62, 139, 144, 164). The total ash and the concentration of individual elements are also influ- 38 THE COMPOSITION OF FUNGUS CELLS Table 2. The Mineral Constituents of Fungi* Rhizopus Penirillium Trametes Morchella lineral japonicus notation suaveolens esculenta (99) (50) (198) (94) K 27.3 4.52 31.52 63.07 Na 12.9 7.89 0.46 0.38 Ca — 26.72 22.57 1.75 Mg 1.42 8.56 6.07 1.77 P 28.7 25.01 4.64 26.13 S 0.28 2.17 19.06 1.78 Si 21.2 0.63 CI — 0.5 0.52 1.37 Al — 20.62 0.26 — ■ Fe 1.84 4.52 — 2.00 Zn 0.18 — — — * As per cent of total ash. enced, probably indirectly through effects on growth or pH, by the type of nitrogen source (64, 143, 194) and by the concentration of nitrogen (78). A low nitrogen supply may be expected to act directly on phosphorus content by restriction of the synthesis of nucleic acids (63). _ It is assumed that only phosphorus, sulfur, and chlorine occur in fungi in organic combination; an attempt to isolate organic com- pounds of magnesium failed (143). Phosphorus is, of course, present in several types of organic molecules: sugar phosphates and adenosine phosphates (22, 92, 113), phosphoproteins (107), nucleic acids (63), and phospholipids (p. 47). None of these has been investigated thoroughly in the fungi. The inorganic phosphate of fungal mycelium includes orthophos- phate and inorganic condensed phosphates (151). Pyrophosphate occurs in Aspergillus niger (106), but most attention has been directed to the metaphosphates, polymers of the general formula (NaP03)n. Volutin, a common visible intracellular inclusion, is probably meta- phosphate (85). Metaphosphates have been found in cells of Asper- gillus niger (96) and, possibly, Neurospora crassa (83). The metaphos- phate of A. niger has a molecular weight of 6000-7000 (86). Fungal enzymes which hydrolyze these condensed inorganic phosphates are considered briefly in Chapter 9. Utilization of the bond energy of these compounds to drive synthetic reactions, although not impossible in principle, has not as yet been demonstrated. THE CELL WALL 3. THE CELL WALL 39 The most serious defect in our knowledge of cell wall composition in the filamentous fungi is the fact that no "pure" wall material is yet available. The major sources of error are the inclusion of extraneous substances in the preparation, and the loss of wall material during purification. We do not have, therefore, for any fungus even a quali- tative picture of the chemical components of the wall. This section, consequently, can only review work in which one or the other major constituent has been studied. When we speak of a chitin wall, we mean a wall with detectable chitin, not necessarily or even probably a wall which is exclusively chitin. Morphologically, in fact, the wall may be a multiple structure with layers of different composition (15, 46). In most fungi the basic material of the cell wall is chitin, a polymer of AT-acetylglucosamine, identical chemically with the chitin of arthro- pods. The history of the study of chitin in fungi is reviewed by Schmidt (152) and by Frey (56). Much confusion exists, however, because of the use of methods which are not critical. The most ac- ceptable method of qualitative identification of chitin is determina- tion of the X-ray diffraction pattern (56, 66, 79, 87, 91). However, if chitin is present only in small amounts, the X-ray method may not reveal it. Other methods — microchemical tests, isolation of derivatives from a hydrolyzate, and determination of the nitrogen in a purified preparation — are usually in agreement with X-ray results, but are not sufficiently discriminating to stand by themselves. Microchemical methods for chitin and cellulose are particularly unsatisfactory (56). The quantitative determination of chitin requires purification, usu- ally by the method of Scholl (153), and determination of the nitrogen of the product. In a survey of 25 fungi, chitin contents from 2.6 to 26.2 per cent of the dry weight have been reported (18); other quanti- tative data are found in Table 1 above, and in the paper of Schmidt (152). The possibility that some protein nitrogen, from resistant pro- teins, is included in these analyses cannot be ignored. On the basis of microchemical tests for chitin, von Wettstein (186) proposed that large taxonomic groups of the fungi differ in the consti- tution of the cell wall. Specifically, he found chitin in the Zygomy- cetes and in the higher fungi — ascomycetes and basidiomycetes; repre- sentative Myxomycetes, Oomycetes and Monoblepharidales were found not to form chitin. This supports a polyphyletic origin of the phycomycetes. o i— r-~ cs o CM o o a, £ o U 5 3 2 S .Va S a a a '**5 00 "3- "3- . ! X- 1 , *> 00 cO CM C> I © 1 3; CO CO C3 - CU H u 3 H Cs •-5 3 \0 Ca O O CO r, o !-i c u 03 u CJ — V -1 T> u o -a V o o +-* — tn ■ — < !h B >- S 3 r-- © o o CO CM r-~ o ca o o to CM CO O CM *-* ©' r-" ©' Q o o 0 c C3 C3 O en CD -a ,u •*— t > • O O O s~' -a .a s S 2 CO < o c CO o O o T3 CJ • — o o o in £2 o .y o Si OJ u o c DC o o X i-) 33 o c 'o ^ c u a 8 O TJ crj O 0 c o u V "D cu s .a -3 33 0 J o c CJ C3 O CJ TJ O o C .2 CJ tC C o a ei CO '- o >. "c3 ■*-» O V o ~ o S CO O o V 'c V T3 ' C V en en Q O 2 ° O S s «j 33 <5 J H 48 BIBLIOGRAPHY 49 occurrence and the chemistry of the cerebrins are reviewed by Celmer and Carter (24). Virtually all fungi appear to form sterols. The amount in myce- lium is usually about 1 per cent or less of the dry weight. Compre- hensive surveys have been made by Pruess and others (132-134): the sterol content was found to be affected by the growth medium, the highest level attained being 1.70 per cent of the dry weight, in Paeci- lomyces varioti. Different isolates assigned to Aspergillus niger vary in sterol content from 0.23 to 1.16 per cent (12). A more recent survey (4) finds that the sterol content of fungi ranges from zero, in Colletotri- chum graminicolum , to 2.2 per cent of the dry weight, in Penicillium ivestlingii. The principal sterol of fungi is ergosterol; a dihydro derivative, fungisterol, occurs in ergot (174). Ergosterol occurs as the palmitate in Penicillium spp. (121) and Aspergillus fumigatus (187). Other sterols have been only tentatively identified (70, 71, 146). 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PROBLEMS OF METHOD Carbon compounds serve two essential functions in the metabolism of fungi, as of all heterotrophic organisms. They supply in the first place the carbon needed for the synthesis of the compounds which go to make up the living cell — proteins, nucleic acids, cell wall ma- terials, reserve foods, and so on; in a typical fungus about 50 per cent of the dry weight is carbon (Chapter 2). Second, the sole source of appreciable amounts of energy is the oxidation of carbon com- pounds, which may account for half or more of the carbon supplied to a culture. Knowledge of carbon nutrition, therefore, is fundamental to an understanding of the physiology of the fungi. Much valuable work has been done in this area, but it will be apparent that the data re- quired for confident generalization are usually still lacking, and that much of our knowledge is based on experiments which are open to criticism on the ground of method. The study of the carbon nutrition of fungi, or of other microorgan- isms, involves general problems of two sorts: conceptual and meth- odological. The concept of utilization may have more than one operational meaning. The commonest usage describes an experiment in which a small inoculum is placed in a medium containing one major source of carbon and all other materials necessary for growth; the organism 55 56 CARBON NUTRITION must initiate and maintain growth at the expense of the test com- pound. A second, more narrow, meaning of utilization is the ability of viable active mycelium to make further growth at the expense of a test compound. Here, the design usually involves providing a small amount of a utilizable carbon source and measuring the additional growth, if any, obtained from a second compound. It is often found that materials which are not by themselves satisfactory carbon sources are used in this kind of experiment; examples in particular fungi in- clude lactose (135), sugar alcohols (150), and cellobiose (142). As discussed later, a compound which is used only in association with another carbon source is probably dependent for its metabolism on the induced synthesis of an adaptive enzyme system or upon the oc- currence of mutants possessing the enzyme system. So long as these two experimental designs are not confused, each has its place. Confusion, however, does arise if an author proposes to use the first design — one compound as the sole source of carbon — but includes other carbon sources in the medium, either as complex ex- tracts or as amino acids. Unless the additional carbon is negligible, and this must be shown by control experiments without carbohydrate, there is the possibility that the experiment measures utilization in its second meaning rather than the one intended. Fortunately, most fungi can be grown in a synthetic medium with an inorganic nitrogen source and with pure growth factors at levels which do not contribute significant amounts of carbon. There are several methodological errors which are common enough to deserve comment. A cluster of such errors is associated with acidity. The initial pH should be determined after sterilization, to guard against the possibility that compounds in the medium are broken down by autoclaving. Some compounds, particularly organic acids, may not enter the cell at the pH chosen, in which case it is necessary to deter- mine whether or not the organism can tolerate a pH at which pene- tration is more likely. Finally, the organism itself may produce or- ganic acids which in a poorly buffered medium lower the pH so far as to restrict growth. Media may be buffered with phosphate for most organisms; for those fungi which cannot tolerate the level of phosphate required for effective buffer action (45), the pH may be controlled by addition of sterile solid calcium carbonate after autoclaving or by in- clusion of an indicator and periodic adjustment of the pH with sterile alkali (58). The only way in which these problems can be detected in a particular instance is to determine the culture pH at frequent inter- vals and to exercise judgment in the interpretation of growth results which may be affected by pH changes. PROBLEMS OF METHOD 57 It is possible that metabolites other than acids may accumulate in inhibitory concentrations; for example, the accumulation of ethanol has been suggested (181) as the explanation of thiamine toxicity to Rhizopus suinus. Another set of problems is associated with contamination of sup- posedly pure chemicals with growth-active substances. There is evi- dent in the literature an unjustified confidence in the label placed on a chemical by the manufacturer. Even compounds rated as "reagent" quality or the equivalent may be contaminated with biologically sig- nificant amounts of other substances, particularly inorganic ions and organic growth factors. Such contamination is particularly likely in a compound derived from natural sources, and most of the important carbon and organic nitrogen sources are so derived. In a highly puri- fied medium one source of carbon may appear superior to another merely because it is contaminated with a required inorganic element in which the basal medium is partially deficient. More commonly, the fungus has an unrecognized vitamin requirement which is met by the less pure of a series of carbon compounds; thus, the early and anomalous finding that Diplodia macrospora grows with starch, mal- tose, or sucrose but not with glucose or fructose is now known to re- flect contamination of the first group of compounds with biotin, re- quired by the fungus as a growth factor (199, 241). Growth factors for fungi have been found in many carbon compounds, e.g., soluble starch (172), glucose (121), galactose (44), and saponin (19). Growth inhibition by contaminants is less commonly encountered but has been reported from the ions present in crude natural materials (153) and from an unidentified alcohol-soluble contaminant of glu- cose and galactose (44). It should not be necessary to point out that such materials as cellu- lose and chitin as they occur in nature may be contaminated with or- ganic materials in sufficient amount to provide the carbon source for visible growth of fungi. It is not, for example, justified to claim that an organism can utilize chitin merely on the evidence that it grows on the exoskeleton of an insect, particularly since insect cuticle is less than 50 per cent chitin (239). Breakdown of sugars during autoclaving constitutes another serious and often unrecognized source of error in studies of carbon nutrition. Growth inhibition from sugar breakdown products occurs in both bacteria and fungi (9, 116, 193). Breakdown is more extensive if the sugar is in contact during autoclaving with phosphate (59) or with amino acids (86, 125). Ketohexoses, e.g., fructose and sorbose, break down (to 5-hydroxymethylfurfural) more rapidly than do the aldo- 58 CARBON NUTRITION hexoses (138), and furfural formation from autoclaved xylose is suffi- cient to inhibit bacterial growth (120). Autoclaving of carbohydrate in the medium has been reported also to cause the formation of growth- promoting substances for bacteria (36) and fungi (114). Oligosaccharides may be partially hydrolyzed during autoclaving, especially at low pH (7, 22), and probably many claims of slight growth on oligosaccharides reflect merely the partial hydrolysis of a non- utilizable sugar to a utilizable monosaccharide. It follows that sugars and other carbon sources should never be autoclaved in a medium. As a general rule, they may be autoclaved separately and then added aseptically to the cooled basal medium. The expedient of intermittent sterilization is no better than autoclav- ing (136). The ultimate recourse is to filter sterilization, and ques- tionable results with a possibly labile sugar should always be checked with this method. It is usual in carbon nutrition experiments to harvest all cultures after a predetermined, often arbitrarily chosen, incubation period. No objection can be made to this procedure if the only purpose of the experiment is to find a practicable growth medium for a specific pur- pose. If, however, it is intended to yield meaningful information on the intrinsic utilizability of compounds, some error may be introduced. One compound may be used with a very long lag, probably adaptive in nature (p. 86); the classification of this compound as utilizable or non-utilizable will depend on the length of the incubation period chosen. On the other hand, if time is allowed for slow utilization of compounds, growth on rapidly utilized substrates may have been com- pleted and followed by extensive autolysis before the cultures are harvested. Ideally, both these difficulties may be met by planning periodic harvests; comparison of compounds can then be made on the basis of two criteria, maximum growth and the time required to reach maximum growth. The data of Lilly and Barnett (116) show for many fungi the gain in understanding which flows from the use of more than one incubation period. Some additional information, and considerably greater confidence in interpretation, may be gained by analysis of the medium at the time of harvest for residual carbon source. This procedure affords an ad- ditional check upon growth evidence, especially if growth is limited. Differences in results between different laboratories are often casually explained as an expression of strain differences or as the result of variation. There is, of course, no doubt that strains of a given species may differ in their nutritional requirements, and there are authenti- cated cases of metabolic changes resulting from spontaneous genetic PROBLEMS OF METHOD 59 change. Nevertheless, in the absence of adequate evidence, these phe- nomena should not be accepted as sufficient explanation of the failure of one laboratory to confirm the work of another. It is clear that ex- tensive recourse to such "explanation" would mean that no study could be confirmed or contradicted by later evidence. From this review of methodological problems, a number of recom- mendations may be formulated. Although not of equal importance and not necessarily applicable to all cases, they should be considered carefully in designing experiments on the carbon nutrition of fungi. 1. Complex natural products which supply carbon should if pos- sible not be included in the basal medium. 2. A control series with only the basal medium should be included in every experiment. Failure to include such a control makes it almost impossible to detect limited utilization of the test carbon compound, since it is not known to what extent impurities in the basal medium support growth. 3. Adequate replication of cultures is necessary, the number to be determined by the variance found in pilot experiments. Probably for most fungi a minimum of four replicates for each harvest is re- quired. 4. The carbon source should always be autoclaved separately from the basal medium and added aseptically to it. If it is found to be labile to heat, it should be sterilized by filtration. 5. The pH of the medium should be determined electrometrically after all ingredients are added, and periodically during growth. 6. The inoculum should be of minimum size, to avoid carry-over of materials from the parent culture. In doubtful cases, serial sub- culture on the same medium may be necessary. All cultures should be inoculated with the same amount (mass or number) of cells. 7. The measurement of growth must be objective and quantitative, e.g., by dry weight, total nitrogen, or turbidity (Chapter 1). 8. For fungi which have rapid rates of growth and autolysis or which grow adaptively on some substrates it may be necessary to construct a growth curve for each carbon source or at least to make more than one harvest. 9. Analysis of the spent medium for residual carbon source is highly desirable, although not always practicable. 10. Possible interactions between carbon and nitrogen sources should be considered; in some cases it may be necessary to study all putative carbon sources in media with different nitrogen sources. 11. When applicable, the distinction between initiation of growth 60 CARBON NUTRITION and maintenance of preformed growth should be a factor in the inter- pretation of results. 12. Reasonable precautions should be taken against complications arising from (a) contamination of the carbon source with impurities which affect growth, or (b) the formation of toxic metabolites during growth. Hydrogen ion is the most common toxic metabolite, and consequently the most important precaution in this connection is the periodic determination of the pH of the culture during the growth period. The most frequent, and perhaps the least excusable, deficiency in experiments on carbon nutrition is failure to meet the criterion of ob- jective and quantitative measurement of growth. As a rule, it is wise to disregard results which are reported in qualitative terms only, al- though the complete absence of growth in such experiments may on occasion be significant. This stricture applies with almost equal force to the evaluation of growth on the basis of linear extension of a colony on agar; this type of growth measurement has its uses (Chapter 1) but is not sufficiently accurate to assess the relative utilizability of differ- ent carbon sources. 2. MONOSACCHARIDES AND THEIR DERIVATIVES Hexoses. Of the 6-carbon sugars, the hexoses, D-glucose is biologi- cally the most important and is utilized for growth by virtually all cultivable fungi. A few forms have been reported not to grow with glucose; the only convincing evidence so far presented is the qualita- tive observation of Schade (175) that Leptomitus lacteus uses none of the common hexoses but grows with acetate or other fatty acids as the sole source of carbon. This pattern resembles that of the "acetate flagellates," e.g., Polytomella caeca, in which it has been suggested (122) that enzymes necessary for the phosphorylation of glucose are lacking. A similar deficiency in Pseudomonas putrefaciens has the same effect (106). For the great majority of fungi and actinomycetes D-fructose and D-mannose are equivalent to glucose for growth. Occasionally, as in Stachybotrys atra (99), one of these may appear superior to glucose for unknown reasons. Particularly among the lower fungi there ap- pear to be numerous exceptions to the general rule that fructose and mannose are equivalent to glucose. In conventional experiments of limited duration it is found that either or both of these sugars fail to support the growth of several members of the Chytridiales (2, 45) and MONOSACCHARIDES AND THEIR DERIVATIVES 61 Blastocladiales (94, 123). Less exhaustive studies suggest that the same is true of some species of the Saprolegniales (17, 234). Of sev- eral reports of failure of higher fungi to utilize fructose, that of Shira- kawa (184) is the most convincing, although the fructose was auto- claved in the medium. Several isolates of Streptomyces fail to grow in an agar medium with fructose as carbon source (14). Considerable light has recently been thrown on the non-utilization of mannose and fructose through the work of Sistrom and Machlis (185) on Allomyces macrogynus, one of the species of the Blastocladiales previously found not to grow appreciably on these sugars in experi- ments of conventional duration. It is now clear that A. macrogynus can grow on both mannose and fructose with a very long period of incubation; the final growth is comparable to that on glucose. In addition, provision of a small amount of glucose to a medium in which the main carbon source is mannose or fructose allows normal growth with no delay. Both of these observations are consistent with the hypothesis that this fungus — and by implication perhaps others — utilizes fructose and mannose only under conditions which permit or encourage the formation of an "adaptive" or induced en- zyme, although other observations remain to be reconciled with this hypothesis. It would seem, therefore, that utilization of these mono- saccharides is dependent on an enzyme system which is constitutive in some organisms and inducible in others; strains of a given species may fall into different groups in this regard. Data from a comprehensive study of 57 fungal species, mostly of the Fungi Imperfecti (116), show that relatively few — about one-fifth of the sample — are completely unable to grow with D-galactose as carbon source in a medium containing asparagine and salts. Many, especially in the genus Fusarium, grow with galactose as well as with glucose. Other fungi, e.g., Helminthosporium sativum, reach the same final dry weight as with glucose but reach it slowly, suggesting that here too an "adaptive" mechanism is at work and that the inclusion of asparagine in the medium may have biased the results by providing a substrate able to support the formation of induced enzymes. Finally, for many fungi galactose is clearly utilized but does not even in a long time period support the same level of growth as does glucose. We may conclude from this and other work that galactose is used by most fungi but is not usually so good a source of carbon as glucose. A sub- stantial number of fungi are unable to utilize it, and it may even be toxic to a few (90, 194). The ability or inability to utilize galactose bears no apparent relation to taxonomic position. On comparative grounds, it seems probable that the ability of an 62 CARBON NUTRITION organism to use fructose, mannose, or galactose depends upon its ability to convert the sugar in question into a phosphor y la ted deriva- tive of glucose able to enter the main respiratory pathways. The metabolic relationships of these lour sugars in yeast and animal cells are shown in the diagram (67): Polysaccharide Galactose Jt I Glucose-1 -phosphate ^ Galactose-1 -phosphate Glucose — -> Glucose-6-phosphate ;=± Fructose-6-phosphate ! I if. i ! si Mannose — * Mannose-6-phosphate Fructose From this diagram it may be suggested that in adaptive growth on fructose, mannose, or galactose, the enzyme actually lacking in the cell and induced by the environment is the specific kinase responsible for the phosphorylation of the free sugar. However, no data are available and other possibilities and other pathways are of course conceivable. The occurrence of mannose- 1 -phosphate in the mycelium of Penicillium chrysogenum (105), for example, suggests a pathway of mannose utilization similar to that of galactose in the scheme outlined above. Studies on the economic coefficient (p. 89) and similar indices of growth indicate that poorly utilizable monosaccarides are such because they are not attacked or do not penetrate the cell, rather than because once attacked they do not provide energy or usable metabolic products. This is especially clear from experiments with Aspergillus niger, for which the small amount of a poor carbon source which disappears from the medium is as efficient in supplying carbon for growth as an equiva- lent amount of glucose (208, 209). The sugars which do not support growth adequately remain, that is, in the medium. Whether this is the result of specific enzymatic deficiencies as suggested above or whether it is to be attributed to slow penetration of the sugar remains to be determined. The two possibilities are not necessarily mutually exclusive if penetration of sugars depends, as it may, on a surface enzymatic reaction. It is perhaps significant that the yeast cell is im- permeable to sorbose, galactose, and arabiriose (171), all of which are generally poor sources of carbon for microorganisms. L-Sorbose, a ketohexose differing from fructose in the configuration of carbon atom 5, supports normal growth of a few fungi and actinomy- cetes (14, 116, 146). Although unequivocal data are not available, it appears not to be utilizable by most forms and it is definitely toxic to MONOSACCHARIDES AND THEIR DERIVATIVES 63 some. The toxicity is evidenced by death of the hyphal tips, followed by branching of the mycelium below the killed portion (116). Fungi, e.g., Neurospora crassa, which normally spread widely on an agar plate grow as small colonies in a medium with sorbose (204). The fundamental basis of sorbose toxicity is not yet known; although often more pronounced in a maltose medium, toxicity to some fungi is equally well expressed in glucose media, so that interference with maltose utilization cannot be the explanation. Sorbose inhibition oc- curs only at relatively high concentrations and is strongly affected by temperature (116). A possible line of approach to the problem of its mode of action may be found in the hypothesis that sorbose interferes with a respiratory pathway; organisms dependent on this pathway would then be inhibited completely, fungi which have alternate path- ways would be less or not at all affected. Heptoses. The naturally occurring 7-carbon sugars have not been tested as carbon sources for fungi, except for the report (195) that Aspergillus niger does not grow with D-mannoheptulose. Pentoses. D-Xylose is the most generally utilizable of the pentoses, and has even been reported to be superior to glucose for some organ- isms (12, 150, 216). Some fungi and actinomycetes grow poorly or not at all with xylose, and species within the same genus may differ markedly (195, 201). It must be remembered that xylose is known to be broken down to furfural during autoclaving, for which reason all reports of its non-utilizability should be regarded with some reserve until confirmed with filter-sterilized xylose. Phymatotrichum omniv- orum makes better growth with alcohol-sterilized than with auto- ciaved xylose (18), but the poor growth of Venturia inaequalis strains on agar with xylose is not improved by filter sterilization of the sugar (113). Both isomers of arabinose occur in nature and are commercially available. Probably, in those publications in which the isomer used is not specified the L-isomer, which is more easily available, was used. Penicilliurn urticae utilizes D-arabinose much less well than it does L-arabinose (57). In most studies, L-arabinose is inferior to xylose and glucose as a carbon source (116a). In a medium containing a second carbon source, however, several fungi, e.g., Endoconidiophora adiposa, utilize L-arabinose with a long incubation period, apparently adap- tive!}' (116). Relatively few studies on rhamnose, a methyl pentose, have been made, but the indications are that it is used by only a few organisms, including StacJiybotiys atra (99) and Streptomyces coelicolor (39). 64 CARBON NUTRITION Although Penicillium urticae grows with D-ribose as carbon source (57), it and the other naturally occurring pentoses and methyl pentoses have not been investigated thoroughly. Early studies, not all satis- factory in their methods, on pentose utilization by fungi are reviewed by Hawkins (80) and by Peterson et al. (155). It seems, in conclusion, that xylose is often a good source of carbon for fungi and that contrary findings should be examined closely. The pathway of utilization of xylose is not known; it seems likely that it enters the phosphogluconate oxidation pathway (Chapter 7) as pen- tose phosphate. L-Arabinose is used by several fungi, probably not by most. The other pentoses and methyl pentoses have been investigated rather infrequently, but they appear in general to be poor sources of carbon or not utilizable at all. Other Monosaccharides. A single study of a 4-carbon sugar (195) indicates that D-threose is not utilized by Aspergillus niger. The L-isomers of the naturally occurring aldohexoses and of xylose do not support growth of fungi, nor do those monosaccharides which do not occur in nature (99, 195, 201). Acetylation or methylation of the ring carbons of glucose yields derivatives which are not utilized by representative fungi (187), although Aspergillus niger has been reported to make a limited growth, after adaptation, with pentaacetyl- glucose (8). Aspergillus niger also grows with 2-deoxyglucose as car- bon source (8). Polyhydric Alcohols. Reduction of the sugars yields sugar alcohols, e.g., D-sorbitol arises by reduction of D-glucose, D-mannitol by reduction of D-fructose or D-mannose. In addition to the hexitols, derived from the hexose sugars, tetritols, pentitols, and heptitols, related respec- tively to 4-, 5-, and 7-carbon sugars, occur in nature. Glycerol may be considered as a reduction product of a 3-carbon sugar. Mannitol is the most generally utilizable of the hexitols, and is equivalent to glucose for many fungi and actinomycetes (39, 100, 144, 180). However, many fungi with generally wide substrate ranges grow only poorly or not at all with mannitol as sole carbon source (65, 129, 152). In the lower phycomycetes, a few forms grow poorly with mannitol (17, 32); most do not utilize it at all. Sorbitol and dulcitol are unavailable to most fungi and actinomy- cetes. From the limited data, it appears that sorbitol, the reduction product of glucose, is more generally used than is dulcitol, which is related chemically to galactose. It may be significant that many or- ganisms able to utilize dulcitol are also able to grow more or less well MONOSACCHARIDES AND THEIR DERIVATIVES 65 with galactose (17, 180, 201). Erythritol is apparently only rarely utilizable, although the data again are extremely limited. Glycerol is often the best carbon source of all for actinomycetes (40, 43, 221). In the fungi the ability to utilize glycerol appears to be specific; thus, among fairly omnivorous saprophytes, Aspergillus oryzae and A. niger make only poor growth (195, 201), but Memnoniella echi- nata grows well with glycerol as sole carbon source (150). Although the data are insufficient for generalization, it may tenta- tively be concluded that the only polyhydric alcohols available to many fungi are glycerol and mannitol. These may be expected on oxidation to yield, respectively, fructose and glyceraldehyde or di- hydroxyacetone, all of which can after phosphorylation enter known respiratory pathways. Sugar Acids. Oxidation of the aldehyde carbon of an aldohexose yields an acid; D-gluconic acid, from D-glucose, is the only one to have been investigated as a source of carbon for fungi. As might be ex- pected from the fact that many strains of Aspergillus niger form and then consume gluconic acid during sugar metabolism (Chapter 6), it is a good source of carbon for A. niger (46) and A. oryzae (201), al- though the strain of A. niger studied by Steinberg (195) could not grow with gluconate as sole carbon source. Several other organisms grow on gluconate (40, 99, 121), but others, e.g., Chalara quercina, do not utilize it (12). Growth with gluconate is probably often limited by an unfavorably high pH resulting from utilization of the neutralized acid. Utilization of 2-ketogluconic acid by fungi is, like that of gluconic acid, highly specific (50) and is probably a function of the respiratory systems available. A single report (87) indicates, without quantitative data, that Asper- gillus niger grows with D-glucuronic or D-galacturonic acids as sole car- bon source. D-Saccharic acid, the dibasic acid resulting from the oxidation of carbon atoms 1 and 6 of D-glucose, has been reported a good source of carbon for Aspergillus niger (46), but a relatively poor source for A. oryzae (201). Glycosides. Limited studies of the methylglucosides indicate, as would be expected from the relative ubiquity of /3-glucosidases (Chap- ter 5), that /?-methylglucoside is much more easily utilizable than the ^-isomer (54, 180). Amygdalin, which is hydrolyzed to gentiobiose and rnandelonitrile, is utilized by several members of the Saprolegnia- 66 CARBON NUTRITION ceae (17); in general it may be expected that the ability of an organism to use a given /^-glycoside will depend on its ability to use the sugar which is split off from it. The cyanogenetic glycosides, however, may not be utilizable by reason of the toxicity of the cyanide liberated on hydrolysis (174). 3. OLIGOSACCHARIDES Chemically, the oligosaccharides are glycosides composed of two or more monosaccharides and are characterized by the nature of the com- ponent sugars, the number ol units, and the type of glycosidic bond. Those which are important in the nutrition of fungi include five disaccharides — maltose, cellobiose, trehalose, sucrose, and lactose — and one trisaccharide, raffinose, which is made up of galactose, glucose, and fructose units. In addition to data on specific sugars, two general problems are relevant at this point: (1) the enzymatic basis of disac- charide utilization, and (2) adaptive growth with oligosaccharides. Maltose occurs in nature as a product of starch hydrolysis and is utilized by virtually all fungi which have been tested, even those which have a limited substrate range. A few fungi are known, on the basis of quantitative studies, to grow poorly or not at all with maltose: Chytridium sp. (45), Polychytrium aggregation (2), Entomophthora (Empusa) sp. (242), and Penicillium digitatum (65, 244). Surprisingly, the two chytrids mentioned make at least some growth with starch; the other fungi use neither starch nor maltose. Maltose is more easily hydrolyzed by autoclaving than are sucrose or lactose (136), but even with this possible source of error it seems that it is very generally available to fungi. Cellobiose, like maltose, is composed of two glucose residues but the linkage is of the /^-configuration. The data are more limited than those for maltose, but indicate that it is almost as widely utilizable as maltose. Indeed, two of the fungi mentioned above which do not grow with maltose do with cellobiose. Tricholoma flavobrunneiim is among the small group of tested fungi which fail to utilize cellobiose, but other species of the same genus grow well with it as sole carbon source (142). The utilizability of cellobiose reflects the general oc- currence of /3-glucosidases in fungi (Chapter 5). Trehalose, a glucose-containing disaccharide produced by fungi and a few higher plants (Chapter 5), appears from the few studies which have been made to be generally available, even to rather fastidious fungi (40, 99, 201, 242). Sucrose, the characteristic sugar of higher plants, is generally a good OLIGOSACCHARIDES 67 source of carbon for fungi but is not so nearly universally available as maltose. Thus, of 57 fungi studied by Lilly and Barnctt (116) only 4 were completely unable to utilize sucrose, but several grew slowly with it. Among the major groups of fungi, failure to utilize sucrose appears to be particularly common in the Chytridiales (Table 1) and the Mucorales (174), less common in other groups. Actinomycetes rarely, if ever, utilize sucrose (13, 221). Lactose, the /3-galactosido-glucose of mammalian milk, is used by far fewer fungi than any of the disaccharides so far mentioned, and may be characterized as a poor carbon source (61, 116). At least some actinomycetes grow with lactose (40, 51, 215). Adaptation to lactose occurs among fungi (p. 87), and whether or not an organism is re- ported as able to utilize the sugar often, therefore, depends on the length of the incubation period chosen. Melibiose, an a-glycoside, has not been investigated often; the few data available (Table 1) indicate that it is not utilizable by many fungi. Data for gentiobiose are similarly too limited to allow general- ization. Growth on amygdalin, the /^-glycoside of gentiobiose, by aquatic fungi (17) presumably indicates gentiobiose utilization. Raffinose attacked at one linkage yields galactose and sucrose, at- tacked at the other yields fructose and melibiose. From the limited data of Table 1 and the more extensive studies of Lilly and Barnett (116), it would appear that the majority of fungi utilize raffinose, but that non-utilization is common. Some doubt is raised with regard to raffinose utilization by the finding (18) that raffinose supports growth of Phymatotrichum omnivorum if autoclaved but not if sterilized by alcohol treatment; a reinvestigation of this problem by comparing filter-sterilized to heat-sterilized raffinose may show that raffinose is broken down by autoclaving at least to the extent that some simple sugars are available to initiate growth and to permit, therefore, the formation of adaptive enzymes which then hydrolyze the unchanged raffinose. The utilization of an oligosaccharide is believed to be preceded by and dependent on its conversion to hexoses or, possibly, hexose phos- phates. The evidence for this generalization may be summarized: 1. Fungi that can use a disaccharide possess enzymes which hydro- lyze it (17, 79). An organism which grows on sucrose, for example, proves on examination to form sucrase. Formation of the enzyme is not, however, a sufficient condition for utilization — a mutant of Neurospora crassa produces lactase (/3-galactosidase) but does not grow with lactose as sole carbon source (112). Chytridium Macrochytrium Polychytrium Allomyces sp. botrydioid.es aggregation javanicus (45) (45) (2) (94) 4 1 0 0 100 100 100 100 101 1 0 0 101 120 109 99 23 0 0 8 1 7 108 0 0 4 1 0 gg CARBON NUTRITION Table 1. Carbon Nutrition of Some Fungi and Actinomycetes* Carbon Source None Glucose Fructose Mannose Galactose Sorbose Xylose L-Arabinose D-Arabinose Ribose Rhamnose Mannitol Sorbitol Dulcitol Adonitol Erythritol Glycerol Maltose Sucrose Cellobiose Lactose Trehalose Melibiose Raffinose Glycogen Starch Dextrin Inulin Cellulose Formate Acetate 0 < 3 Propionate Oxalate Lactate 17 9 0 10 116 0 103 9 4 0 0 100 137 0 3 4 0 0 5 2 5 2 0 0 94 194 50 106 + + 3 3 3 + 0 0 Succinate 0 <3 0 Fumarate Malate Tartrate 0 < 3 Citrate 0 < 3 Gluconate * All data adjusted by taking growth on glucose as 100. Organisms arranged in order: phycomycetes, ascomycetes, basidiomycetes, Fungi Imperfecti, acti- nomycetes. Tr = trace of growth; + = visible growth, not measured, OLIGOSACCHARIDES 69 Table 1 (continued) Carbon Source None Glucose Fructose Mannose Galactose Sorbose Xylose L-Arabinose D-Arabinose Ribose Rhamnose Mannitol Sorbitol Dulcitol Adonitol Erythritol Glycerol Maltose Sucrose Cellobiose Lactose Trehalose Melibiose Raffinose Glycogen Starch Dextrin Inulin Cellulose Formate Acetate Propionate Oxalate Lactate Succinate Fumarate Malate Tartrate Citrate Gluconate Allomyces javanicus Blastocladia Brevilegnia Saprolegnia macrogynus pringsheimii gracilis delica (123) (32) '(17) (17) 0 5 0 0 100 100 100 100 0 88 81 102 0 97 94 0 0 7 86 0 0 7 0 6 0 0 0 7 0 0 0 0 52 0 89 29 0 0 0 0 39 86 14 107 101 88 107 0 99 83 14 0 0 0 5 5 6 61 0 76 0 50 76 93 54 95 76 100 98 87 86 86 0 6 5 40 0 37 5 70 CARBON NUTRITION Table 1 (continued) Pytophthora Pythiogeton Chalara Coprinus Carbon Source cactorum sp. quercina sp. (129) (33) (12) (100) None 0 3 2 34 Glucose 100 100 100 100 Fructose 50 104 119 107 Mannose 38 96 127 Galactose 63 10 115 76 Sorbose Xylose 42 64 215 52 L-Arabinose 55 7 46 56 D-Arabinose Ribose Rhamnose 58 13 Mannitol 63 ±10 13 111 Sorbitol 92 Dulcitol 55 2 Adonitol Erythritol 52 2 Glycerol 102 5 15 Maltose 125 99 133 105 Sucrose 133 101 94 84 Cellobiose 101 119 Lactose 83 36 4 72 Trehalose Melibiose 5 27 Raffinose 63 100 96 Glycogen Starch 125 98 6 112 Dextrin 63 217 120 Inulin 83 77 Cellulose + 23 Formate 0 Acetate 0 Propionate Oxalate 0 Lactate 0 Succinate 0 15 Fumarate Malate 0 Tartrate 0 Citrate 0 Gluconate 0 2 OLIGOSACCHARIDES 71 Table 1 (continued) Psalliota Stereum L'slilago rusanum oxysporum Carbon Source bispora gausapatum violacea f. nicotianae (216) (82) (19) (243) None 12 5 Glucose 100 100 100 100 Fructose 95 104 99 104 Mannose 104 109 Galactose 73 89 63 78 Sorbose 59 Xylose 132 123 3 135 L-Arabinose 109 43 5 68 D-Arabinose 14 Ribose 74 Rhamnose 41 7 39 Mannitol 20 93 41 Sorbitol 90 24 Dulcitol 27 Adonitol Erythritol 7 Glycerol 24 24 48 Maltose 15 160 95 138 Sucrose 68 158 88 113 Cellobiose 78 108 Lactose 11 19 Trehalose 111 Melibiose 23 Raffinose 126 8 120 Glycogen 112 Starch 20 160 10 59 Dextrin 25 105 Inulin 23 70 9 Cellulose Formate Acetate 11 Propionate Oxalate 100 Lactate Succinate Fumarate Malatc 116 Tartrate 32 Citrate Gluconate 72 CARBON NUTRITION Table 1 (continued) Carbon Source None Glucose Fructose Mannose Galactose Sorbose Xylose L-Arabinose D-Arabinose Ribose Rhamnose Mannitol Sorbitol Dulcitol Adonitol Erythritol Glycerol Maltose Sucrose Cellobiose Lactose Trehalose Melibiose Raffinose Glycogen Starch Dextrin Inulin Cellulose Formate Acetate Propionate Oxalate Lactate Succinate Fumarate Malate Tartrate Citrate Gluconate Memnoniella Penicillium Sclerotium echinata digitatum delphinii (150) '(65) (151) 100 100 100 110 108 113 90 108 105 99 145 102 76 105 95 98 90 50 100 170 140 45 35 40 25 10 65 46 85 104 105 21 152 105 92 147 105 101 120 64 45 2 1 21 28 195 191 43 OLIGOSACCHARIDES 73 Table 1 (continued) Carbon Source None Glucose Fructose Mannose Galactose Sorbose Xylose L-Arabinose D-Arabinose Ribose Rhamnose Mannitol Sorbitol Dulcitol Adonitol Erythritol Glycerol Maltose Sucrose Cellobiose Lactose Trehalose Melibiose Raffinose Glycogen Starch Dextrin Inulin Cellulose Formate Acetate Propionate Oxalate Lactate Succinate Fumarate Malate Tartrate Citrate Gluconate Stachybotrys Streptomyces atra coelicolor (99) (40) 17 100 100 241 79 66 100 128 84 0 26 94 143 25 65 22 9 91 100 82 0 27 28 20 25 0 166 135 131 62 78 34 72 81 84 59 90 56 66 107 53 32 113 + 0 22 33 tr. tr. tr. 60 tr. 47 69 3 60 0 20 tr. 24 9 82 74 CARBON NUTRITION 300 200 &0 E x: Dp E c 120 1 i / i / Young — / mycelium — — / /^ Older / mycelium — Vy~~ 1 1 I 1 40 80 120 Time, hours 160 Figure 3. The formation of /3-galactosidase (lactase) by resting mycelium of Neurospora crassa. Arrows indicate time of exposure to the inducing sugar. Redrawn from O. E. Landman, Archives of Bio- chemistry and Biophysics Vol. 52, p. 93-100 (1954), by per- mission of Academic Press, Inc. themselves support growth only after a long lag, may be explained by postulating that the glucose allows sufficient growth so that an enzyme essential to fructose or mannose utilization can be synthesized. Naturally, some energy must be available for the enzyme synthesis; for resting cells endogenous reserves may be adequate, but with a spore inoculum an exogenous energy source may be necessary. Extensive data on the utilization of mixed carbon sources have been reported by Lilly and Barnett (116). Several fungi, e.g., Sordaria fimicola and Thielaviopsis basicola, behave in accordance with the foregoing postulate — they grow on a disaccharide, melibiose for exam- ple, only if a utilizable monosaccharide is available. However, results with Gliomas tix convoluta are not entirely explicable on this basis, unless we postulate, without proof, that the sugar which supplies energy for enzyme synthesis must be present in relatively high concen- tration. These experiments are complicated by the inclusion of asparagine, which supplies some carbon, in the basal medium. The results of Steinberg (194) on the utilization of mixed carbon sources by Aspergillus niger are not satisfactorily explained by the hypothesis of enzyme induction. Here a mixture of two unutilizable compounds supports good growth. It is clear, from these results and from those mentioned in the preceding paragraph, that the interaction of different carbon sources requires further study. EFFICIENCY OF GROWTH 89 In relation to utilization of mixed carbon sources, it should be noted that preferential utilization of one compound is common, e.g., the utilization of acetate before glucose (154) and the utilization of soluble sugars before more complex carbon compounds (G2, 109, 188). Fungi in nature live as a rule in a dilute solution with more than one carbon source available or potentially available. These conditions do not very closely resemble a single-substrate pure culture. Lignin and cellulose, for example, may be much more rapidly decomposed by the action of induced enzymes than they are when given to an organ- ism in culture as the sole source of carbon. The ecological importance of the ability of fungi to synthesize new enzymes — if this ability proves to be general — justifies much more study of the problem. It is to be hoped, however, that such studies will be guided, more than they have been in the past, by the wealth of experience accumulated in the investigation of induced enzymes in other microorganisms. 10. EFFICIENCY OF GROWTH Several indices of the over-all efficiency of growth in fungi have been proposed. Of these, the economic coefficient has been most frequently determined. It may best be represented as: Mycelial dry weight, gm X ioo Carbohydrate consumed, gm Some authors have used the ratio or its reciprocal without multiplica- tion by 100. As an approximate index of the efficiency of carbon utilization, the economic coefficient is satisfactory. More elaborate indices, which take account of energy quantities more directly, have been devised (202, 206, 208, 210). The economic coefficient can be seen by inspection to be maximal when respiratory carbon dioxide and soluble metabolic products are minimal in quantity. Since both of these are affected by cultural conditions, no one value or even range of values can be set down as characteristic of the fungi as a whole. Fungi grown on dilute media for no longer a time than is necessary to utilize the carbohydrate usu- ally have an economic coefficient in the range 20 to 35; a few higher values have been reported (119, 161, 205, 209). Very low values (77, 228, 230) reflect probably the production of significant amounts of soluble carbon compounds in high-carbohydrate media. The economic coefficient, although unaffected by temperature (209), 90 CARBON NUTRITION is strongly influenced by substrate (205), metal deficiencies (29, 158, 167, 182), and nitrogen nutrition (157, 211). The influence of age is variable (28, 155). As one would expect, increasing the glucose sup- plied decreases the economic coefficient (119, 159, 195). All of the factors just listed as influencing the economic coefficient almost certainly act by changing the amount of soluble metabolic products formed; it is a possibility not so far explored that the loss of carbon as C02 is affected, but this seems doubtful. A low economic coefficient should suggest in the first instance a search for soluble carbon compounds in the medium. 11. CONCLUSION The most important single facet of carbon nutrition is the high degree of specificity; differences between species and strains are the rule in the fungi. Nevertheless, a search for some general principle has been and should be pursued. One such is to relate carbon nutrition to phylogeny. It has often been pointed out, e.g., by Garrett (69), that the major groups of fungi differ with respect to cellulose and lignin utilization. The Zygomycetes do not use either of these materials; in the ascomy- cetes and the Fungi Imperfecti, cellulose utilization is common; the basidiomycetes contain most of the known lignin-utilizing fungi in addition to many species able to grow on cellulose. This progression toward utilization of more complex materials would be more impres- sive if the capacity to break down cellulose were not widespread in the Chytridiales and perhaps in the Oomycetes. The phycomycetes, usually conceded to be the most primitive major group of fungi, have certain nutritional and metabolic characteristics which, taken together, separate them rather clearly from the higher fungi : 1. The fungi which produce large amounts of lactic acid from carbohydrate are all phycomycetes (Chapter 6). 2. The fungi able to grow at low oxygen tensions are usually phy- comycetes (Chapter 1). This ability may, of course, be related to the ability to form lactic acid. 3. Requirements for organic sulfur among fungi occurring in nature are known only in the phycomycetes (Chapter 9). 4. The ability to use the lower fatty acids appears to be more com- mon in the phycomycetes than in other groups (p. 81), although the data are still rather limited. CONCLUSION 91 It may be that the appropriate theoretical framework for carbon nutrition is more the ecology than the phylogeny of fungi. The char- acteristics of the phycomycetes outlined above can be understood as related to the aquatic habit of most of the forms so far studied just as easily as they can be interpreted in terms of taxonomy or phylogeny. The carbon nutrition of the mycorrhizal basidiomycetes is another example. The evidence is incomplete and not entirely satisfactory, but it appears that this group is restricted in its carbon nutrition to simple sugars, starch, and the pectic substances (74, 91, 130, 131, 170). These fungi stand, therefore, in contrast to the litter-decomposing and wood-rotting basidiomycetes, which use cellulose, lignin, or both. At least one mycorrhizal basidiomycete, however, does form an adaptive cellulase (142). The order Mucorales is cited often as a group of "sugar fungi" un- able to utilize cellulose (26). This characterization appears to be sound, although relatively few of the Mucorales have been adequately investigated. Ecologically, however, the "sugar fungi" include a num- ber of the Fungi Imperfecti, especially species of Aspergillus and Penicillium, found in soils high in fresh organic matter and in carbo- hydrate-containing materials. The line between "sugar fungi" and cellulose-utilizing fungi cuts therefore across taxonomic boundaries, since different species of Aspergillus, for example, are found in one or the other group. In Table 1 are collected some data on the carbon nutrition of different fungi, although it cannot be said that the organisms chosen are in any way typical. The organisms of wide substrate range in- clude a large group of soil saprophytes and plant parasites — Asper- gillus oryzae, Brevilegnia gracilis, Fusarium oxysporum f. riicotianae, Memnoniella echinata, Penicillium digitatum, Phytophthora cacto- rum, Psalliota bispora, Stachybotrys atra, and Streptomyces coelicolor. In comparison, the aquatic phycomycetes in the list appear more fastidious; the contrast between the soil-inhabiting Brevilegnia gracilis and the closely related aquatic Saprolegnia delica is particularly in- structive. On the other hand, the plant parasite Chalara quercina has a much narrower nutritional range than the aquatic Pythiogeton sp. The most restricted of all in the group are Entomophthora coronata, parasitic on insects, and the aquatic Polychytrium aggregation. The fungi parasitic on animals might be expected, by analogy to bacterial parasites, to be nutritionally specialized. The available data indicate that these fungi are not in fact particularly fastidious with respect to carbon sources (140, 200). One incidental problem may be mentioned here. Throughout this 92 CARBON NUTRITION chapter, it has been assumed that carbon sources are utilized by path- ways involving hexoses or hexose phosphates as intermediates. How- ever, the data of Table 1 afford several instances in which glucose is inferior to some other carbon compound, xylose, gluconic acid, disac- charides, or polysaccharides. None of these cases has been studied. The superiority of xylose or gluconate could mean that the phospho- gluconate oxidation pathway (Chapter 7) is more active than others. Or, a compound may be superior to glucose for accidental reasons — pH relations, accumulation of intermediates, contamination with metals or growth factors, etc. This second explanation has been suggested earlier for the superiority of particular disaccharides and polysaccharides. It must be admitted, however, that the question is still open. Looking at the whole field of carbon nutrition of fungi, one is struck with the gaps in our knowledge. Certain groups — the Mu- corales and the Peronosporales are examples — have been more or less neglected. Comparative studies of species within a genus, particularly species of different habitat, might prove most valuable. The bio- chemical basis of nutritional capabilities is largely a matter of in- ference at present; failure to utilize fructose or mannitol, for example, probably has a simple enzymatic basis. As just discussed above, in- duced enzyme synthesis in the fungi is still largely a matter for specu- lation. 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E. 1950. Advances in Enzymol. 10: 443-486. 4\ Carbon Metabolism Part I Introduction T _l-he sheer bulk of the reported investigative work on carbon metabolism dictates some subdivision of the topic for the sake of convenience. In this and the two succeeding chapters we are con- cerned with the principal transformations of carbon compounds brought about by fungi, excluding those primarily important in the metabolism of nitrogenous compounds (Chapter 8) and a few which are better considered in conjunction with respiration (Chapter 7). Broadly considered, the physiological problems of carbon metabo- lism are two: the conditions under which a compound is utilized, and the conditions under which a compound is manufactured by the cell. These lead to two corresponding biochemical problems, the pathway of breakdown and the pathway of biosynthesis. The natural occur- rence of compounds and processes is also of interest. For any class of compounds the information is usually incomplete; thus, the physiology of acid formation is better known than are the biosynthetic pathways involved, and very little solid information on the breakdown of the acids is available. Many interesting compounds are known so far largely as curiosities, since neither their physiological significance nor their biosynthesis has been explored; it seems warranted, however, to include at least some examples of these in a discussion of fungus physiology. Because so much work has been done on the metabolites which accumulate in large amounts, it is salutary to recall that probably most fungi in culture form cell material and carbon dioxide exclu- sively. Thus, of 40 basidiomycetes investigated by Whi taker (21), 99 00 CARBON METABOLISM I only 3 produced significant amounts of soluble carbon compounds. The classical studies of Birkinshaw, Raistrick, and others were car- ried out on common saprophytes growing in a medium relatively high in carbohydrates; even under these conditions numerous species of Penicillium, Aspergillus and other genera were found to form only carbon dioxide and cell substance. It is safe also to assume that fungi growing in soil do not accumulate the same products which they do in pure culture. Carbon balance studies provide essential information as to the type of metabolism involved. The methods of Birkinshaw and Rais- trick (6) are still valuable, and others have been developed more re- cently (18). Table 1 illustrates a typical carbon and oxidation-reduc- tion balance. Determinations of carbon balances include many on members of the Fungi Imperfecti (2, 4, 5, 12, 15, 20), and a few on the lower phycomycetes (8, 13) and on the higher fungi (3, 18, 21). Table 1. Carbon and Oxidation-Reduction Balances in Aspergillus Niger (18) Oxidation- Over-all Reduction Oxidation- Carbon, Value Reduction mM mM per Mole* Value Substrate utilized Glucose 38.0 228.0 0.0 0.0 Ammonium chloride 1.96 0.0 0.0 Oxygen 109.36 2.0 218.6 Total 228.0 218.6 Products formed Carbon dioxide 88.20 88.20 2.0 176.4 Citric acid 16.13 96.78 3.0 48.4 Extracellular polysac- charide (as glucose) 2.51 15.06 0.0 0.0 Cell Carbohydrates (as glucose) 2.93 17.58 0.0 0.0 Chitin 0.114 3.65 -6.0 -0.68 Protein nitrogen 0.765 5.92 -1.78f -2.72 Lipids Trace Total 227.2 221.4 Carbon recovery = 227.2/228.0 = 99.7% Oxidation-reduction balance = 221.4/218.6 = 1.01 * See original paper for calculation. f Per atom nitrogen. INTRODUCTION 101 It is not always appreciated that a carbon balance implies an oxidation- reduction balance, and that some error must have been made if the oxidized products cannot be accounted for by the reduced products. The use of resting cells in metabolism studies is another valuable technique, but its limitations are not always recognized. The essential feature of the method is that cells are grown in a complete medium, washed, and put in contact with, usually, a single compound. This compound may be a putative precursor of a fungal metabolite which is of interest, or one may be concerned with the dissimilation of the compound itself. In a short space it is impossible to describe all the pitfalls of this method, but some of the more important may be sum- marized: 1. The previous history of the cells may influence the results ob- tained, at least quantitatively (1, 14, 19). 2. The thick, inhomogeneous mat of a surface culture is probably always less desirable than the more nearly homogeneous mycelium from a shaken or aerated culture. 3. The purpose of the method is defeated if a source of nitrogen is included; generally the cells contain and liberate some nitrogenous compounds. 4. The endogenous metabolism must be taken into account or even, if possible, manipulated, e.g., by starvation. Thus, it is possible that an added compound may simply protect an endogenously formed metabolite from utilization (7) and appear, therefore, to be a precursor of it. 5. Probably the technique is of more value in the study of the dis- similation of a compound than it is in the study of synthetic pathways. A compound, e.g., acetate, has so many metabolic channels open to it that its effect on a synthesis is difficult to interpret. Similar problems arise in studies of dissimilation, but in general the interpretation is clearer, provided the products account for the bulk of the compound disappearing. It has been observed (16) that the weight of "resting cells" may increase during incubation with sucrose; presumably this is a manifestation of oxidative assimilation (Chapter 7). The use of the resting cell technique is discussed by Foster (11), who offers the particularly valuable suggestion that in precursor studies a known low level of carbohydrate — just sufficient to allow minimal syn- thesis of the product — should be supplied. The details of particular enzymatic methods are beyond our scope here; a compendium of such methods has recently appeared (9). Two common problems which arise repeatedly in the fungi may, however, 1 02 CARBON METABOLISM I be mentioned. First, it is not always taken into account that the typical preparation contains many enzymes in addition to the one under study. Thus, the appearance of isomaltose during the hy- drolysis of starch by amylase was once taken as evidence for the structure of the starch molecule; it is now known that it merely de- notes the presence of a contaminating transglycosidase (17). A second common difficulty in enzymatic work is the tendency to read too much into experiments with intact cells. Thus, the appear- ance of unsaturated fatty acids in the medium during the metabolism of saturated fats by intact cells is certainly prima-facie evidence for the existence inside the cells of a fatty acid dehydrogenase; it is not, however, a demonstration of the enzyme and should not be described as such. Ex hypothesi, an intracellular enzyme can be demonstrated unequivocally only in a cell-free preparation, although properly de- signed experiments with whole cells may establish a high probability that the enzyme is present. The variability of fungi is a real problem which arises in connection with virtually all phases of metabolism. However, the amount of variation should not be exaggerated and should not be used as a crutch for a failing hypothesis. Complete loss of metabolic capacities is, of course, well known to be inducible by mutagenic agents and presumably, therefore, may occur as a result of spontaneous mutation. Most such mutants, however, are non-viable except under carefully adjusted conditions of culture. It appears that quantitative changes are more common than qualitative; an organism may change in the amount of a given substance formed but still form some. Exceptions will occur and must be taken into account, but the progress of a cumu- lative science requires that the material foundation be assumed con- stant unless the evidence clearly proves that it is not. Variability may result from changes in the growth rate, such as those described among geographical isolates and cultural variants of Fomes annosus (10), rather than from a loss of metabolic capacities. If two strains have the same metabolism but different growth rates, analysis at one arbitrary time for a metabolite may give an exaggerated picture of the differences between them. Thus, if a given compound is first formed and then utilized after exhaustion of the principal carbon source, a rapidly growing strain may complete the process of reutilization at a time when a more slowly growing strain is still in the phase of rapid formation of the compound; it would appear from analysis at this single time that the rapidly growing strain was deficient in the synthesis. To circumvent this difficulty it is necessary to study cultures at the same physiological age — judged by the amount of BIBLIOGRAPHY 103 growth or disappearance of substrate — rather than at the same chron- ological age. The physiologist often, perhaps inevitably, overlooks problems of taxonomy and morphology. Particularly in rather difficult genera like Penicillium and Aspergillus, the careful description of cultures with at least some indication that the species has been correctly named is of definite value to the next student of the problem. The question of morphology arises in a different context in the fungi, in that conditions of growth or special treatments employed in preparing cells may drastically alter the ratio of different kinds of cells, e.g., the ratio of spores to mycelium or, even more important, the ratio of living to dead cells. It should not be necessary to labor this point. BIBLIOGRAPHY 1. Bernhauer, K. 1928. Biochem. Z. 197: 287-308. 2. Birkinshaw, J. H., J. H. V. Charles, A. C. Hetherington, and H. Raistrick. 1931a. Phil. Trans. Roy. Soc. London Ser. B 220: 55-92. 3. Birkinshaw, J. H., J. H. V. Charles, A. C. Hetherington, and H. Raistrick. 1931b. Phil. Trans. Roy. Soc. London Ser. B 220: 99-125. 4. Birkinshaw, J. H., J. H. V. Charles, H. Raistrick, and J. A. R. Stoyle. 1931a. Phil. Trans. Roy. Soc. London Ser. B 220: 27-54. 5. Birkinshaw, J. H., J. H. V. Charles, H. Raistrick, and J. A. R. Stoyle. 1931b. Phil. Trans. Roy. Soc. London Ser. B 220: 93-98. 6. Birkinshaw, J. H. and H. Raistrick. 1931. Phil. Trans. Roy. Soc. London Ser. B 220: 11-26. 7. Butkewitsch, W. (Butkevich, V. S.), E. V. Menzhinskaia, and E. I. Troffimova. 1934. Mikrobiologiya 3: 319-342. 8. Cantino, E. C. 1949. Am. J. Botany 36: 95-112. 9. Colowick, S. P. and N. O. Kaplan (eds.). 1955. Methods in Enzymology, Vol. 1. New York: Academic Press, pp. 835. 10. Etheridge, D. E. 1955. Can. J. Botany 33: 416-428. 11. Foster, J. W. 1949. Chemical Activities of Fungi. New York: Academic Press, pp. 648. 12. Hockenhull, D. J. D. 1950. /. Exp. Botany 1: 194-200. 13. Ingraham, J. L. and R. Emerson. 1954. Am. J. Botany 41: 146-152. 14. Kluyver, A. J. and L. H. C. Perquin. 1933. Biochem. Z. 266: 82-95. 15. Lockwood, L. B., J. J. Stubbs, and C. E. Senseman. 1938. Zentr. Bakteriol. Parasitenk. Abt. II, 98: 167-171. 16. LvofF, S. and G. M. Toupizina. 1938. Compt. rend. acad. sci. U.R.S.S. 21: 307-311. 17. Pan, S. C, L. W. Nicholson, and P. Kolachov. 1953. Arch. Biochem. Biophys. 42: 421-4.", I. 18. Shu, P. and J. A. Thorn. 1952. Can. J. Botany 30: 252-265. 19. Thies, W. 1930. Zentr. Bakteriol. Parasitenk. Abt. II, 82: 321-347. 20. Wells, P. A., A. J. Moyer, and O. E. May. 1936. /. Am. Chcm. Soc. 58: 555- 558. 21. Whitaker, D. R. 1951. Can. J. Botany 29: 159-175. 5 . Carbon Metabolism Part II The Metabolism of Carbohydrates and Related Compounds T .he principal topic of this chapter on carbon metab- olism is the metabolism of oligosaccharides and polysaccharides, anticipated to some degree by the discussion of their role in carbon nutrition (Chapter 3). The related compounds included are the amino sugars and the polyhydric alcohols; discussion of the monosac- charides is limited to a few synthetic reactions. 1. THE METABOLISM OF MONOSACCHARIDES The breakdown of monosaccharides by fungi has been considered separately, as respiration, in Chapter 7. A second aspect of monosac- charide metabolism is the synthetic one, in which the sugar or a deriva- tive of it is incorporated into larger molecules. As an example, glucose in the glucolipid ustilagic acid is found by isotope studies to be in part incorporated as such and in part resynthesized from triose before incorporation (30). In this case and in the formation of glucose polysaccharides no modification of the basic glucose molecule need be assumed. However, the organism may synthesize a complex molecule containing sugar moieties which were not provided in the culture medium; examples include galactose in polysaccharides (Chapter 2) and amino sugars in certain antibiotics (p. 122). In these processes it is reasonable to assume that the new sugar or a derivative of it, e.g., a phosphate ester, is made and then incorporated into the larger 104 OLIGOSACCHARIDES 105 molecule, but there is no evidence directly on this point in the fungi and actinomycetes. Free sugars appear in the mycelium (130, 227) and in the culture medium (84, 85), but their significance is not known. The phosphate esters of the monosaccharides are hydrolyzed by phosphatases (phosphomonoesterases). The phosphatases active on glycerophosphoric acid may be presumed to act also on hexose phos- phates. No direct study of these reactions in fungi has been made, but there is no doubt that at least the common saprophytic fungi and actinomycetes can hydrolyze hexose and pentose phosphates; commer- cial enzyme preparations made from fungi contain acid phosphatases (179) and have been used in many laboratories for the dephosphoryla- tion of sugar phosphates. Phosphatases are considered in more detail in Chapter 9. 2. THE METABOLISM OF OLIGOSACCHARIDES The literature on the enzymatic breakdown of disaccharides and trisaccharides by fungi is extensive but, as in so much of fungus physi- ology, relatively few types have been studied. Most of our informa- tion comes from work on the Fungi Imperfecti, the wood-destroying basidiomycetes, and the Saprolegniaceae. Many other groups of fungi have not been investigated and must, therefore, be excluded from any generalization. The most significant recent advance in our approach to the break- down of oligosaccharides is the concept of transglycosidation. Until the formulation of this concept the breakdown of sucrose, for example, was visualized as a simple hydrolysis: G12H22O11 + H20 -+ G6H1206 + C6H1206 (1) Sucrose Glucose Fructose Since sucrose is made up of glucose and fructose units linked by a glycosidic bond, this hydrolytic reaction may be schematized: Fr— Gl -> Fr + Gl (2) It now appears that at least some and possibly all of the so-called hydrolytic enzymes are in fact group transferases, capable of transfer- ring a sugar residue to any suitable acceptor. The acceptor may be water, in which case Equation 2 describes the reaction, but other sugars or alcohols can also serve as acceptors. In a particular case, the action of yeast invertase, the fructose residue of sucrose is believed to be transferred as follows (72): 106 CARBON METABOLISM II Fr— Gl + Enzyme (E) ±=; Fr— E + Gl (3) Fr— E + Fr— Gl t=; Fr— Fr— Gl + E (4) Fr— E + H20 -> Fr + E (5) The fructose-enzyme complex is, of course, hypothetical but it is neces- sary to explain the observed reactions. Because of the irreversibility of the fifth reaction and the great excess of water present, in a long- term experiment virtually all of the fructose residues are transferred to water and the result is described grossly by Equation 2. The technique of paper chromatography applied in short-term experiments has been essential to the elucidation of this type of group transfer. The characteristic of transglycosidase action on disaccharides is the transient appearance of new oligosaccharides. Disaccharides. Most of the work on this topic has been concerned with the breakdown of sugars, especially the common disaccharides such as sucrose, maltose, and lactose. Synthesis of disaccharides occurs in the fungi, the best known case being trehalose (p. 108). Sucrose is attacked by an enzyme variously known as invertase, saccharase, glucosaccharase, and sucrase; the name sucrase is preferred. The first indication of its presence in fungi came from the work of Bechamp (18, 19). As might be expected from the frequent utilizability of sucrose as a carbon source (Chapter 3), the enzyme is widely distributed in the fungi. Sucrase is somewhat rare in the Mucorales — again in agreement with nutritional data — but does occur in some species (93, 203). Sucrase is found in some isolates of Streptornyces (101, 110), not in any of five species of dermatophytes (229). Fungus sucrase may be entirely intracellular (148) but is usually found also in the medium, at least after the onset of autolysis (58). It occurs in spores (71, 141); in Myrothecium verrucaria and Aspergil- lus luchuensis there is evidence that it or a sucrose phosphorylase is located at or near the spore boundary (142). This boundary location is thought to be characteristic of sucrase in the yeast cell (62, 155). It has long been argued that the predominant sucrase of fungi is a "glucosaccharase" differing from the "fructosaccharase" of yeast (121); the history of this controversy is reviewed by Neuberg and Mandl (156). The question has now been re-examined in the light of the finding that both yeast and fungus sucrases are probably transfructosi- dases. It appears that the two sucrases differ, but not in the way formerly thought, and that it is not necessary to postulate a glucosac- charase (15, 66). Transfructosidase activity has been demonstrated in several fungi, OLIGOSACCHARIDES 107 Enzyme Activity Substrate Transfructosidase Sucrose Raffinose Transglucosidase Maltose Isomaltose Cellobiose />-Nitrophenyl /3-glucoside Transgalactosidase Lactose Table 1. Transglycosidase Activity in Fungi Fungi Aspergillus spp. (9, 10, 16, 66, 169, 170) Aspergillus spp., Penicillium spinulosum (15) Aspergillus spp. (47a, 169, 173, 174), Peni- cillium chrysogenum (202) Aspergillus oryzae (171, 172) Aspergillus niger (11, 12), Chaetomium globosum (48), Myrothecium verrucaria (119) Aspergillus spp. (113, 226) Penicillium chrysogenum (226) Aspergillus oryzae (244, 245) acting on sucrose or raffinose (Table 1). A fructose-enzyme complex is postulated, as in Equations 3, 4, and 5 above. In Penicillium spinulosum there is no evidence for any sucrase other than the transfructosidase, but in Aspergillus oryzae there is probably a second enzyme acting on sucrose (16). Paper chromatography of the enzymes of A. oryzae similarly suggests that more than one sucrase is present (112). Sucrase, acting as it does on /3-fructofuranosides, is instrumental in the metabolism of raffinose and stachyose. Mandels (143) presents indirect evidence for the non-hydrolytic metabolism of sucrose by spores of Myrothecium verrucaria, possibly to be credited to a sucrose phosphorylase of the type known in bacteria. Maltose is used as a source of carbon by almost all fungi (Chapter 3). Correspondingly, beginning with the work of Bourquelot (33), we find that the enzyme maltase is found in almost every fungus and actinomycete investigated, although quantitative differences between strains are common (127). It has been claimed that Polyporus betulinus lacks maltase (138), and the chytrids which are unable to grow on maltose (Chapter 3) may be found to lack the enzyme. Maltase is an a-glucosidase and is found in bacteria, animals, and higher plants. The earlier separation of fungus from yeast maltase as a "glucomaltase" is probably not valid, but the two maltases are not identical (89). As with sucrase, recent investigations have estab- lished in fungi the existence of a transglycosidase acting on maltose to form a series of transient oligosaccharides (Table 1). The existence of a specific hydrolase for maltose may be doubted, but it is not yet known whether the transglucosidase of Aspergillus niger and A. oryzae, the only fungi so far studied, is of general occurrence as a single 108 CARBON METABOLISM II enzymatic entity. Transglucosidases which act on maltose to form polysaccharides are known in bacteria but have not yet been reported from fungi. The disaccharide lactose is split to its constituent hexoses by the enzyme lactase, a /?-galactosidase. The enzyme has been reported in several fungi and actinomycetes, but is apparently not of such general occurrence as either sucrase or maltase. Some of the early work on lactase in commercial enzyme preparations and in basidiomycete sporophores may be open to question because of the possibility of bacterial contamination. The adaptive formation of lactase in Neurospora crassa has been proved (Chapter 3). A purified enzyme from Aspergillus oryzae acts as a transgalactosidase; galactose residues are transferred to alcohols, to lactose itself to form a trisaccharide, or to water to yield free galactose (Table 1). Whether all action of lactase can be credited to this enzyme is not yet known. Cellobiose is formed during the hydrolysis of cellulose. Although it is common to refer to the enzyme splitting it as cellobiase, there is no reason to think that this enzyme differs in any way from /3-glucosi- dases acting on other /3-glucosides. Specific assays with cellobiose as substrate have been made infrequently, but it seems that most fungi can split it to its constituent glucose residues (191). Resting and growing cells of Aspergillus niger form new oligosaccharides from cellobiose (11, 12), and additional evidence for transglycosidase action on cellobiose has been obtained with other fungi (Table 1). Cellobiose is synthesized by Ustilago zeae as a component of ustilagic acid (128). Trehalose, an a-glucosido-glucose, is not attacked by the usual a-glucosidases but rather by a special enzyme, trehalase, found in animals, plants and fungi (89). Early work on trehalase in fungi is reviewed by Czapek (59); this and more recent data indicate the enzyme is common (203, 256). The spores of Aspergillus luchuensis contain measurable amounts of the enzyme (139). In Lycogala sp., a myxomycete, trehalase of the fruit body disappears during ripening (108). Trehalose is the only disaccharide to be formed free in appreciable amounts by fungi. The discovery of the sugar in ergot (Claviceps purpurea) by Wiggers (254) and early reports of its occurrence in other fungi are reviewed by Zellner (264) and Obaton (164). Trehalose is especially common in the basidiomycetes but is found also in many other fungi (34, 36). Usually it constitutes 2 to 3 per cent of the mycelial dry matter (108, 193, 227). In all probability, trehalose in fungi has the role of a translocatable OTHERGLYCOSIDES 109 reserve carbohydrate, formed early in metabolism and used up later (43); under some conditions reutilization may not occur during the life of the fungus (164). An adequate picture of the course of trehalose synthesis and breakdown is not yet available. In yeast, trehalose is synthesized as its phosphate from glucose and a glucose phosphate, with uridine diphosphate glucose as coenzyme (68, 125); whether the same mechanism is operative in the filamentous fungi remains to be seen. Melibiose, an a-galactosido-glucose occurring naturally as a con- stituent of raffinose, is split by an a-galactosidase (melibiase) known in yeast and higher plants and reported from Aspergillus spp. (100, 230). Trisaccharides and Tetrasaccharides. Raffinose, composed of galac- tose, glucose, and fructose, is split by enzymes from most fungi which have been tested. This is to be expected, in view of the fact that sucrase splits raffinose to melibiose and fructose. The complete hydrolysis of raffinose requires both sucrose and melibiase; melibiase alone yields sucrase and galactose. In all likelihood, hydrolysis of raffinose by unspecified fungal enzymes should usually be attributed to sucrase (2, 15). Melezitose is hydrolyzed, at least partially, by several fungi (15, 122). Detailed studies of the reaction are lacking; complete hydrolysis requires an enzyme able to attack the disaccharide turanose. Gentianose is completely hydrolyzed to glucose and fructose by an enzyme preparation from Aspergillus niger (40, 41); the effective preparation has been shown (42) to contain a /3-glucosidase acting on the gentiobiose moiety of gentianose. Stachyose, a tetrasaccharide composed of galactose, glucose and fructose in the ratio 2:1:1, is split by enzymes from Penicillium spinulosum and Aspergillus spp. completely to its constituent sugars (15, 228). Presumably sucrase and an a-galactosidase are required. 3. THE METABOLISM OF OTHER GLYCOSIDES The oligosaccharides, just considered, are one class of glycoside, in which the glycosidic bond connects two monosaccharide residues. The same bond can be formed between a sugar and certain other molecules; the non-carbohydrate moiety of the resulting compound is then termed an aglycon. Information on the metabolism of glycosides of this type by fungi is restricted to a few compounds, in all of which glucose or mannose is the sugar involved. The /3-glucosides have received more attention than any others in 110 CARBON METABOLISM II this general category. The enzyme /^-glucosidase (formerly termed emulsin) occurs in almost all of the fungi and actinomycetes which have been examined for it (31, 38, 39, 101, 147, 201, 229), although it is not always found in the medium (191) and may not be so nearly universal as, for example, maltase or amylase. Fusarium oxysporum var. lycopersici forms detectable amounts of the enzyme within the living infected plant (60). Although it is common practice to speak of a single /3-glucosidase hydrolyzing all /3-glucosides — including the /3-linked disaccharides cellobiose and gentiobiose — it seems more likely that a group of related enzymes is involved (238) or that at least there are distinct com- ponents of the enzyme (111, 112). The /3-glucosidases of Aspergillus niger and A. oryzae, for example, act at different rates on different synthetic /3-glucosides (151). The enzyme of A. niger has been purified about 10,000-fold (160, 161). During the action of /?-glucosidase on synthetic /3-glucosides, the glucose residue can be transferred to alcohols, indicating trans- glucosidase activity similar to that with cellobiose as substrate (Table 1). Alpha-glucosides, in principle at least, should be attacked by maltase, an a-glucosidase. However, maltases from different sources have differ- ent behavior toward, e.g., methyl-a-glucoside, and the maltase of fungi seems to be of minimum activity toward this substrate (89). A crude preparation from Aspergillus niger hydrolyzes methyl-a-glucoside slowly (65), and in some fungi there is an a-glucosidase distinct from maltose (15), but the problem has been too little studied for any definite conclusion to emerge. Aspergillus niger cultivated with methyl-a-galactoside forms the corresponding a-galactosidase (182). An enzyme of Streptomyces griseus converts mannosidostreptomycin to streptomycin (175). The enzyme has been found to split synthetic methyl-a-mannoside (99) and may therefore be termed an a-manno- sidase. The same action, with methyl-a-mannoside as substrate, is known in Aspergillus niger (98). 4. THE METABOLISM OF POLYSACCHARIDES The Breakdown of Starch and Glycogen. These two related polysac- charides are hydrolyzed by amylases (diastases), of which there are in nature two general types (154): 1. Alpha-amylase (endoamylase, dextrinizing amylase, liquefying amylase), found in animals and microorganisms. Attack on starch, at POLYSACCHARIDE S 111 least in its early phases, results in the formation of dextrins, reducing sugars appearing only later. 2. Beta-amylase (exoamylase, saccharifying amylase) of resting seeds. Attack on starch liberates maltose immediately. It has been repeatedly found, with highly purified or crystalline preparations, that the amylase of Aspergillus oryzae is of the a-amylase group (49, 73, 235). So also, apparently, is the amylase of Streptomyces spp. (213). The crude amylase of A. oryzae contains more than one component, as judged by electrophoretic data (82). The enzyme of Rhizopus delemar, termed glue amylase (177, 178), can be separated from a-amylase and resembles /^-amylase in certain respects. There seems, however, no reason to believe that a typical ^-amylase occurs in the fungi (127, 129, 146). Table 2 summarizes some of the properties of the purified amylase of Aspergillus oryzae and its differences from other a-amylases. Amylase activity, defined as the ability of cell-free materials to hydrolyze starch or glycogen, is virtually universal in the fungi and actinomycetes. Penicillium digitatum does not form detectable amylase in culture (103), and in view of its inability to grow on starch (Chapter 3) is presumably deficient for the enzyme. Early studies on fungal amylase are summarized by Thaysen and Galloway (230), and the industrial technology of the enzyme is reviewed by Langlykke et al. (123). Table 2. Properties of Alpha-amylases (74) Source of Crystalline Alpha-amylase Activity per mg N Activity per mg protein Nitrogen, per cent Optimum pH for activity Optimum pH for stability Isoelectric point Absorption maximum (m/x) Saccharogenic/dextrinogenic ratio Activation by Ca Activation by CI Energy of activation Human Aspergillus Bacillus Pig Saliva, Malt oryzae sub t His Pancreas Pancreas 2350 2400 3600 4000 6200 315 310 500 630 980 13.4 12.9 15.8 15.8 15.8 4.7-5.4 5.5-5.9 5.3-6.8 6.9 6.9 4.9-9.1 5.5-8.5 4.8-8.5 7.0-8.5 4.8-11 5.6 circ. 4.2 ? 5.3 5.3 280 280 280 280 280 9.8 + 9.8 9.8 9.8 9.6 — + + + 7050 10650 13350 0 to 15° 13500 13350 112 CARBON METABOLISM II 1 r Dry weight 16 Time, days Figure 1. Amylase liberation by Aspergillus oryzae. Redrawn from Crewther and Lennox (58), by permission of the Australian Journal of Biological Sciences. Amylase is, with some exceptions (64), found in the culture medium as well as in the cells. The amount of enzyme is usually increased by inclusion of starch in the growth medium (92, 210) and is affected by the nitrogen source (210) and by small amounts of salts (69). Figure 1 illustrates the time course of appearance of extracellular amylase in Aspergillus oryzae; it is to be noted that the peak of amylase activity in the culture filtrate is reached only after the onset of autolysis. It has been reported (28) that the culture filtrate of Streptomyces microflavus forms gentiobiose during the hydrolysis of starch. The Breakdown of Cellulose. Since many fungi utilize cellulose as a carbon source (Chapter 3), it is to be expected that an enzyme or enzymes are released from the cell and hydrolyze cellulose to soluble sugars. Cellulose-hydrolyzing preparations free of living cells have indubitably been made from several fungi (83, 191, 214), and dis- tinguished from enzymes acting on lichenin and hemicelluloses (90). However, two important limitations apply to most of the reported work on cellulase: (1) native cellulose is relatively resistant to attack, and most assays of "cellulase" have therefore utilized various types of modified cellulose; (2) most data derive from studies with crude cellulase preparations contaminated with other carbohydrases and with interfering proteins (232). It is difficult, therefore, to assign more than a descriptive value to much of the work so far reported. POLYSACCHARIDES 113 Crude cellulase preparations from several fungi are alike in that their optimum pH is in the acid range, 4.5 to 5.5 (90, 163, 204); the optimum for the enzyme of Stachybotrys atra is, however, higher, pH 6.5-8.0 (230a). Studies on Tricholoma furnosum indicate that the enzyme is formed only by cells in contact with cellulose or lichenin, and that the enzyme in a culture is predominantly extracellular (163). Cellulose is apparently attacked at several points along the chain of glucose residues, inasmuch as in the early stages of attack on cellulose there is a decline in optical density without a corresponding liberation of sugars (162, 163a). Other characteristics of fungal cellulases are summarized by Siu (214). For the present, a general statement on the nature of cellulase is not possible. The classical view (183) postulates at least two enzymes, a "cellulase" splitting the long polymeric chain to cellobiose residues, and a cellobiase (/?-glucosidase) splitting the disaccharide to glucose. Although substantial evidence exists for the appearance of cellobiose as an intermediate (119, 159a, 250), the process is probably more complex. Two theories, both based on work with Myrothecium verrncaria, have been proposed recently. The first of these postulates a minimum of three enzymes as required for cellulose hydrolysis (83, 91a, 150, 190, 191), with only one of them characteristic of the cellulolytic organisms and the others occurring also in non-cellulolytic fungi. In sharp distinction to this view, other studies on the same organism (251, 253) have resulted in the isolation of an apparently single enzyme, purified to electrophoretic homogeneity, with a molec- ular weight of 63,000. This single enzyme hydrolyzes cellulose to glucose without the obligatory participation of cellobiose as an in- termediate, although cellobiose may be formed during the process. The enzyme acts, at different rates, on cellobiose, cellotriose, and higher oligosaccharides of this group (252), and is inhibited by sulfhydryl reagents (14). It is obvious that further work on the enzyme will be necessary before any general theory can be formulated. The Breakdivn of Hemicelluloses and Pentosans. The hemicellu- loses are poorly defined plant constituents, particularly of wood and gums. The principal components of the hemicelluloses are polysac- charides and polyuronides. The literature reports essentially two types of work, that on hemicellulose fractions of unknown composi- tion, and that on more or less purified polysaccharides derived from hemicellulose. Crude hemicellulose fractions from wood are attacked by enzymes of Aspergillus oryzae, yielding free sugars and a soluble polysaccharide Ill CARBON METABOLISM II (166, 107). As might be expected, the wood-destroying basidiomycetes are especially active in the hydrolysis of hemicelluloses (31, 189); the gross utilization of hemicelluloses has been mentioned earlier (Chapter 3). The only other fungi which have been studied are some members of the Saprolegniaceae (21, 201), and of the genus Phytophthora (147), all of which form reducing sugars from the crude hemicellulose of date endosperm. The most abundant of the pentosans are xylans; the rarer arabans are found in association with pectic substances. The limited literature on fungus metabolism of xylans is reviewed by Voss and Butter (240). Enzymes (xylanases) attacking xylan have been found in Aspergillus oryzac (90) and Glomerclla cingulata (96). The xylanase of Chae- tomium globosum is formed in appreciable amounts only if the organism is grown on xylan, and consists of an exocellular component converting xylan to xylobiose and an intracellular enzyme hydrolyzing thexylobiose (217, 218). Arabans — pentosans made up largely of L-arabinose — are hydrolyzed by a crude enzyme preparation from Aspergillus oryzae (67). Simpson (212) surveyed the action of 112 isolates of 24 genera on a purified pentosan of unspecified composition. An active hydrolysis was brought about by the culture filtrate of about half of the isolates tested; the most active filtrates were from cultures of strains of Aspergillus, Fusarium, Tricothecium, and Trichoderma. Of the 54 active strains, only 6 formed the enzyme during growth on glucose; in the rest, growth on the pentosan — replaceable in some instances by free pentoses — appeared to be necessary for enzyme synthesis. In the genus Streptomyces, 5 of the 19 isolates tested formed an extracellular enzyme active on the pentosan. A purified arabogalactan from Larix is split by an enzyme from Aspergillus niger (189). Mannans are hydrolyzed by an enzyme of Chaetomium globosum which is apparently distinct from the enzyme acting on xylans (217). The chemistry of the substrates is poorly known and the nature of the enzymes involved in their hydrolysis is uncertain. In particular, it is not possible from the data available to decide on the possible identity or non-identity of enzymes which have been studied by different workers. It is generally accepted that the enzymes attacking the pentosans and the crude hemicelluloses are distinct from cellulose- hydrolyzing enzymes and from lichenase (180), but beyond this point specificity is not known. The Breakdown of Pectic Substances. Perhaps nowhere in the field of carbon metabolism is the confusion so great as it is in the POLYSACCHARIDES 115 study of the pectic materials. Historically, interest in the pectic enzymes was first stimulated by the discovery (13, 247) that crude enzyme preparations from plant pathogenic fungi macerate plant tissues by destruction of the middle lamella, the cementing material between cells. First thought to be a cellulase, the tissue-macerating enzyme has also been termed cytase. Other early studies are reviewed by Brown (46). The insoluble pectic materials as the) are found in plants are now called protopectin (116). Neither the structure nor the composition of protopectin is known. The corresponding enzyme protopectinase (pectosinase) is defined in principle as the enzyme converting proto- pectin to water-soluble pectinic acids (255). Operationally, the enzyme is defined by its ability to macerate blocks of living plant tissue. It is found in pathogenic and non-pathogenic fungi belonging to all of the major taxonomic groups (Table 3). Table 3. Occurrence of the Pectic Enzymes in the Fungi* Enzyme Occurrence "Protopectinase" Aspergillus spp. (61, 222), Botrytis spp. (46, 237), Byssochlamys julva (17, 105), Chaetomium globosum (222), Daedalea flavida (32), Fomes jraxineus (152), Fusarium spp. (51, 149, 211), Gloeosporium fructige- num (Glomerella cingulata) (149), Lenzites sepiaria (263), Macrophomina phaseoli (185), Metarrhizium sp. (222), Moniliafructigena (237), Mucor racemosus (93), Phytophthora erythroseptica (149), Polyporus spp., Polystictus spp. (32), Pythium debaryanum (4, 97), Rhizoctonia sp. (258), Rhizopus spp. (94, 95), Stereum purpureum (145), Trametes spp. (32). Aspergillus spp. (81, 114), Botrytis cinerea (80), Fusa- rium spp. (87, 241), Penicillium spp. (114, 176), Sclerotinia cinerea (61), Sclerotiutn bataticola,\ Thiela- viopsis basicola] (231). Pectin-polygalacturonase Aspergillus spp. (6, 64, 81, 114, 208), Botrytis cinerea (81, 114, 257), Byssochlamys Julva (102, 192), Col- letotrichum phomoides (185a), Fusarium spp. (47, 185a, 211, 241, 243), Mucor spp. (185a), Neuro- spora spp. (64, 197), Penicillium spp. (64, 176, 185a, 243), Rhizopus nigricans (185a), Stachybolrys atra, Syncephalastrum racemosum, Thamnidium elegans, Tri- choderma spp. (64, 185a), Verticillium albo-atrum (205). * Including only the three major enzymes; for other enzyme activities, see text. f Assayed by hydrolysis of half-calcium salt of methyl-D-tartaric acid (157). Pectin-methylesterase IK', CARBON METABOLISM II The indefinite nature of the substrate, the crudity of the enzyme assay, and the fact that other enzymes may solubilize the pectic materials of plants (116) are compelling reasons to doubt the real existence of protopectinase. This doubt is reinforced by the finding (102) that a partially purified pectin-polygalacturonase from Byssoch- lamys fulva causes disintegration of tobacco tissue. Although it is possible, of course, that the preparation was contaminated with a protopectinase, it seems more probable that pectin-polygalacturonase has the ability, usually ascribed to protopectinase, to macerate tissue. It is significant in this connection that all protopectinase preparations examined contain pectin-polygalacturonase (115). The present nomenclature of the pectic enzymes was first clearly formulated in 1927 (61) and fully worked out only in 1914 (117). Consequently the earlier work on these enzymes employs different and often highly individual nomenclature. In particular, the "pectinase" of the older literature is usually synonymous with what is now called protopectinase, i.e., the tissue-macerating enzyme. Pectinase as now understood is a synonym for pectin-polygalacturonase. The nomencla- ture used here is that of Kertesz (116). The pectinic acids are colloidal macromolecules composed of a-D- anhydrogalacturonic acid units linked together through 1 ,4-a-glycosidic bonds. The carboxyl groups of some of the galacturonic acid residues are esterified with methyl alcohol. Certain pectinic acids of specific properties are designated pectins. Pectinic acids are obtained from the crude plant pectic materials by partial hydrolysis. Excluding protopectinase from consideration, the known and postulated pectic enzymes of fungi are of three types, depending on their action on pectinic acids. They are: 1. Pcctin-methylesterase, catalyzing the hydrolysis of the methyl ester groups of pectinic acids, and converting pectinic acids to pectic acids (polygalacturonic acids). 2. Pectin-polygalacturonase, hydrolyzing the pectic acids to free D-galacturonic acid. 3. Pectin-depolymerase, a less well-known enzyme thought to cause a partial hydrolysis of pectic acids to smaller units without the ap- pearance of free galacturonic acid. Pectin-methylesterase (pectase, pectinesterase, pectinmethoxylase) was originally denned (61) as an enzyme converting soluble pectin (pectinic acid) into insoluble calcium pectate. Its action may be schematized as: (R— CO— O— CH3)„ + (n)H20 -» (R— COOH),, + (w)CH3OH (6) POLYSACCHARIDES 117 The enzyme occurs in commercial enzyme preparations of pectin- polygalacturonase (75) and in the fungi listed in Table 3. The enzyme of Fusarium oxysporum var. lycopersici appears to differ from that of commercial preparations from other fungi (241), and the latter in turn differs from the pectin-methylesterase of higher plants (145). Production of extracellular pectin-methylesterase by Botrytis cinerea and Aspergillus niger occurs if pectin is the growth medium but only to a negligible degree with glucose as the carbon source (3, 80, 81). The extracellular enzyme of Penicillium chrysogenum is formed only if pectin, pectic acid, or chemically related substances are in the growth medium (176). Recently, the role of pectin-methylesterase in the pathogenicity of fungi to plants has received attention. The third major category of the pectic substances is that of the pectic acids, derived from pectinic acids by de-esterification. They are colloidal polygalacturonic acids with molecular weights ranging from 25,000 to 100,000. The molecule consists of D-galacturonic acid units and may be visualized as follows: coo- coo- coo- The enzyme hydrolyzing the pectic acids has been named variously pectinase, pectolase, and polygalacturonase. The most unambiguous name appears to be pectin-polygalacturonase (116). It acts on the de-esterified portions of pectinic acids or on the pectic acids resulting from complete de-esterification of pectinic acids. It is often con- taminated with pectin-methylesterase, and the mixture of the two enzymes will, of course, act on pectinic acids both by de-esterification and by hydrolysis. The product of pectin-polygalacturonase activity on a polygalacturonide is, eventually, free D-galacturonic acid. The properties of the enzyme have been studied extensively by Matus (144). Table 3 lists the fungi which have been shown to form the enzyme, as assayed on pectinic acid or polygalacturonic acids, i.e., excluding assays of tissue-macerating enzyme. Two comments are apposite to the problem of the occurrence of the enzyme in fungi. First, it is probable that the fungi producing protopectinase always form pectin- polygalacturonase or that the two enzymes are identical. If either of these hypotheses is true, then the sources of protopectinase given in Table 3 must be included, adding numerous phycomycetes and 118 CARBON METABOLISM II basidiomycetes to a list which is predominantly composed of ascomy- cetes and imperfect fungi. Second, the fact that many fungi form pectin-polygalacturonase only if pectinic acids or related materials are present in the medium means that a complete survey of representative fungi should use such a medium; in addition, in any such survey the pH of the medium should be kept below 6.0 by reason of the rapid inactivation of the enzyme at higher pH values (116). From the data available and in the light of these considerations, it seems likely that most fungi are able, at least under favorable conditions, to elaborate pectin-polygalacturonase. The pectin-polygalacturonase of fungi differs in its properties from the corresponding enzymes of yeast and higher plants (118, 133). The enzyme is highly specific to pectic acids, failing to hydrolyze other galacturonides or other uronides (131). Separation from pectin- methylesterase has been accomplished (131, 136). It is not yet possible to assert that pectin-polygalacturonase is a single enzymatic entity. One view (114) is that it is a single enzyme acting by random scission of the pectic acid molecule to form shorter galacturonic acid chains and eventually free galacturonic acid. On the other hand, it has recently been suggested that three different enzymes are involved in Aspergillus foetidus (6, 63). The pectin-polygalacturonase of Penicillium chrysogenum and of Fusarium spp. appears to be elaborated adaptively, in media with pectin as carbon source but not in glucose media (47, 176), but the same enzyme in other fungi is formed in glucose media (64, 80, 81). All these experiments were carried out by growing cells from a spore inoculum in media with or without the presumed inducing substrate; hence, a possible effect of mutation or selection was not rigorously excluded. Other enzymes, depolymerases, acting on pectins have been described in plant parasitic and saprophytic fungi (17, 19a, 88, 195, 209). These enzymes share in common the characteristic that only certain of the glycosidic linkages of the substrate are attacked, resulting in the liberation of short-chain polyuronides. The existence of such enzymes in fungi has been challenged (192, 197), but the evidence, especially that from work on Neurospora crassa (195) is strong. Assay methods for the pectic enzymes are reviewed by Kertesz (116); cup plate assays have been devised (53) and may be particularly useful in survey studies. There is some doubt (116) that assay of pectin- methylesterase by hydrolysis of the half calcium salt of methyl-D- tartaric acid (157) is valid. It seems that the immediate problem in the study of the pectic POLYSACCHARIDES 119 enzymes, as in that of so many other enzymes, is to determine the action of single enzymes on substrates of definite and known com- position. Once such information is available, there will be some hope of successfully analyzing the more complex situation in nature. The Breakdown of Other Polysaccharides. It is generally con- sidered that a special enzyme, inulase, catalyzes the breakdown of the plant fructosan inulin. The enzyme was discovered in Aspergillus niger by Bourquelot (37) and has since been found in many other fungi (25, 59, 79). It has often been reported that cultivation on inulin as carbon source increases the amount of enzyme (25, 184, 261), but some inulase is always formed whatever the carbon source. No systematic study of the enzyme or its occurrence has been made; some properties of fungal inulase have been determined by Pigman (179), and it is possible that two different enzymes are responsible for the activity (153). On the other hand, there is some evidence that inulase ;..', is a transfructosidase active also on sucrose (155a). A bacterial dextran, composed of anhydroglucose units linked by 1,6-crglucosidic bonds, is hydrolyzed by a dextranase present in the mycelium and culture fluid of several fungi, e.g., Penicillium funicu- losum, Verticillium coccorum, and Spicaria violacea (106, 234). The enzyme is formed only if dextran is present in the medium. Limit dextrin, the polysaccharide fraction remaining after starch hydrolysis by «-amylase, is hydrolyzed by a limit dextrinase from Aspergillus oryzae; the enzyme has been crystallized (235). A commercial enzyme preparation, probably made from an Aspergil- lus sp., hydrolyzes a galactogen of snail eggs, yielding d- and L-galactose (248). Lichenin, the polysaccharide of Irish moss (Chondrus crispus) resembles cellulose except for its high proportion of 1,3-glycosidic bonds. It is hydrolyzed by an enzyme found in animal and plant material (180) and in Aspergillus oryzae (200) and Merulius lacrymans (181). Lichenase is probably distinct from cellulase and xylanase (78, 90). The mucopolysaccharides are complex polysaccharides yielding amino sugars on hydrolysis (76); examples include hyaluronic acid, chitin, agar, and unidentified cell wall materials. Among the enzymes attacking these materials the bacterial hyaluronidase and lysozyme are fairly well known, but the mucopolysaccharases of fungi are not. Chitinases are known among actinomycetes and both higher and lower fungi (49a, 233); the enzyme or enzymes responsible for chitin hydroly- sis in Streptomyces sp. are adaptive (194). The lytic enzymes of 120 CARBON METABOLISM II actinom\cetes, e.g.. tlie enzyme system of Strepto?7i\ces olbus (134. ict on cell wall polysaccharides and can therefore tentatively be classed with the mucopolysaccharases. Enzymes of this type have been reported frequentlv from actinomycetes (114a. 249) and must be present in those fungi in which the chitin breaks down during autolysis (216). I i tain marine actinomvcetes liquefv agar (107, 219). The enzyme responsible has been called gelase: it is evidently either rare or absent in terrestrial fungi and actinomycetes. since agar is used so generalh in culture media that gelase-producing organisms would have long since been detected if they were common. The Synthesis of Polysaccharides. The known fungal polysac- charides are reviewed elsewhere (Chapter 2); it will be recalled that they include chitin. polyhexoses, and more complex polysaccharides. The most common hexose component is glucose, the next most com- mon galactose: since all of the galactans are formed in media with glucose or sucrose as carbon source, it seems probable that a galacto- waldenase is present, as in yeast, catalyzing the conversion of glucose to galactose (124). The metabolic origin of the polysaccharides of fungi has been little investigated, but may be expected to follow the pathways known in other organisms. The two best known mechanisms of polysaccharide synthesis in bacteria are. in outline: CuHsA, — (CHtoOs). - (n)C6H120 (7) Disaccharide Polysaccharide Monosaccharide (n)Glucose-l -phosphate ±^ Polysaccharide + phosphate (8) The first of the>e is a transghcosidation. of which the action of levansucrase is an example: the second is catalyzed by a phosphorylase and is exemplified b\ glycogen formation in the animal. Levan s\nthesis in Aspergillus sydowi spores may be a transghcosidation; it can be effectuated by a broken-cell preparation, and the levan is formed during growth on sucrose but not during growth on monosac- charides _ The polyglucose of Torulopsis rotundata, on the other hand, is probably synthesized b\ a phosphorylase (139). Evidence on polysaccharide biogenesis in the fungi is so far limited to these two The polysaccharides of some fungi are formed more abundantly in acid than in neutral media (27, 140. 207;. but the reverse is true of lerotiose formation (1). In yeast, inhibition of polysaccharide formation by alkalinity is believed to result from an inhibition of AMINO SUGARS '21 phosphor, lase (224). Presumably, other environmental factors are influential; on a priori grounds it is to be expected that the con- centration of available carbohydrate and the carbon: nitrogen ratio of the medium are important. Oxygen is required for the synthesis of polysaccharide by Aspergillus niger (2< The function of fungal poh saccharides is almost certainly as a reserve food., and the data of Ergle (70 on glycogen utilization dur: . the germination of sclerotia bear out this contention. This view of polysaccharides as one form in which fungi can store energy was first clearly stated by Chrzaszcz and Tiukow '/y in opposition to an earlier view of polysaccharide formation as a symptom of a deranged metabo- lism. It should be noted that under some cultural conditions the polysaccharide formed may never in fact be utilized., cell death and autolysis intervening before the carbohydrate supply becomes limiting and before the reserves are attacked. Several fungi pathogenic to man form polysaccharides, often as- sociated with a capsule and often immunologically active. Those of Coccidioides immitis and Cryptococcus 'Torulor./ spp. are listed in Chapter 2: others are reviewed by Xickerson 158). 5. THE METABOLISM OF AMINO SUGARS The only amino sugar which has been studied extensively is r> glucosamine | 2-amino-D-glucose), interest in which arises primarily from the fact that its N-acetyl derivative is the basic unit of chitin, the cell wall material of most fungi Chapter 2). Conceivably, the conversion of glucose to glucosamine could proceed with glucosone. CfTOH— CHOH — CO— CHO; as intermediate and glucosone is formed by Aspergillus parasiticus, at least under some conditions 29 . However, in Xeurospora crassa extracts glucosamine is formed by transamidation from glutamine (126) as folio- Hexose-6-phosphate — glutamine -^ glucosamine-6-phosphate — glutamic acid (9) It appears from other work that the hexose phosphate involved directly in the reaction is fructose-6-phosphate 26). Glucosamine appears in chitin and in animal mucopolysaccharides as the N-acetyl derivative. Introduction of the acetyl group involves aceu 1-coenzvme A in both animals and Xeurospora crassa (52, 12 in N. crassa it is not certain whether free glucosamine or its phosphate is the compound acetylated. Amino sugars are frequently found as constituents of antibiotics. 122 CARBON METABOLISM II including cordycepin of Cordyceps militaris (20) and many of the actinomycete antibiotics (22, 56). Whether they are related to toxicity is uncertain, but the possibility is at least suggested by the number of instances uncovered. 6. THE METABOLISM OF POLYHYDRIC ALCOHOLS The compounds of this class that have been studied in connection with the carbon metabolism of fungi include the sugar alcohols, glycerol, and a few glycols. Mannitol and glycerol are the most im- portant. Sugar Alcohols. Mannitol was first discovered in fungi by Bracon- not (44, 45), and subsequent work during the nineteenth century revealed its general occurrence in the sporophores of basidiomycetes and ascomycetes (36, 59, 264). Other analyses of cellular material show that mannitol occurs in mycelium, e.g., of Aspergillus elegans (91) and Rhizopus japonicus (130), in sclerotia (70), and in spores (223, 242, 265). Mannitol may also appear in the medium, and very high yields — from 20 to 50 per cent of the glucose consumed — are found in the culture medium of Aspergillus sp. (23) and Byssochlamys fulva (186). Mannitol appears to function physiologically as a reserve food (164, 165); its general utilizability as a carbon source (Chapter 3) is con- sistent with this function. It is formed in culture from pentoses, hexoses, and glycerol (57, 260). The biosynthesis has not been studied, but it is a reasonable hypothesis that mannitol is formed by the reduc- tion of fructose or a fructose phosphate. Extracts of Stachybotrys atra carry out the reversible reaction (54): Fructose + DPNH + H+ ±=; Mannitol + DPN+ (10) Provided that a hydrogen donor is available, this reaction could synthesize mannitol. Consistent with a reductive origin of mannitol, mannitol formation is increased by restriction of the oxygen supply (23). D-Sorbitol has been reported formed by Boletus bovinus (132), and preformed mats of several basidiomycetes dehydrogenate sorbitol to L-sorbose (239). Other sugar alcohols which are formed by fungi include: D-volemitol (35), D-arabitol (77, 182a, 259), z'-erythritol (168, 220, 262, 265). Some of the sugar alcohols account for an appreciable fraction of the carbon metabolized; thus, D-threitol (/-erythritol) con- stitutes 13 per cent of the dry weight of the mycelium of Armillaria POLYHYDRIC ALCOHOLS 123 mellea (24). Intact cells of Fusarium lini oxidize erythritol to erythru- lose (86), and it is likely that similar reactions occur in those fungi which themselves accumulate sugar alcohols. Glycerol. The formation of glycerol by fungi is mentioned in Chap- ter 7 as an indication of the functioning of the Embden-Meyerhof respiratory pathway. The available data (86, 187, 188) are consistent with the metabolism of glycerol via this pathway, i.e.: Glycerol -» triose phosphate — » pyruvic acid (11) No enzymatic studies have, however, been made, and the role of phosphorylated intermediates is only conjectural. Glycerol phosphates are hydrolyzed by phosphomonoesterases (phosphatases), which also attack other organic phosphates (196). Hydrolysis of organic phosphates at acid pH is brought about by mycelial extracts of Actinomyces Israeli (104), and of fungi (5, 55, 58, 198, 199). In Trichophyton rubrum cytochemical tests suggest the presence of another phosphomonoesterase active at alkaline pH values (159), and enzymes of this type (alkaline phosphatases) have been reported in other fungi (50, 236). These and other phosphatases are considered elsewhere (Chapter 9). Baba (7, 8) isolated an unusual metabolite, monoallyl phosphoric acid, CH2=CH — CH2— OPOsH2, from cultures of Aspergillus niger metabolizing glycerol, and showed in addition the presence of an enzyme, a phosphatase, able to dephosphorylate the compound. Glycols. 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Leipzig: W. Engelmann, pp. 257. 265. Zellner, J. 1910. Monatsh. Chem. 31: 617-634. 6. Carbon Metabolism Part III Organic Acids, Lipids, and Other Compounds T, .he final group of carbon metabolites of the fungi has as its most important members the organic acids and the lipids. Brief consideration must also be given to other carbon compounds of various types. Many of the miscellaneous compounds included here have not been studied from a physiological point of view. Neverthe- less, they are involved in metabolism and therefore in physiology; they will not remain indefinitely as biochemical curiosities. Space does not permit an exhaustive listing of the aromatic and other com- pounds known to be produced by fungi; many more will be found in the reviews cited later. 1. THE METABOLISM OF ALIPHATIC ORGANIC ACIDS The fungi are perhaps best known in microbiology for their toler- ance of acidity — although this is much less general than usually thought — and for the ability of some to accumulate non-volatile organic acids. This section considers the metabolism of the organic acids, exclusive of the higher fatty acids, the keto acids of the respira- tory pathways, and those acids which are basically ring compounds. Sugar Acids. Acids derived by direct oxidation of either or both terminal carbon atoms of a sugar may be grouped together as the sugar acids. Of these, the only one to be formed in quantity is gluconic acid. The reaction was discovered by Molliard (379) in Aspergillus niger, and subsequent work has shown that at least small amounts of gluconate are formed by a variety of fungi (Table 1). So 131 132 CARBON METABOLISM III Table 1. The Formation of Organic Acids from Carbohydrates by Fungi and Actinomycetes* Acid Gluconic acid HOCHo— (HCOH)4- -COOH Mannonic acid HOCH2— (CHOH)4— COOH Galactonic acid HOCH2— (CHOH)— COOH Glucuronic acid J HOOC— (HCOH)4— CHO Saccharic acid HOOC— (CHOH)4— COOH 2-Ketogluconic acid HOCH2— (CHOH)3— CO— COOH Xylonic acid J HOCH2— (CHOH)3— COOH Arabonic acid J HOCH2— (CHOH)3— COOH Lactic acid CH3— CHOH— COOH Organisms Mucor plumbeus, Rhizopus spp.f (465); Ceratostomella ulrni (166); Coniophora cerebellar Merulius lacrymans,\ Polypo- rus vaporarius (465); Aspergillus spp. (80, 192, 321, 379); Dematium pullulans (422); Fumago vagans (80); Fusarium lini (399); Penicillium spp. (80, 321); Sclerotium delphinii (414) Aspergillus niger (321); Penicillium luteo- purpurogenum (13) Aspergillus niger (321) Penicillium spp. (298, 474); Stereum hirsu- tum (356); Ustulina vulgaris (567) Aspergillus niger (126) Penicillium brevi-comp actum (474) Fusarium lini (265) Fusarium lini (265) Allomyces arbuscula (292); Apodachlya sp. (23); Blastocladia pringsheimii (106); Chytridium sp., Macrochytrium botrydio. ides (152); Monoblepharella sp. (374); Mucor rouxii (132); Mucor spp. (502) > Pythiogeton sp. (107); Rhipidium sp. (231); Rhizopus spp. (453, 541); Sapro- myces elongatus (231); Botrytis cinerea (224); Monilia spp. (377, 457); Actino- myces spp. (184, 291); Streptomyces spp. (171, 424, 562) * Exclusive of the keto acids, the higher fatty acids, and acidic ring compounds. Organisms are listed by groups: phycomycetes, ascomycetes, basidiomycetes, Fungi Imperfecti, actinomycetes, in that order. t Identification doubtful. X See text. § In sulfite-inhibited culture. ALIPHATIC ORGANIC ACIDS 133 Table 1 (continued) Acid Citric acid CHs— COOH HO— G— GOOH CH.COOH Fumaric acid HOOC— CH CH— COOH Fumaryl-DL-alanine HOOC— CH CH3 CH— CO— N— CH H COOH Succinic acid HOOC— CHo— CH2— COOH Malic acid J HOOC— CHr- CHOH- -COOH Acetic acid CH3COOH Organisms Mucor piriformis (548); Coniophora cere- bella (75); Merulius spp. (30); Asper- gillus spp. (354); Botrytis sp. (444a, 465); Fusarium graminearum (159); Mo- nilia jormosa (458); Penicillium spp. (134, 136, 546, 547); Sclerotium del- phinii (414) Circinella sp., Cunninghamella sp., Mucor sp. (207); Rhizopus spp. (177, 207); Neurospora sitophila (spores) (535); Aspergillus spp. (465, 496, 513, 549, 550); Caldariomyces fumago (Fumago vagans) (141); Monilia jormosa (458); Penicillium griseo-fulvum (432); Strepto- myces coelicolor (145) Penicillium resticulosum (68) Blastocladia pringsheimii (106); Mucor spp. (196, 502); Rhizopus sp. (56); Neuro- spora sitophila (spores) (535); Fomes annosus, Merulius spp. (400) ; Aspergillus terreus (70, 433); Botrytis cinerea (224); Clasterosporium sp., Fumago vagans (80); Fusarium spp. (351); Monilia spp. (377, 457); Penicillium spp. (81, 410); Strep- tomyces coelicolor (145) Rhizopus spp. (56, 503); Neurospora sito- phila (spores) (359); Merulius spp. (30); Aspergillus spp. (52, 70, 496, 550); Botrytis cinerea (224); Fusarium graminearum (159); Penicillium spp. (228, 298, 465) Mortierella spp. (416); Mucor mucedo (415); Neurospora crassa (418); Fomes annosus, Merulius spp. (402) ; Polyporus anceps (415); Fusarium spp. (225, 351); Monilia Jormosa (457); Sclerotium del- phinii (414) ; Memnoniella echinata (419) ; Actinomyces spp. (291); Streptomyces spp. (416, 421) 134 CARBON METABOLISM III Add Ethyl acetate CH3COOCH:( 11 Oxalic acid HOOC— COOH Itaconic acid COOH I C=CH- I CH2 I COOH r.'V-Aconitic acid CH— COOH II C— COOH I CHo— COOH Itatartaric acid COOH HO— C— CHoOH I CHo I COOH Tartaric acid X HOOC— (CHOH)r- COOH Glyoxylic acid OHC— COOH Glutaric acid J HOOC— (CH2)3— COOH T;tblc 1 (continued) Organisms Endoconidiophora moniliformis (241); Peni- cillium digitatum (72) Rhizopus nigricans (545); Xeurospora crassa (418); Sclerotinia spp. (33, 545); Armil- laria mellea (261); Coniophora cerebella (75); Corticium centrifugum (277); Fomes annosus (401); Marasmius chordalis (478); Merulius spp. (401); Polyporus lumulosus (437); Poria vaillantii (402); Aspergillus spp. (67, 515, 545); Botryti* cinerea (224, 545); Dcmatium pullulans (34); Fusarium spp. (159. 357); Peni- cillium spp. (134, 156, 545); Sclerotium delphinii (414); Trichophyton tonsurans (533) Aspergillus itaconicus (314, 315); A. terreus (105, 349); Ustilago zeae (263) Aspergillus niger (455) Aspergillus terreus mut. (492) Fusarium spp. (357) Aspergillus niger § (87); Botrytis cinerea (224) Aspergillus niger (26) ALIPHATIC ORGANIC ACIDS 135 Table 1 (continued) Acid Organisms Glutaconic acid X Aspergillus niger (26) HOOC— CH=CH— CH2— COOH Dimethylpyruvic acid Aspergillus niger § (272, 438, 439) (CH3)2— CH— CO— COOH Ethylene oxide Aspergillus jumigatus (67, 368); Monilia a,/3-dicarboxylic acid formosa, Penicillium viniferum (458) O / \ HOOC— CH— CH— COOH far as is now known, the accumulation of large amounts of the acid is restricted to certain strains of Aspergillus spp. and Penicillium spp., and many fungi yield no gluconate by classical isolation procedures (321, 372,' 465). Because of the potential industrial importance of the process, the conditions of gluconate synthesis by Aspergillus niger have been in- tensively studied. It is found generally that a relatively high pH favors the formation of gluconate over that of citrate (101, 221, 513). Calcium carbonate is customarily added to the medium to control the pH: interference with metabolism by precipitated calcium gluconate can be minimized by the addition of boric acid or borax to the medium (386). As in other acid syntheses, but to a more pronounced degree, the accumulation of gluconate is favored by aeration (464, 554). It is also favored by a relatively high glucose concentration (46, 269) and a limiting amount of phosphate (269). Under appropriate conditions almost complete conversion of glucose to gluconate occurs (221). The biochemical basis of gluconic acid formation is probably the action of enzyme glucose aerodehydrogenase (glucose oxidase) (Chapter 7), although Dematium pullulans is reported to form gluconate (422) and to lack the enzyme (212). The first product in the reaction is glucono-8-lactone, which may be found in the medium with the free acid (80). Gluconate is further utilized by fungi (46, 514), but the path of metabolism is not known. The formation by Aspergillus niger of other hexonic acids — man- nonic acid from mannose and galactonic acid from galactose — has been reported (Table 1), but the process has not been studied further. Penicillium chrysogenum forms lactobionic acid from lactose (151a). Glucuronic acid is reported to be formed in traces by fungi (Table 1), although an unequivocal identification is still lacking. Saccharic acid is found in cultures of Aspergillus niger (126, 539). The appear- 1 36 CARBON METABOLISM 111 ance of pcntonic acids in cultures of Fusarium lini (265) has not been confirmed and the method of identification used leaves something to be desired. Lactic Acid. The formation of lactic acid in fungi is restricted almost entirely to phycomycetes (Table 1), and all fungi which con- vert a major fraction of the carbohydrate to lactate are in this group. The early history of the problem and the cultural conditions favoring lactic acid accumulation are reviewed by Foster (200), Lockwood et al. (353), and Ward et al. (541, 542). The optical isomer formed by fungi is usually L-lactic (dextro- rotatory) (536, 541), although formation of the d- and DL-forms has been reported (453, 457, 501). As discussed in Chapter 7, the anaerobic formation of lactate by Rhizopus oryzae conforms to the postulate that glucose is broken down by the Embden-Meyerhof sequence of reactions to yield lactate, ethanol, and carbon dioxide in equimolar amounts; the mechanism by which the equimolar ratio is maintained is not yet known. More lactate is formed aerobically than anaerobically, and the biosynthesis of this "extra" lactate is not understood. If it is assumed that all of the glucose used is converted to pyruvate, the aerobically produced lactate must arise from it or from ethanol, perhaps with 4-carbon acids as intermediates (109). Some carbon dioxide enters into the lactate molecule (226). Lactic acid production in high yields is obtained from cultures which are aerated either by shaking (54) or by the rotary drum method (542). It should be noted that Sakaguchi and co-workers (460) could not confirm the stimulation of lactate formation by aerobiosis. Lactate-forming organisms other than Rhizopus spp., that is, the lower phycomycetes and Streptomyces, do not, so far as is known, form "extra" lactate aerobically. Again in contrast to RJiizopus, some lower phycomycetes anaerobically convert 85 to 100 per cent of the glucose consumed to lactic acid (106, 231, 292). Evidently the bio- chemical basis here differs in important respects from that of Rhizopus; the difference recalls that between the homofermentative and the heterofermentative lactic acid bacteria. Lactic acid is not an end product of aerobic metabolism but is con- sumed after exhaustion of the principal carbon source (106, 171). An aerobic lactic dehydrogenase is known in Penicillium chrysogenum (111), but the enzymatic basis of lactate utilization in those fungi which accumulate the acid is not known. The oxidation of lactic acid by resting cells of P. chrysogenum is suppressed by glucose (278). ALIPHATIC ORGANIC ACIDS 137 Citric Acid. The ability of fungi to accumulate citric acid was first discovered by Wehmer (546, 547) in species of "Citromyces" (Penicilliam). Later (155) it was found that higher yields are obtained from selected strains of the black-spored Aspergilli, usually referred to as Aspergillus niger. Certain of these strains are presumably used for the commercial production of citric acid. Although the acid is probably a metabolic intermediate of aerobic metabolism in all fungi (Chapter 7), it appears in significant amounts only in a few forms (Table 1), primarily species — or, more correctly, certain strains of species — of Penicillium and Aspergillus. Most strains of A. niger have the potentiality for the accumulation of citric, gluconic, and oxalic acids; particular strains under a given set of conditions may form predominantly one acid or a mixture of two or three (48, 58). Strain differences contribute to the failure of different laboratories to agree on the details of the citric acid process, but the basic physiological events are presumably the same in all strains. Selection may increase the yield of citrate (169, 307), and, correspondingly, a strain may change its capacity as the result of mutation (218, 331). No adequate genetic analysis has been made; probably the typical strain is a heterocaryon with nuclei of different genetic types which segregate in spore formation (28). The biosynthesis of citrate by Aspergillus niger was investigated extensively during the period 1920 to 1940, largely by growth and replacement culture experiments which by themselves do not dis- criminate among the various possible metabolic pathways. Theories based on this work, all obsolete or correct only by chance, are re- viewed by Foster (200) and Walker (538). As discussed in Chapter 7, it now seems that fungi, like other organisms, utilize the citric acid cycle in aerobic respiration; the evidence, which is admittedly still incomplete, comes from enzymatic studies and from isotope experiments with citrate formation in Aspergillus niger. At least one other pathway of citrate (or isocitrate) metabolism is known in fungi (405) and may be found in the future to make a significant contribution to citrate synthesis. If we postulate the ubiquity of the citric acid cycle in fungi, accumulation of citrate is best regarded as an unusual modification, under genetic and en- vironmental control, of a process which occurs in all cells under aerobic conditions. Figure 1 is a simplified diagram of some of the known and probable relationships of organic acids in Aspergillus niger and, with some ex- ceptions, very likely in all fungi. The salient and physiologically significant implications of the scheme may be summarized: 38 CARBON METABOLISM III Amino acids Formate Glucose *" Gluconate Pyruvate Fumarate Lactate 1 ? C02 ' ^^CO 2 Oxalate Acetyl- coenzyme A >- Acetate > Lipids 0 N Glycolate Oxalacetate ? • ' ' '' , ' Citrate Succinate + Glyoxalate ' i Malate cfs-Aconitate Oxalate Itaconate Isocitrate ^C02 Succinate T a-Ketoglutarate Amino acids C02 Figure 1. The interrelations of the organic acids in metabolism. 1. Citric acid is shown as arising by the condensation of oxalacetate and acetyl-coenzyme A; an origin from succinate and glyoxalate is a second possibility (405). 2. In order for citrate to accumulate, oxalacetate must therefore be provided; in Figure 1 its only indicated source is the carboxylation of pyruvic acid. Whether this mechanism is quantitatively adequate for the high yields of citrate which have been reported (553) is not finally certain, although there is no doubt that carbon dioxide does enter into the citrate molecule in substantial amount (369). 3. Citrate may be oxidized further, i.e., it is not an end product. This is borne out both by studies of the time course of citrate ac- cumulation (47, 58a, 213) and by isotope studies (110, 337). 4. Especially during growth, the carbon of citric acid cycle inter- mediates is drawn upon for amino acid and lipid synthesis. The ALIPHATIC ORGANIC ACIDS 139 particular reactions shown in Figure 1 may not all occur in fungi, but there is no doubt that fatty acid and amino acid skeletons are built up from intermediates of carbohydrate oxidation. It is significant that strains of Aspergillus Jiiger which do not accumulate citrate have a high growth rate (185), and that highly acidogenic mutants are rela- tively slow growing (218). 5. Carbon may also be drawn off into gluconic or oxalic acid in Aspergillus niger and into still other acids in other fungi. These biochemical relationships explain many of the known physio- logical requirements for citric acid accumulation. In the first place, the concentration of available carbohydrate must be high (354); usu- ally glucose at 15 to 20 per cent is employed. Under these conditions there is an excess of carbon over that required for energy and for the synthesis of cellular material; this excess will then, we think, accumu- late wherever genetic and environmental factors permit rapid forma- tion but only slow breakdown of a metabolite. Such a metabolite may be directly on a major pathway or on a "shunt," diversionary, pathway (200). The requirement for a high carbohydrate concentration has its counterpart in the usual finding that highest yields of citrate are ob- tained if other major nutrients — nitrogen, phosphorus, magnesium, sulfur— are at levels which limit growth (103, 155, 308, 443). Data which contradict this generalization are, however, often reported, and there are undoubtedly secondary physiological effects of nutrients (472, 473). Minor elements affect citric acid production, but contradictory re- sults have been reported. There is no doubt, on the one hand, that zinc, iron, manganese, and copper are essential to growth and acid formation (518) or, on the other, that high concentrations of metals may lower yields (420). Between these two extremes, it appears from recent work that the best yield of citric acid and other acids is obtained at concentrations of the minor elements which allow some growth but are at the same time limiting to growth (130). Increases of zinc, for example, may result in a greater total yield but the percentage of conversion of glucose to acid is lowered (129). Perhaps the clearest example is the inhibitory effect of iron on citrate accumulation; the inhibition disappears in a medium in which growth is limited by low phosphate (472). The optimum level of any given nutrient, affected as it is by the strain used, the conditions of culture, and the other nutrients present, is a matter for individual determination. The important general Ill) CARBON METABOLISM III 160 Time, hours 360 Figure 2. Growth and chemical changes in the course of citric acid synthesis by Aspergillus niger in shake culture. Redrawn from P. Shu and M. J. Johnson, Industrial and Engineering Chemistry, Vol. 40, p. 1202-1205, July 1948. Copyright 1948 by the American Chemical Society and reprinted by permission of the copy- right owner. principle that growth and acid formation are to a degree competitive should not be obscured by these secondary effects, important as they may be for other reasons. The time course of citric acid formation in submerged aerated cul- ture is illustrated in Figure 2, from which it is evident that there is a phase of growth with little acid production, followed by a phase of acid formation and slow growth. Maximum yields obviously require that a balance be struck between growth and acid formation; at some point there is enough cell material to convert the residual glucose with maximum efficiency into product. These considerations apply in principle to all acid syntheses, and in fact to all syntheses of non- protoplasmic materials. Reference to Figure 1 makes it clear that many different carbon compounds, entering the metabolic pathway at different points, may be expected to provide carbon for citrate synthesis, and this proves to be true (354). There are so many possibilities here that experi- ments in which a carbon compound is added to a preformed mycelium in an attempt to trace biochemical pathways generally fail — almost any compound can be fed into the citric acid pool. ALIPHATIC ORGANIC ACIDS 141 Oxygen is required for citrate synthesis, both in surface culture (169) and in the more recently developed submerged processes (308, 172). Too vigorous aeration may, it has been suggested (186), sweep out the carbon dioxide rapidly enough to reduce citrate formation. The probably essential role of carbon dioxide, mentioned above, is supported by experiments showing that increases in the carbon dioxide of the atmosphere increase the yield of citrate (259). Measurements of the oxidation-reduction potential during the cul- ture cycle suggest that there is an optimum potential for citrate forma- tion; above the optimum, Aspergillus niger forms gluconic acid rather than citric, and below it ethanol is formed (332). Virtually all studies of citric acid formation indicate that a low cul- ture pH is essential to good yields; generally in surface culture an initial pH of about 2.0 is recommended for Aspergillus niger, 3.0 — 4.0 for Penicillium spp. (169, 213). In aerated cultures of A. niger a somewhat higher initial pH — about 4.5 — appears to be optimal (472). At pH values above the optimum gluconic and oxalic acids tend to replace citric (213, 383). The mechanism of this pH effect is unknown; it has a counterpart in the effect of pH on the balance of metabolic products formed by the lactic acid and the coliform bacteria. Metabolic poisons affect citric acid formation and have been used in attempts to elucidate the biochemistry of acid formation. The results are ambiguous, as is to be expected from the multiplicity of reactions which compete with or supply carbon to the citrate pool and from the fact that most poisons have several sites of action. The effect of slightly growth-inhibitory concentrations of the lower ali- phatic alcohols on the yield of citric acid is very striking (383, 455) and may prove, on further investigation, to be specific. Fumaric Acid and Related Acids. Four of the 4-carbon dicarboxylic acids — oxalacetic, malic, fumaric, and succinic — may be considered together inasmuch as they are readily interconvertible (see Figure 1). Oxalacetate is unstable and does not accumulate appreciably. Prob- ably all the other acids of this group can be detected by sufficiently sensitive methods in the cells or culture fluid of fungi during aerobic metabolism. For this reason the listing of fungi which produce these acids is somewhat deceptive; it is more a history of the interest of investigators than a survey of acid formation. Fumaric acid is formed in small amounts by many fungi (Table 1); certain of the Mucorales, especially Rhizopus spp., are the only fungi in which it accounts for a major fraction of the glucose con- sumed. Aspergillus fumaricus as originally isolated formed fumaric acid in large amounts (549), but the strain on examination ten years 142 CARBON METABOLISM III later proved to have lost the capacity to form the acid from glucose except under special circumstances (513, 550). Fumarate is also found in the sporophores of basidiomycetes (575, 576). Penicillium resticulosum in glucose medium forms fumaryl-DL- alanine, in which the amino group of alanine is joined in a peptide linkage with one carboxyl of fumarate (68). Succinic acid accumulation is rarely so great as that of the other acids considered thus far. Resting cells of Blastocladia pringsheimii accumulate succinate equivalent to about 10 per cent of the glucose metabolized (106), but the amounts formed by most organisms listed in Table 1 are much smaller. Malic acid has only rarely (56, 465) been reported to be formed in large amounts from glucose, although it is produced by a variety of saprophytic fungi (Table 1). Most of the identifications of malic acid, it should be noted, rest on color tests, the use of which is often difficult and uncertain; re-examination by enzymatic methods would be profitable. In fumaric acid synthesis, one of the most striking physiological aspects is the effect of zinc, which increases growth and glucose con- sumption and decreases, by as much as 80 per cent, the formation of fumarate (208). Fumaric acid formation is thus a symptom of retarded growth in the presence of an abundance of carbohydrate. By analogy with acid formation in Aspergillus niger, it is likely that any metal deficiency has the same effect (129, 130), but the role of other metals is still uncertain and iron above its optimum level seems to have little influence (208). Carbon dioxide has been shown by isotope studies to enter into the molecule of fumaric acid (204, 205) and succinic acid (144). Succinate formation by Streptomyces coelicolor and Blastocladia pringsheimii is enhanced by carbon dioxide (106, 144), but the relation of exogenous carbon dioxide to fumarate formation is uncertain (206, 207). The 4-carbon dicarboxylic acids under discussion probably are formed by more than one biochemical mechanism. Four such mecha- nisms have been suggested, namely: 1. Oxidative formation in the citric acid cycle. Bulk formation by this mechanism has not been proved. 2. The reductive carboxylation of pyruvic acid, either to oxalace- tate or directly, by the agency of the "malic enzyme," to malic acid. This is the best known and perhaps the major pathway; evidence for its role comes from studies on the incorporation of isotopic carbon dioxide and on the physiological effects of carbon dioxide mentioned above. 3. The direct condensation of two 2-carbon fragments in the Thun- ALIPHATIC ORGANIC ACIDS 143 berg-Wieland reaction. This pathway has been proposed in fungi on the basis of both chemical and isotope data (30, 49, 102, 202, 203, 450). As noted elsewhere (Chapter 7), the evidence is not conclusive, al- though there is of course some mechanism for the conversion of acetate and other 2-carbon compounds to larger molecules. 4. The cleavage of isocitric acid directly to succinic and glyoxylic acids (405). This system is probably identical with one known in bac- teria (477); its functions during glucose metabolism and its quantita- tive importance in fungi are not yet known. Formic and Acetic Acids. These two volatile acids are more char- acteristically bacterial than fungal metabolites, and under normal con- ditions of culture neither accumulates in such large amounts as do the non-volatile acids so far considered. Formic acid has not been reported to accumulate during carbohy- drate utilization, but several fungi form it during the metabolism of lactic and pyruvic acids (133, 454). These experiments suggest the well-known transacetylase reaction: Pyruvic acid + coenzyme A — » acetyl-coenzyme A + formic acid (1) The entrance of formate, or a derivative, into metabolism is demon- strated by isotope results (302) and by direct utilization of formate (137, 262). It has been suggested that formate may enter through the sequence of reactions: Glycine + formate -» serine -> pyruvate (2) Until active cell-free preparations are studied these reactions of formate must remain hypothetical in the fungi. Acetic acid is found in traces in the medium of many fungi and actinomycetes; the list in Table 1 omits non-specific determinations of "volatile acids" (54, 73). Larger amounts of acetate are formed by Streptomyces fradiae (421) and by Fusarium spp. (351). As also shown in Table 1, two fungi form ethyl acetate from carbohydrate. In view of the central role of acetate and acetyl-coenzyme A in metabolism we may assume their presence even if isolable amounts are not formed; this generalization is borne out by isotope dilution studies on Penicillium chrysogenum (368) and Streptomyces coelicolor (144). In particular organisms acetate may be formed from pentose (225), from ethanol by oxidation (398, 415), or, possibly, from glycine by deamination (562). Direct studies have shown that exogenous acetate is oxidized by Merulius niveus (30), Penicillium chrysogenum (278), and Zygorhynchus moelleri (382). 114 CARBON METABOLISM III The higher fatty acids, above acetic, are considered integrally with the metabolism of lipids. Oxalic Acid. Oxalic acid is a common metabolic product of fungi (Table 1) especially ol the Agaricales. Species of Penicillium and Aspergillus accumulate the largest amounts but substantial oxalate production occurs in a few other fungi, e.g., Sclerotium delphinii (414). Sporophores of basicliomycetes often contain oxalate (17, 575, 576). The physiology of oxalate formation deserves more study. The acid is formed from various carbon sources (49); the most favorable cultural conditions include a high carbohydrate concentration, ade- quate aeration (49, 545), a limited supply of inorganic nutrients (129, 447), and, most important, a relatively high initial pH (46, 49, 299). The enzymatic basis of the pH requirement has been elucidated by Shimazono (470): at low pH an enzyme, oxalic acid decarboxylase, is formed and converts oxalate to formate and carbon dioxide. This finding suggests, for the higher basicliomycetes at least, that oxalate is a normal metabolite and accumulates under conditions of an enzyme deficiency. Cultures of Aspergillus niger utilize oxalate pro- vided that the pH is below 5.5 (27). In Corticium centrijugutn oxalic acid production is increased by a growth-limiting deficiency of thiamine (391). This relation is con- sistent with the general view that oxalic and other acids are formed maximally when available carbon cannot be used for growth. The biochemical origin of oxalic acid has been investigated rather frequently, but none of the pathways proposed has been proved (4, 5, 403). Recent results with isotopic carbon are consistent with the origin of oxalate from oxalacetate (88, 138a, 201, 338). The possibility that glycine, which in different organisms is metabolically related to other 2-carbon compounds, is involved has not yet been explored. Progress on the problem depends on the preparation of active non- cellular preparations. Itaconic and Itatartaric Acids. Itaconic acid was discovered by Kinoshita (314, 315) to be formed by a strain which he designated Aspergillus itaconicus n. sp. This strain has lost its capacity to form more than small amounts of the acid, but strains of Aspergillus terreus have been found to produce large yields (105, 384). Other strains of the same species form no itaconate, and ultraviolet-induced mutants yield more or less acid than the parent culture (349). The optimum conditions for itaconic acid formation are rather different from those which give best growth of the organism (348, 350, ALIPHATIC ORGANIC ACIDS 145 352, 384). Distinctive features include the very low pH optimum — about 1.9 for shaken cultures — and the pronounced effects of sodium chloride and magnesium sulfate under certain conditions. The low pH optimum of the processes has been shown conclusively (334) to be determined by the fact that the enzyme system for itaconate formation is present only during cultivation at low pH. Itaconic acid is formed in vitro from ds-aconitic acid by the enzyme cis-aconitic decarboxylase (41); since n's-aconitate is a component of the tricarboxylic acid cycle, the formation of itaconic acid may be regarded as an alternate metabolic pathway for glucose carbon (Figure 1). A mutant of Aspergillus terreus, obtained by ultraviolet irradiation, forms a mixture of itatartaric acid and its lactone (492). The struc- ture (Table 1) has an obvious resemblance to that of itaconate, and it is reasonable to suppose that the two acids are biochemically related. Other Acids. Tartaric acid has been reported in small amounts in cultures oiFusarium spp. (Table 1), but the criteria of identification were not wholly satisfactory. Using color tests and copper reduction as criteria, Bernhauer and Bockl (51) found that most strains of Aspergillus niger form tartrate during incubation of preformed mycellium with ethanol. The problem merits study with the more sensitive methods now available for isolation and determination of acids. Glyoxylic acid (OHC — COOH), often accompanied by glycolic acid (HOCH2 — COOH), is formed in traces during carbohydrate break- down by some fungi (Table 1); it has also been reported as being formed during the metabolism of acetate (55, 125, 403), ethanol (136), and citrate (125). It should be noted that substances giving the color reactions for glyoxylic acid are found in sterile autoclaved culture media (157); it has not always been clear that adequate controls have been included in experiments on the occurrence of glyoxylate. Glyoxylate has been proposed as an intermediate in the oxidative formation of oxalate from acetate by the following reactions (403): Acetate — » glycolate — > glyoxylate -» oxalate (3) It is not yet certain that these reactions occur. The fact that glyoxylic and glycolic acids replace glycine in the nutrition of a mutant of Nenrospora crassa (566) suggests a second pathway for their utilization and formation. Finally, the ability of fungi under some conditions to split isocitrate to succinate and glyoxylate and to oxidize glycolate to glyoxylate has been shown unequivocally (405). Glyoxylate is also, it should be noted, a product of the action of allantoicase (Chapter 8). HG CARBON METABOLISM III The other acids listed in Table 1 — glutaric, glutaconic, dimethyl- pyruvic, and ethylene oxide a,/J-dicarboxylic acids — have as yet no established physiological or biochemical significance. The first two have been reported only once, from xylose cultures; if their formation can be confirmed, a route from pentose to a-ketoglutaric acid is a pos- sibility. Dimethylpyruvic acid accumulates in cultures poisoned with bisulfite. Suggestions on the biochemical origin of dimethyl- pyruvic acid are reviewed by Birkinshaw (63). Ethylene oxide a,/?-dicarboxylic acid ( R— CO— CH2— COOH (4) Fatty'acid /3-Keto acid R— CO— CH2— COOH -^ R— COOH + CH3COOH (5) Acetate Coenzyme A is required at two points: in the "activation" of the original fatty acid, forming an acyl-coenzyme A, and in the formation of acetate, which appears as acetyl-coenzyme A. Repetition of reac- tions 4 and 5 leads ultimately to complete conversion of the long- chain fatty acid to 2-carbon compounds. The process is reviewed in detail by Green (245) and by Chaikoff and Brown (114). Evidence for /3-oxidation in fungi derives almost entirely from studies on the breakdown of fats and fatty acids by growing or resting 1 54 CARBON METABOLISM III cells. Interest in the problem was first aroused by the finding that methyl ketones are present in certain of the fungal cheeses and in edible oils attacked by fungi (482, 494). In a number of experiments it has been shown that Penicillium glaucum converts a fatty acid to a methyl ketone of one less carbon atom, i.e., R_CH2— CHo— COOH -* R— CO— CH3 + C02 (6) This reaction has been demonstrated in experiments on many fatty acids (2, 509, 511), e.g.: CH.3— CHo— CHo— COOH -> CH3— CO— CH3 (7) Butyric acid Acetone CH3— (CH2)6— COOH -* CH3— (CH2)4— CO— CH3 (8) Octanoic acid Methylamyl ketone CH3— (CH2)10— COOH -* CH3— (CH3)8— CO— CH3 (9) Dodecanoic acid Methylnonyl ketone The same general reaction is found in Penicillium notatum and in Aspergillus spp. (149, 380, 569). Thaler and co-workers (510) propose the following sequence of reactions in the formation of methyl ketones from fatty acids: R-CH2- CHo— COOH -» R— CH=CH— COOH Fatty acid a, ^-Unsaturated fatty acid -> R—CHOH— CHo— COOH /3-Hydroxy fatty acid -* R— CO— CHo— COOH -> R— CO— CH3 + C02 (10) /S-Keto fatty acid Methyl ketone The evidence for this sequence is that methyl ketones arise during the growth of Penicillium glaucum on «,/?-unsaturated acids and /?-hy- droxy acids. Thus, methylpropyl ketone is formed from hexanoic acid, 2-hexanoic acid, and /3-hydroxyhexanoic acid. Further, fungi are known to form /3-keto acid decarboxylases (309). This scheme, up to the formation of the /3-keto acid, agrees with our current understanding of the process in animal cells, but until en- zymatic studies are made, the identity of the two systems remains hypothetical. The last step — formation of the methyl ketone — does not go on appreciably in the normal animal cell, which instead forms, as mentioned above, a 2-carbon compound and a new fatty acid. This suggests that methyl ketone formation by fungi is an abnormal or at least unusual reaction, perhaps to be attributed to toxicity of the substrate fatty acid (494). It is reasonable to suppose that complete oxidation of fatty acids in the fungi follows the animal system, with liberation of acetyl-coenzyme A or free acetate as the last step. The LIPIDS 155 strongest evidence for this supposition is in the finding by Webley and others (544) that the oxidation of ^-substituted phenyl fatty acids by Nocardia opaca, an actinomycete, yields end products in conformity with predictions based on /J-oxidation. Comparable studies on the fungi have not yet appeared. Indirect evidence for /^-oxidation in the fungi is provided by the incorporation of fatty acid carbon into the side chain of penicillin (380, 516). The most effective precursors are fatty acids which by their structure are not susceptible to complete /3-oxidation. It has been argued from simultaneous adaptation experiments that Streptomyces aureofaciens oxidizes octanoate by some mechanism other than /3-oxidation (276). However, it is now known that the adaptation technique is unreliable in fatty acid oxidation, since the adaptive system may be that which activates the fatty acid; failure of octanoate-adapted cells to oxidize butyrate may therefore mean only that activation of the two acids is carried out by different enzymes. Fatty acid oxidase systems probably occur in fungi and actinomy- cetes (387, 543). In addition, mycelial extracts of Penicillium glaucum and of Aspergillus spp. contain a lipoxidase, capable of oxidizing un- saturated fatty acids (387). The Metabolism of Steroids and Triterpenes. Sterols occur in small amounts in the lipid fraction of most fungi; ergosterol appears to be the compound of this group most often formed by fungi, but other sterols have been tentatively identified (Chapter 2). Cultural conditions which favor sterol formation are reviewed by Bernhauer (50). It appears that sterol and fat synthesis are competitive reactions; the effect of pH on their formation (428) is perhaps the strongest evidence for this competition. Biochemical studies on yeast and on fungi indicate that ergosterol is formed in large part from 2-carbon compounds, although isovaleric acid may also be involved (146, 318, 407). The amount of sterol formed varies among strains and among single spore cultures of a given strain (556). The breakdown of exogenously supplied steroids has been studied in two connections: (1) the utilization of steroids for energy and carbon, and (2) partial and limited transformations of the steroid molecule. Cholesterol is oxidized in soil (521) by actinomycetes and by Gram- negative bacteria; it is not clear which organisms are responsible for steroid breakdown in natural soils (462, 523, 525). Some fungi are apparently able to grow with ergosterol as carbon source (462), and some of the products of the action of Proactinomyces erythropolis on ] -1(5 CARBON' METABOLISM HI steroids have been identified (522, 524, 526), but most of the pathway of steroid oxidation is unknown. Specific microbiological transformations of steroids, i.e., reactions at single specific points on particular steroid molecules, have been studied extensivelv, first with bacteria and yeast (364. 532), later with actino- mvcetes (271, 329, 330, 555) and fungi (423). The pharmacological importance of these processes has stimulated since 1952 a very large volume of experimental work, for which the fungi and actinomycetes have been the best material. The known processes mav be classified as follows ^557): 1. H\droxylations, specific introduction of the — OH group. Ex- amples are shown in Table 2: it will be noted that among the most frequent positions of attachment are 6/?, 11a, 11/3, and 17a. Table 2. The Hvdroxvlation of Steroids by Fungi and Actinomycetes* Or^anU::. Steroid Position Hydroxylatedf rgillus spp. 68, 11a, 17a, 21 -halothecium roseum -". 11a, la Colletotrichum antirrhini 15a Coniothyrium sp. 11.8 Cunninghamella blakesleeana 118 Curvularia spp. "a, 88 (?), 11/3, 14a Dactylium dendroid, I Ha, l~a Didymella todakii 16a Gibberella baccata 15a Hdicostylum piriform 68, 8£, 11a, 14a Lenzites abietina 6,8, 150 Mucor spp. 68, 88 (?), 14a X-rurospora crassa 38 or 9a Ophiobolus herpoirk 21 Pinicillium spp. § 11a Peziza spp. a Phycomyces blakesleeanus :■ 15/3 ProactinomyccS roseus $ Rhizopus spp. 68, 7/3, 8|, 11a ".ptomyces spp. 68, 8£ or 9| (?), 118, 16a Trichotfucium roseum 68, 17a * Except as noted, from Wettstein (557). A more complete list appears in the review of Eppstein et al. (182a). f The specific compounds attacked, and often the yield of product, are given in the original papers cited by Wettstein (557). | Dulaney et al. (1~3). 5 Dulaney et al. (172). LIPIDS 157 2. Hydrogenation of steroid aldehydes or ketones by bacteria, yeast, Streptomyces spp.. and Rhizopus nigricans. 3. Hydrogenation of ring carbon atoms by Clostridium sp., Strepto- myces sp., and Rhizopus nigricans. 4. Dehydrogenation of steroid secondary alcohols b\ bacteria and actinomycett^. 5. Epoxydation ol steroids containing an isolated double bond, by Curuularia lunata and Cunninghamella blakesleeana. 6. Dehydrogenation in ring A. with or without degradation of the side chain, by Fusarium solani, Calonectria decora, Cxlindrocarpon radicola, and other fungi. 7. Oxidation of the side chain only, e.g., by Gliocladium caten- ulatiun. 8. Ring cleavage with lactone formation, e.g., bv Pejiicilliufn chrysogemim. Specific enzvme systems must, of course, be responsible for these transformations, and some knowledge of animal and bacterial enzymes is available (244, 577). The 6/3-hydroxylation of progesterone by Aspergillus ochraceus is accelerated by zinc: it has been suggested that the metal is required for the synthesis of an induced enzyme which carries out the reaction (17- Steroidal sapogenins, e.g., diosgenin, appear not to be hydroxylated by fungi or other microorganisms (376). Chemical studies on several basidiomycetes have brought to light a new series of fungal metabolites, the tetracyclic triterpenes. As terpenes they are related to the carotenoids, but their structure also bears an obvious relation to that of the sterols; they are not, however, true sterols. Similar compounds are known in higher organisms, e.g.. betulin in plants and squalene in animals. The structure of eburicoic acid, formed bv several Hxmenomvcetes (151, 220, 281), may be taken as an example of the group: HOOC HO— CI 158 CARBON METABOLISM III Other compounds of this type include the closely related poly- porenic acids A, B, and C, isolated from Polyporus spp. and Poria cocos (91, 92, 151, 154, 257, 260). The triterpenoids are biosyntheti- cally related, as judged by the occurrence of all three polyporenic acids in one species (154) and by the fact that three strains of Poria cocos form polyporenic acid B and a fourth forms eburicoic acid (151). In Polyporus eucalyptorum eburicoic acid constitutes at least 20 per cent of the dry weight of the mycelium (220) and must therefore be con- sidered a major metabolite. The chemistry of these compounds has been reviewed by Jones and Halsall (306); triterpenoids occur also as lichen products (24). The antibiotics cephalosporin P1 and helvolic acid are tetracyclic compounds and may be related to the steroids, but their complete structure is not known (100). The Metabolism of Carotenoids. The carotenoid pigments, which appear among the unsaponifiable lipids, include: (1) the true caro- tenes, e.g., /3-carotene and lycopene, and (2) oxygen-containing deriva- tives, e.g., rhodoviolascin. All may be considered as built up of C I eight isoprene units, the carbon skeleton of which is: C=C — C=C. In addition to the colored carotenoids, colorless polyenes are found in the fungi and will be considered with the pigments. The formula of ^-carotene illustrates the essential features of the carotenoid pigments: CH3 CH3 \ / C GH3 GH3 / \ I I CH2 C— CH=CH— C=CH— CH=CH— C=GH— CH=CH— CH I II CH2 C — CH3 \ / CH2 CH3 CH3 \ / GH3 CH3 C I I / \ =C— CH=CH— CH=C— CH=CH— C CH- II I CH3 — C CH2 \ / CH2 LIPIDS 1J^ Carotenoids are of widespread but not universal occurrence in the fungi, including representatives of all the major taxonomic groups (232, 236, 264). Table 3 lists the carotenoids of a few fungi which Table 3. Carotenoids of Some Fungi Organism Carotenoids Allomyces javanicus 7-carotene,* j8-carotene, lycopene, phytofluene (178, 529) Phycomyces blakesleeanus a-carotene, /3-carotene, * 7-carotene, ^-carotene, \ lyco- pene, neurosporene, phytofluene, phytoene (233, 310) Neurospora crassa /3-carotene, 7-carotene,* 0-carotene, lycopene,* spiril- loxanthin (rhodoviolascin),* lycoxanthin, neurospo- rene,* phytofluene, phytoene, unidentified com- pounds (264) Dacrymyces stillatus a-carotene, /3-carotene,* 7-carotene (?), £-carotene,t torulene, cryptoxanthin, zeaxanthin, phytofluene, phytoene (?) (234) Puccinia spp. /3-carotene, 7-carotene (?), lycopene (293) * Quantitatively important. f See Haxo (264) on fungal ^-carotene. have been intensively studied. In contrast to higher plants, /3-carotene is not so nearly universal in the fungi, and acidic carotenoids, e.g., torularhodin, are more common (232). The intracellular distribution of fungal carotenoids has not been systematically investigated. In certain of the higher fungi, carotenoid pigments occur both in rod-shaped structures, apparently part of the mitochondrial system, and in oil droplets in the cytoplasm (266, 267). The carotenoids of the male gametangia of Allomyces spp. are found in cytoplasmic oil droplets (178), those of Rhodotorula rubra in granules in the pericapsular region (127). The physiological conditions of carotenogenesis are to some degree specific for individual organisms. In Phycomyces blakesleeanus, the time course of carotene formation (Figure 5) indicates that the bulk of the carotene is formed after growth has been completed and that carotenoid formation is independent of lipid formation. Independ- ence of fat and carotene synthesis also appears from their divergent pH requirements (238). Carotenogenesis is affected, of course, by carbon and nitrogen nutrition, but the data do not afford a basis for generalization. Cultivation of Phycomyces blakesleeanus at low temperature decreases total carotene synthesis but does not affect the 1()0 CARBON METABOLISM III Figure 5. Growth, fat forma- tion, and carotenogenesis in Pliycomyces blakesleeanus. Re- drawn from Goodwin and Will- mer (238), by permission of the Cambridge University Press. Time, days proportions of the different carotenoids formed (215), as it does in Rhodotorula rubra (392). Oxygen is required for carotene formation by Neurospora crassa (572), and presumably this is true of other fungi. The mycelial carotenoids of Neurospora crassa do not develop in complete darkness, although the conidial carotenoids do; this problem has been studied by Zalokar (572, 573). Very brief exposure to light is sufficient to activate pigment synthesis, which is proportional to light intensity up to a saturation value. The action spectrum of light activation is consistent with, but does not, of course, prove the participation of a flavoprotein as the photoreceptor. Calculated quantum yields are so high as to make it necessary to postulate that activation is an indirect process. Carotenogenesis in other fungi is stimulated by light but does not require it (131, 219). Synthesis by green plants does not require light. Carotenoid formation is inhibited by streptomycin, chloram- phenicol, and isonicotinic hydrazide at concentrations which do not inhibit growth (235). Diphenylamine inhibits carotenogenesis in both bacteria and fungi (233, 528). Its effect is to reduce the absolute and relative amounts of the more unsaturated carotenoids, e.g., /3-carotene, and to increase the relative amounts of the more saturated, e.g., phytofluene and neurosporene. The biosynthesis of the carotenoids may be considered under three LIPIDS 161 heads: (1) the origin of the isoprene skeleton, (2) the mode of con- densation of isoprene units to form the 40-carbon chain of the carotenoids, and (3) the relation of the different carotenoids to one another. On grounds of comparative biochemistry and especially from studies on higher plants (304), the most likely route of synthesis of the iso- prene skeleton involves the following steps (236, 346): 1. Formation of acetoacetic acid from acetate and coenzyme A. 2. Condensation of acetoacetate with acetyl-coenzyme A to form ^-hydroxy, /3-methylglutaryl-coenzyme A. 3. Conversion of /^-hydroxy, /?-methylglutaryl-coenzyme A to p- methylcrotonyl-coenzyme A. This process involves more than one reaction and includes a decarboxylation. The condensation of two 5-carbon units to yield a 10-carbon chain, followed by successive reactions with new 5-carbon units, could in principle yield the 40-carbon chain of the carotenoids (346). Evidence for the foregoing scheme in the fungi is somewhat limited, but adequate to establish it as a working hypothesis. Caroteno- genesis in fungi is stimulated by /?-methylcrotonaldehyde (237) and by pantothenic acid and pantethine, which enter into the coenzyme A molecule (249). The labeling of the ^-carotene of Mucor hiemalis grown on C14-acetate is qualitatively consistent with the proposed mechanism (247, 248, 248a). That fungi form carotenoids during cultivation on acetate (463) cannot be urged as proof for the hypoth- esis, although again the fact is consistent with it. The final set of problems is associated with the relationship of the carotenoids to one another. From genetic data on higher plants, Porter and Lincoln (427) proposed a unilinear sequence of formation of the carotenoids by successive dehydrogenations, that is, that the unsaturated hydrocarbons are derived from the saturated, as follows: Tetrahydrophytoene -> phytoene -» phytofluene -> ^-carotene -» neurosporene — > lycopene — » y-carotene — » /3-carotene — > a-carotene. Although this scheme fits the behavior of mutants of Chlorella vulgaris (138), Rhodotorula rubra (89) and Neurospora crassa (264, 469), it cannot be regarded as proved. Goodwin (232, 233) has argued, largely on the basis of inhibitor studies, that the more saturated polyenes, e.g., phytofluene, are formed by one pathway, the less saturated, e.g., /3-carotene, by another, both from a common precursor. The effects of added methylheptenone and /?-ionone have been interpreted as ] (39 CARBON M I T ABOLISM III supporting the second hypothesis (361). It must be concluded that the question is still open. It is usually assumed that the oxidized carotenoids, neutral and acidic xanthophylls, arise by oxidation of the hydrocarbon polyenes (264). Although /?-ionone increases carotenogenesis in Pliycomyces blakes- leeanus, isotope evidence indicates that the molecule is not, as one might expect, incorporated as such into the /^-carotene structure (182). Two functions have been suggested for the carotenoids: a direct role as receptor for the light stimulus in phototropism, and an un- defined role in sexual differentiation. Phototropism is discussed later (Chapter 11), and it is concluded that there is no satisfactory proof of the direct involvement of carotenoids. Although the carotenoid pigments and the colorless polyenes of Allomyces javanicus are restricted to the male gametangia (178, 529), an essential role in the sexual process is rendered less probable by the finding (527) that diphenylamine inhibits carotenogenesis in A. javanicus without appreciably affecting the sexual reaction. It seems likely that the carotenes are side products of a metabolism which is in some respects at least distinctive to the male gametangium. The (+) and (— ) strains of some of the Mucorales show quantitative differences in carotenoid content; studies, as reviewed by Goodwin (232), have been confined to the fact itself, and it would appear that in Pliycomyces blakesleeanus the difference between (+) and (— ) strains is not consistent (219). Zygospore formation or merely pairing of compatible strains in some of the Mucorales is accompanied by in- creased formation of carotene (29, 270a). The most serious deficiency in the study of fungal carotenoids is the failure to determine whether or not carotenes occur in association with proteins or lipoproteins. Such an association is theoretically necessary if they are to function in metabolism, e.g., in the reception of light stimuli. Carotenoproteins are well known in animals (234), and the now classical studies on rhodopsin (147, 537) provide a model for the investigation of other pigments. The Metabolism of Hydrocarbons and Hydrocarbon Derivatives. It has been mentioned (Chapter 3) that hydrocarbons support growth of some actinomycetes and fungi. The oxidation of n-dodecane and other hydrocarbons by Proactinomyces opacus can be demonstrated manometrically (Figure 6); the organism also oxidizes long-chain saturated fatty acids, which may be intermediates in hydrocarbon oxidation. Details of the breakdown of hydrocarbons are not known; LIPIDS 163 300 2 200 c a> 00 >> X O 100 60 120 Time, minutes 180 Figure 6. The oxidation of hydrocarbons by Proactinomyces opacus. Redrawn from Webley and DeKock (543), by permission of the Cambridge University Press. ZoBell (577) reviews our knowledge of the process in bacteria. Inter- mediates appearing during the oxidation of naphthalene by Nocardia sp. suggest that benzenoid compounds may be formed (520). Hydrocarbons produced by fungi fall into two groups: ethylene and its derivatives, and the substituted hydrocarbon polyenes and poly- enynes. From the biological effects of volatile products of Penicillium digitatum it seemed probable that the organism produces ethylene (59, 378). This has been confirmed (571); ethylene is probably pro- duced also by Blastomyces dermatitidis (394) and by Fusariurn oxy- sporum v. lycopersict (167). However, the ethylene produced by fungus-infected plant tissues is believed to be a host product, stim- ulated quantitatively by infection, rather than a fungus metabolite (559). An interesting volatile hydrocarbon, 2-methylbutene-2 (trimethyl- ethylene), is formed by uredospores of Puccinia graminis tritici and is perhaps responsible for self-inhibition of germination of the rust spores (199). Several antibiotics produced by fungi and actinomycetes are in two 1 64 CARBON METABOLISM III related classes, the polyenynes and the polyenes (406). The polyenynes, characterized by a system of conjugated triple bonds, include, among compounds of known structure, diatetrynes 1 and 2 of Clitocybe diatreta (10), agrocybin of Agrocybe dura (305, 312), and mycomycin, produced by Nocardia acidophilus (113). Some have nitrogenous substituents, e.g., diatetryne 2: HOOC— CH = C H— C^C— Ce=C— C=N Mycomycin, on the other hand, is an acid containing no nitrogen: HC=C— C=C— CH=C=CH— CHrrCH- CH=CH— CH2— COOH Acetylenic compounds are also known in higher plants and in other fungi than those named here (9, 11, 97). In an unidentified basidio- mycete, the formation of polyacetylenes early in the culture cycle is followed by their disappearance; this may be interpreted as a re- utilization of the compounds (98). The conjugated polyenes include a number of antibiotics the complete structure of which is not known; the polyene structure is inferred from their ultraviolet absorption spectra (28a, 530, 534, 558). All are fungistatic agents. The polyenynes, except for mycomycin, have been found only among basidiomycetes; the known polyene antibiotics, on the other hand, are produced primarily by actino- mycetes. Grifolin, formed by Grifola confluens (275), is a substituted hydrocarbon with one pair of conjugated double bonds. Species of Corticium produce two yellow pigments, both containing a system of conjugated double bonds comparable to that of the carotenoid pigments (183, 246); other aspects of their structure, how- ever, relate them to atromentin and thelephoric acid, considered later with the aromatic compounds. The sporophore of Lactarius spp., and probably the ascocarp of Peziza sp., contain a rubberlike polymer, a as-polyisoprene (491). 3. THE METABOLISM OF AROMATIC COMPOUNDS Aromatic Compounds of Fungi. The fungi resemble the higher plants and differ from the bacteria in that many species convert carbohydrate to aromatic compounds not known to be essential metab- olites. These compounds have been reviewed in detail by Birkin- shaw (63), by Raistrick (430), and, in lichens, by Asahina and Shibata (24). Any classification of them must of necessity be based on their chemical structure and may unintentionally obscure physiological and biosynthetic relations. Thus, a substituted benzene and the quinone AROMATIC COMPOUNDS 165 Table 4. Representative Aromatic Compounds Produced by Fungi Group* I Compound Methyl cinnamate CH=CH— COOCH3 Anisaldehyde CHO OCH3 Gentisyl alcohol OH CH.OH %X Homoprotocatechuic acid CH,COOH ^N OH OH 3,5-Dihydroxyphthalic acid OH COOH COOH HO Gallic acid COOH HO OH OH Organism(s) Lentinus lepideus (74) Trametes suaveolens (64) Daedalea juniperina (69) Polyporus benzoinus (77) Penicillium spp. (66, 93, 176, 180) Polyporus tumulosus (437) Penicillium brevi-compactum (142, 408) Phycomyces blakesleeanus (44) * See text. Ifirt CARBON METABOLISM III Table 4 (continued) Group* I Compound Gladiolic acid (tautomeric) OCH3 CH3 I COOH Organism(s) Penicillium gladioli (253, 254) CHO CHO Cyclopolic acid OCH3 CH3 I COOH Penicillium cyclopium (84) HO CH2OH CHO Cyclopaldic acid OCH3 CH3 I COOH Penicillium cyclopium (84) HO CHO CHO Chloramphenicol Streptomyces venezuelae CO— CHC12 (444) N02— < \ / NH I -CHOH— CH— CHoOH Mycophenolic acid CH3 I HOOC— CH2— CH2— C=CH— CH, OH CH3O CO CH2 Sulochrin OCH3 I Penicillium spp. (6, 65, 83, 142) O Oospora sulphur ea-ochracea (395, 396) OH CO S^ HO COOCH3 HO CH3 AROMATIC COMPOUNDS 167 Table 4 (continued) Group* Compound II 4-Methoxy-2,5-/?-toluquinone O CH3 CH30 O Organism(s) Coprinus similis, Lentinus degener (12) Spinulosin CH Aspergillus fumigatus, Peni- cillium spp. (16, 79, 94) Oosporein CH HO OH HO Oospora colorans (328), Chaetomium aureum CH3 (347) OH Atromentin HO O HO- \ / / \ Pa.xillus atromentosus (325) -OH O OH Thelephoric acid O O A r Thelephora spp., Hydnum ferrugineum (326), Loba- ria retigera (lichen) (23) HO- _/ \_/ \_ CH=CH— CH=CH— COOH COOH OH 168 CARBON METABOLISM III Table 4 (continued) Group* Compound Organism(s) III Chrysophanic acid CH3 Penicillium islandicum (289) CHoOH IV Ravenelin OH (^\/°\r/%/ CH3 OH O Pinselic acid CH3 OH ,o /w\/^ OH OH 6 COOH V Javanicin CH30 OH O Dermocybe sanguined (327) Aspergillus quadrilineatus (290) Helminthosporium ravenelii (389, 431) Penicillium amarum (390) CH2— CO— CH3 CH3 Fusarium javanicum (20. 21, 61a) OH O AROMATIC COMPOUNDS 169 Group* VI Stipitatic acid O Table 4 (continued) Compound OH Organism(s) Penicillium stipitatum (68, 150, 165) HO COOH VII Puberulonic acid O OH V HO HO Lecanoric acid CH3 Penicillium puberulum (25, 81) CO' Parmelia spp. (lichens) (24) ^ -CO— o- -OH HO OH Gyrophoric acid CH3 CO— O— CH3 Gyrophora esculenta and other lichens (24) CH3 OH I COOH -co— o- OH HO OH VIII Nidulin CI CH3 Aspergillus nidulans (163) CI HO /V -co— o- -o -0CH3 \^ CI CSH 5X110 170 CARBON METABOLISM III formed from it by oxidation arc more closely related biologically than are two differently substituted benzene compounds. The fol- lowing division of the aromatic products synthesized by fungi, taken largely from Raistrick (430), should be considered as a formal classifica- tion only. Examples of the major types are illustrated in Table 4. Group I, substituted benzenes, is the largest single group of aromatic compounds, and presents a bewildering array of possible biological interrelations. Some of the members of this group are soluble metab- olites of the culture filtrate, others occur in the mycelium, and still others are associated with reproductive structures (214, 243). Members of Group II, substituted benzoquinones, often have an obvious metabolic relation, as oxidation products, to phenolic com- pounds of Group I. Many are derivatives of toluquinone. Anthraquinones, Group III, are pigments and are known also in lichens and in higher plants (280). The fungal anthraquinones may be considered as derivatives of chrysophanic acid. Group IV, the xanthones, is represented in the fungi by only a few certain examples. Rubrofusarin, formed by Fusarium spp. (389), is probably the same methyl xanthone as asperxanthone of Aspergillus niger (355). Group V comprises the naphthoquinones. In addition to javanicin, other fungal products of this type have been identified as products of Fusarium spp. (104, 451) and other fungi (40, 161). Solanione (552) and oxyjavanicin (21) are probably identical with, respectively, javani- cin and fusarubin (63). Derivatives of tropolone, Group VI in Table 4, are only quasi- benzenoid, inasmuch as the ring contains seven carbons. Such higher plant products as thujaplicin and colchicine are tropolones (412). An origin of tropolones from phenolic compounds has been suggested (371). The depsides, Group VII, are known so far only from lichens; it may be presumed, by analogy with other lichen products (112), that the fungus component of the lichen is responsible for their formation. Nidulin illustrates the depsidones (Group VIII). The lichen depsi- dones are similar; some, e.g., gangaleoidin and diploicin, are chlo- rinated, most are not (24). The physiological importance of the aromatic compounds is largely unknown, most effort to date having been concentrated on problems of identification. In what follows, three topics will be briefly con- sidered: pathways of biosynthesis, enzymatic transformations, and the possible metabolic significance of the aromatic compounds. AROMATIC COMPOUNDS 171 The Biosynthesis of Aromatic Compounds. Although not all of the aromatic compounds of fungi have been proved to be formed from carbohydrate — some have been reported only from sporophores in nature — most of them are probably so formed; this is evidenced, for example, by the very high yields often obtained on synthetic media (95, 128, 252). The origin of the aromatic ring from glucose has been investigated most successfully in connection with the biogenesis of the aromatic amino acids and p-aminobenzoic acid in bacteria. The current view of this process may be diagrammed as follows (162): HO COOH HO COOH "V" ! OH OH 5-Dehydroquinic acid HO OH OH Quinic acid COOH ^N COOH HO "Zi5 OH O ! OH OH OH 5-Dehydroshikimic acid Shikimic acid In this scheme, an unknown compound "V" arises from carbohydrate and is converted through the known materials shown to a second un- identified compound "Zj", which is in turn ultimately transformed into the aromatic amino acids and p-aminobenzoic acid; the known later steps in the process are reviewed in Chapter 8. The initial steps of the reaction sequence are not known; preliminary evidence (162) suggests that the carbon chain of glucose is ruptured before incorporation of glucose carbon into shikimic acid. A mutant of Neurospora crassa which requires p-aminobenzoic acid, tyrosine, phenylalanine, and tryptophan for growth is similar in these requirements to the bacterial mutants which have provided much of the evidence for the scheme just outlined, and appears to be blocked in the conversion of dehydroshikimic acid to shikimic acid (507). It accumulates protocatechuic acid, which is then oxidized by ring fis- sion (p. 174). Protocatechuic acid is known as a metabolic product of Phycomyces blakesleeamis growing on a glucose-asparagine medium 172 CARBON METABOLISM III (44) and is also formed by many fungi during the oxidation of quinic acid (53, 57). Isotope studies on Neurospora crassa indicate that, as in bacteria, 3- and 4-carbon fragments derived from glucose — presumably via the phosphogluconate oxidation pathway (Chapter 7) — combine to form shikimic acid (506). As discussed later, currently available information at least encour- ages the speculation that all aromatic compounds arise analogously to protocatechuic acid in N. crassa, i.e., that the normal pathway to aromatic amino acids is blocked or saturated and that the carbon of shikimic acid or its precursors is diverted to the synthesis of new aro- matic structures. However, in N. crassa protocatechuic acid is oxi- dized with ring fission and does not give rise to other aromatic com- pounds. Another possibility has been considered by Ehrensvard (176), that pyrones might be intermediates in the conversion of glucose to aromatic compounds by Penicillium urticae. Evidence so far is limited to the action of pyranes, e.g., dehydracetic acid, on the syn- thesis of aromatic compounds. Close structural relations among the aromatic compounds are numerous, both within and between species. Many are discussed by Raistrick (430); a few examples may be cited: 1. Simultaneous formation by one organism of a phenol and the corresponding quinone (14, 180). 2. Occurrence in the same organism of a compound and its dihydro derivative, e.g., cyclopolic and cyclopaldic acids (Table 4). 3. Formation of a compound and a hydroxy or methoxy derivative of it, e.g., gladiolic and cyclopaldic acids (Table 4) and methyl cin- namate and methyl-p-methoxycinnamate (74). The ability of fungi to introduce hydroxyl groups into a ring has been noted above in connection with steroid metabolism. 4. The close structural relationship, already mentioned, of all of the fungal anthraquinones, found in such diverse genera as Aspergil- lus, Cortinarius, Helminthosporium, and Penicillium (289). A pos- sible origin of the anthraquinones from benzophenone is discussed by Tatum (505). The foregoing examples refer to more or less static relations. In a dynamic and more conclusive experiment it has been shown that the ratio of patulin to gentisyl alcohol is controlled by the concen- tration of iron in the medium (Figure 7). Similarly, Penicillium urticae in a low-zinc medium forms predominantly 6-methylsalicylic AROMATIC COMPOUNDS 1.5 173 1.0 2.0 5.0 10 Ferrous sulfate, mg per liter (logarithmic scale) 20 Figure 7. The effect of iron on the formation of patulin and gentisyl alcohol by PenicilUum sp. From data of Brack (93). acid, but at higher concentrations of zinc this compound is replaced by a mixture of gentisyl alcohol, patulin, and toluquinone (176). This type of analysis, in which an environmental modification causes a shift in metabolic products, is extremely promising. PenicilUum brevi-compactum produces several related phenols. Both the temporal sequence of appearance of these compounds (409) and the observation that the organism when fed one compound pro- duces others (230) support the proposition that these structurally re- lated molecules are related biologically as well. A role of the polyphenol oxidases in the conversion of phenols to other compounds is probable but has not been investigated in the fungi; Mason (371) reviews several theories on the function of these enzymes in the origin of aromatic products of higher plants. Enzymatic Transformations of Aromatic Compounds. The major known types of attack on aromatic compounds may be summarized as: (1) the oxidation of aliphatic side chains, (2) the oxidation of phenols, (3) the oxidative fission of the aromatic ring, and (4) the hydrolysis of depsides. The side chain of phenylacetic acid is oxidized by PenicilUum chrysogenum; benzaldehyde is formed during the oxidation and is 174 CARBON METABOLISM III thought to he an intermediate (279). The process therefore resembles the oxidation of substituted benzenes by bacteria (479) and higher plants (313), in which benzoic acid is the ultimate product: CH2COOH CHO COOH I I I ^\ ? fii rl (11) Phenylacetic acid Benzaldehyde Benzoic arid However, the entire sequence is not known in fungi, and the forma- tion from phenylacetate of 2-hydroxyphenylacetate and 2,5-dihydroxy- phenylacetate point to other possible modes of attack by fungi and actinomycetes (297, 320, 544). Benzoic acid is formed in culture by Penicillium roseo-purpureum (427a). The oxidation of vanillin and ferulic acid by fungi appears to be of the same general type as that of phenylacetate, the side chain being oxidized in one or more steps to a carboxyl group (268). There occurs in bacterial metabolism an oxidative fission of the aromatic ring of catechol to yield m-m-muconic acid (480): OH I OH + 02 -> HOOC— CH=CH— CH=CH— COOH (12) Catechol CiJ-eir-muconic acid This over-all reaction has been hypothecated for Aspergillus niger (57), and Penicillium sp. accumulates a's-cw-muconate during the oxi- dation of catechol (333). The aromatic ring of phenylacetic acid is split by Penicillium chrysogenum, but the details of the process are not known, except that an induced enzyme system is responsible and that compounds of the tricarboxylic acid cycle appear in the medium during oxidation (294, 296). It has been mentioned that a mutant of Neurospora crassa accumu- lates and then oxidizes protocatechuic acid. The ultimate product is /3-ketoadipic acid and the enzymatic steps involved are similar to but not identical with those known in Pseudomonas fluorescens (250, 481). The first step is the conversion of procatechuic acid to cis, cis-fi-cax- boxymuconic acid, analogously to the reaction of Equation 12, by an inducible oxidase system (250, 251). The oxidation of phenols to quinones is a special problem, and much more data are available on it than on other phases of aromatic metabolism. AROMATIC COMPOUNDS 175 The enzymes responsible for the oxidation of phenols are the poly- phenol oxidases, of which two have been separated: 1. Tyrosinase, which oxidizes both monophenols (tyrosine, p-cresol, etc.) and polyphenols (catechol, pyrogallol, etc.). Oxidation of tyro- sine leads, by a complex series of enzymatic and non-enzymatic re- actions, to the formation of black pigments, the melanins (Chapter 8). 2. Laccase, which oxidizes polyphenols but not monophenols. In contrast to tyrosinase, laccase is sensitive to carbon monoxide and has a somewhat different substrate range. Both enzymes are copper proteins. The characteristic reaction, often used for assay, is the oxidation of catechol to o-benzoquinone: + ±02-> +H20 (13) Catechol o-Benzoquinone Although other polyphenol oxidases probably occur in fungi (108, 286), they have not been characterized. Most assays do not, indeed, discriminate between laccase and tyrosinase. Interest in the polyphenol oxidases has been stimulated by the Bavendamm reaction (35) of the wood-rotting fungi: on an agar medium containing gallic or tannic acid the fungi causing white rots of wood and capable of utilizing lignin form a colored zone. The brown rot fungi in general do not form the zone. Several surveys of the wood-rotting fungi have established that the correlation between zone production and type of rot is high, although not perfect (86, 160, 397). The usual substrates do not distinguish between tyrosinase and laccase, but work with specific substrates and on the distribution of the enzymes between cells and medium indicates that extracellular laccase, rather than tyrosinase, is responsible for the Bavendamm re- action (274, 335, 343, 344). Extracellular polyphenol oxidases are formed also by other basi- diomycetes, including parasitic and mycorrhizal species and both coprophilic and litter-inhabiting saprophytes (340). Fungi other than basidiomycetes have been less well studied; tyrosinase is found in Glomerella cingulata (500) and Neurospora crassa (210, 287) and — judged by color reactions — in Streptomyces scabies (282) and in Sclero- tinia fructicola and Macrosporium sarcinaeforme (445). 176 CARBON METABOLISM III Polyphenol oxidases in the fungi are often present only at particular times in the culture cycle, especially during autolysis (188, 500), or only in particular morphological structures (342, 344). The medium used affects the production of the enzymes, both positively and negatively. Laccase formation by Polyporus versicolor is increased non-specifically by constituents of the medium and specifically by certain aromatic compounds which probably act as inducers (189, 190). Both carbon source and growth temperature affect enzyme synthesis in Glomerella cingulata (499). Negative effects of an interesting type have been found in studies on Neurospora crassa (287): in a medium containing the usual level of sulfate a sulfur-containing dialyzable tyrosinase in- hibitor is formed, the result being that the conventional assay detects tyrosinase only in cultures grown in a low-sulfate medium. This finding requires that earlier negative reports be re-evaluated. The tannins are esters of polyhydroxy phenols with aromatic acids, and are hydrolyzed by tannase, e.g., HO HO OH HO -CO— O— C /> -» 2HO— i HO COOH HO Digallic acid Gallic acid The enzyme, an esterase, also hydrolyzes other similar esters (498). The tannase of Aspergillus niger is inducible (322); its properties and purification are summarized by Dyckerhoff and Armbruster (175), and partial purification has been reported (519). Although most studies have been made on the tannase of A. niger, the enzyme has been reported in a few other fungi (36, 448, 574). The Significance of Aromatic Compounds. Some of the aromatic compounds bulk rather large in the economy of the organism, amount- ing to an appreciable fraction of the glucose consumed (140) and of the dry weight produced (128). Those which have been studied in relation to the history of the culture appear to be first formed and then utilized (93, 95), to be more than inactive waste products of metabolism. Possibly the most likely metabolic significance of these compounds is as products of an unbalanced nutrition and metabolism. Most of them have been isolated from fungi grown on media very high in carbon; thus, the glucose Czapek-Dox medium often used has a car- bon : nitrogen ratio of 60. Under these conditions, it is not surprising AROMATIC COMPOUNDS 177 that carbon may flow into new metabolic pools simply because of an insufficiency of nitrogen. We may visualize this type of process schematically: Glucose ^;A±^B^;C±^D±^E... (15) \\ \\ / A' B' — NH2 w w A" B" If compound D, for example, cannot be synthesized because of a rela- tive nitrogen deficiency, enzymatic equilibria and factors of permeabil- ity and solubility will determine which of the potential alternative pathways is followed. In the specific instance of the benzenoid com- pounds and their oxidation products, it is imaginable that the syn- thesis of aromatic amino acids stops because nitrogen is no longer available, and that a precursor of these amino acids, e.g., shikimic acid, is then diverted to the synthesis of new compounds. This view of the origin of aromatic compounds puts them in the same class as organic acids, fats, and polysaccharides, as products of what have been termed metabolic shunts (200). Physiologically, the model proposed above requires that the ratio of metabolic product to mycelial nitrogen should increase with increasing glucose concentra- tion, at least over a certain range. This has not been tested; the ratio of product to dry weight of mycelium (243) is less informative, because of the likelihood that carbon components of the mycelium may also be products of metabolic shunts. It has often been suggested that fungal quinones play a specific role in respiration, and the hypothesis is an attractive one. The frequent simultaneous occurrence of the reduced and oxidized forms in one organism suggests that the system could transport electrons and so function in terminal respiration. However, satisfactory proof of such a role in the intact cell has not been brought forward. Addition of quinones to a culture often elicits a respiratory response (214, 389), but this can only be taken as an indication that the compound could function, not that it does in fact function. A respiratory role for the polyphenol oxidases of higher plants has often been proposed, and the question is still open. In Glomerella cingulata it appears that tyrosinase cannot be an essential respiratory enzyme, inasmuch as it is not detectable during the growth phase (500). A pyridine nucleotide-menadione reductase occurs in Aspergillus niger (564), and a pyridine nucleotide-quinone reductase in other fungi (565). In principle, either of these could participate in a 1/8 CARBON METABOLISM III coupling mechanism between substrate dehydrogenation and a poly- phenol oxidase system transferring electrons to oxygen. Many of the quinonoid compounds of fungi are more or less in- hibitory to other organisms, and it has been suggested (341) that this property may confer an ecological advantage. Whether highly re- active compounds like the quinones could persist long enough in soil to inhibit competing bacteria and fungi is, however, somewhat doubtful. 4. DERIVATIVES OF PYRONE Several metabolic products may be considered together as deriva- tives of 1,4-pyrone, and as related structurally to the pyranose form of glucose. Kojic acid is typical: CO CHOH / \ / \ HO— C GH HOHC CHOH II II II HC C— CH2OH HOHC CH— CH2OH \ / \ / o Kojic acid Glucopyranose Kojic acid was first discovered, as a product of Aspergillus oryzae, by Saito (452); its structure was determined by Yabuta (568). Many species of Aspergillus, but only one of Penicillium, produce it in cul- ture (71, 227, 242, 303). Kojic acid is weakly antibacterial (381). Kojic acid is produced from most common carbon sources (71, 311), although strains differ somewhat in this regard (404). The number of compounds providing carbon for kojic acid synthesis is less for a preformed mycelium than for growing cells (504). The data of Figure 8 show that, as would be expected, there is over a certain range an inverse relation between nitrogen supplied to a culture and kojic acid production per unit mycelial weight. These data can best be interpreted as showing that kojic acid synthesis per cell is maximal when growth is limited by a nitrogen deficiency, in accordance with the principle that metabolites accumulate when carbon is supplied in excess of that which can be incorporated into cellular material. The formation of kojic acid is sensitive to pH; the yield from a preformed mycelium declines virtually to zero as the pH is raised from 2.2 to 6.0 (319). Ethylene chlorhydrin increases the formation of DERIVATIVES OF PYRONE 179 20 Figure 8. Growth and kojic acid formation by Aspergillus flavus as functions of nitro- gen supply. Drawn from tabular data of May, Moyer, Wells, and Herrick (373). Mycelium 0.1 1.0 10 Ammonium nitrate, gm per liter (logarithmic scale) kojic acid by Aspergillus flavus, but the effect appears to be on the entire metabolism of the organism rather than specifically on kojic acid synthesis (374). The very high yields of kojic acid — up to 57 per cent of the glucose consumed by a growing culture (373) and up to 78 per cent of that used by preformed mycelium (319) — -suggest that the acid may be formed directly from glucose. This is borne out by the isotope studies of Arnstein and Bentley (18). Degradation of kojic acid derived from glucose- 1-C14 shows that the bulk of the acid is formed from glucose without splitting of the carbon chain. The occurrence of small amounts of C14 in other positions is adequately explained by the postulate that a minor fraction of the carbon of kojic acid is derived from compounds of the phosphogluconate oxidation respiratory path- way (19, 283). The details of the conversion of glucose to kojic acid are not known; Arnstein and Bentley (18) discuss some possibilities. The fact that a preformed mycelium does not require added phosphate for kojic acid synthesis (242) is irrelevant to the problem of whether or not phos- phorylative steps are involved in synthesis, since the endogenous phos- phate reserves of the cell would without question be adequate for a cyclic process. Patulin is a pyrone derivative, the structure of which is largely known (181, 563). It has been isolated several times independently tinder different names — clavacin, claviformin, expansin, and clavatin — from species of Penicillium and Aspergillus; its history and synonymy 180 CARBON METABOLISM III are reviewed by Raistrick (430). As mentioned earlier, the effects of metals on patulin production indicate that it is biosynthetically related to some of the aromatic compounds of fungi. Alternariol is a dibenzo-l,2-pyrone formed by Alternaria tenuis after long-continued growth on a glucose medium (435). There is no reason to believe that it is metabolically related to the 1,4-pyrones. 5. DERIVATIVES OF FURAN A number of fungal metabolites are structurally furan derivatives. Most of these are derivatives of tetronic acid and have in common the structure: HO— C=C— Rj ! I r2— G C=0 \ / o One organism, Penicillium charlesii, forms five related compounds of this type: y-methyltetronic acid, carlic acid, carlosic acid, carolinic acid, and carolic acid (140, 143). Other compounds of the same general class are terrestric acid of Penicillium terrestre (82), dehydro- carolic acid of P. cinerascens (94), and penicillic acid of Penicillium spp. and Aspergillus spp. (6, 78, 227). L-Ascorbic acid, well known as a product of higher plants, has been isolated from the culture filtrate of Aspergillus niger (223). Its rela- tion to the tetronic acids is shown by its structure: HO— C G— OH I I CHoOH— CHOH— C C=0 \ / O Ascorbic acid Studies on higher plants indicate that ascorbic acid is synthesized from glucose without rupture of the carbon chain and that L-galactono-y- lactone is an important intermediate (367). It is also striking, in view of the formation of oxalic acid by Aspergillus niger, that oxalic acid is an end product of ascorbate metabolism in the guinea pig (99). Re- ports of ascorbic acid in fungi other than Aspergillus niger are sum- marized by Van Lanen and Tanner (531). An ascorbic acid oxidase of the higher plant type, containing cop- per, has not been reported from fungi. Myrothecium verrucaria BIOLOGICAL METHYLATION 181 spores oxidize ascorbate, but the reaction is believed to be mediated by a new enzyme, probably located on the spore surface (365, 366). Two other derivatives of furan — not derivatives, however, of tetronic acid — are known: 2-hydroxy-5-furan carboxylic acid, from Aspergillus spp. (496, 497) and zymonic acid, from yeasts (493). The thiophen or thiofuran ring differs from the furan in that a sulphur atom replaces the oxygen. A single compound of this type, junipal, is known in the fungi, from Daedalea juniperina (69). 6. BIOLOGICAL METHYLATION The transfer of intact methyl groups from one molecule to another has been most intensively studied in animal materials. The synthesis of methionine may be taken as an example: CH2— SH I CH2 I (GH3)3— N— CH2— COO- + H2N— CH— COOH -+ Betaine Homocysteine CH2 — S — CH3 CH2 H2N— CH— COOH + (CH3)2— N— CH2— COOH (16) Methionine Dimethylglycine In this reaction, details of which are neglected in the above over-all formulation, betaine functions as the methyl donor. Methionine can in turn donate a methyl group to other molecules, e.g., in the stepwise methylation of aminoethanol to choline. Studies on Neurospora crassa mutants which require methionine show that the final step in methionine synthesis is the conversion of homocysteine to methionine, as in Equation 16 above (285). The methyl donor in the fungus system is not known. In a particularly important application of biochemical genetics, Horowitz (284) showed that Neurospora crassa synthesizes choline by the stepwise methylation of aminoethanol, that is: Aminoethanol — > monomethylaminoethanol — > dimethylaminoethanol -» choline (17) Here it is to be presumed that methionine or formate is the source 182 CARBON METABOLISM III of methyl groups. The ultimate source for those fungi — perhaps all naturally occurring fungi— which do not require preformed methyl groups in the medium is, of course, carbohydrate; that is, most or all fungi can synthesize methyl groups from the products of glucose metabolism. Early in the nineteenth century, it was recognized that an arsenical poisoning of humans by volatile compounds is associated with the action of fungi on wallpaper or other building materials containing arsenical pigments. The history of this problem is reviewed by Chal- lenger (117); the toxic material was finally identified as trimethylar- sine (121). The ability of certain fungi to form methylated com- pounds of arsenic and other elements has since been investigated vigor- ously by the group at the University of Leeds. Some examples of the products identified are listed in Table 5; these and others are reviewed Table 5. Methylated Products of Fungi Substrate Na methylarsenate Na cacodylate Arsenous oxide Diethylarsonic acid K tellurite Na selenate Na selenite Ethyl disulfide Inorganic sulfate Product Trimethylarsine Trimethylarsine Trimethylarsine Methyldiethylarsine Dimethyl telluride Dimethyl selenide Dimethyl selenide Methyl ethyl sulfide Methyl mercaptan, dimethyl sulfide Organism Scopulariopsis brevicaulis (121) S. brevicaulis (121) S. brevicaulis (121) S. brevicaulis (120) S. brevicaulis, Penicillium spp. (62) Aspergillus niger (170) Penicillium spp. (62) Scopulariopsis brevicaulis (123) S. brevicaulis (123) S. brevicaulis (124) Schizophyllum commune (76, 119) by Challenger (118). It should be noted that the methods used permit only identification of the final product. Obviously, many steps in addition to methylation must be assumed in the conversion of the highly oxidized selenate ion, for example, to the reduced dimethyl selenide. Evidence is now at hand that these products do in fact arise by transmethylation. Isotope experiments (122, 170) show that the methyl groups of dimethyl selenide and trimethylarsine originate from methionine in Scopulariopsis brevicaulis and Aspergillus niger. Betaine does not serve as a methyl donor; carbon from formate and choline enters the system, probably through methionine. BIOLOGICAL CHLORINATION 7. BIOLOGICAL CHLORINATION 183 Chloride ion is commonly present in culture media simply as an accompaniment of essential ions. With the discovery of geodin and erdin in Aspergillus terreus (433, 434), it was realized that chlorine may be incorporated into organic compounds by fungi. Subsequent investigations have shown many chlorinated metabolites; a partial list includes: 1. p-Methoxy-tetrachlorophenol of Drosophila subatrata (8). 2. Chlorinated depsidones, e.g., nidulin (Table 4). Chlorinated depsidones are also known in the lichens (24); they are presumably synthesized by the fungus component. 3. Griseofulvin, a complex benzenoid compound of uncertain struc- ture, produced by Penicillium spp. (96, 256, 411). 4. Chlortetracycline (aureomycin), an antibiotic of Streptomyces aureofaciens (487). 5. Chloramphenicol (Chloromycetin) (Table 4). Here the chlorine is attached to a side chain carbon; chloramphenicol is also unusual in its nitrobenzene structure. 6. Nalgiolaxin, a chlorinated anthraquinone of Penicillium nal- giovensis, occurring in association with its dechloro analogue nalgio- vensin (436). 7. Caldariomycin of Caldariomyces fumago (Fumago vagans), prob- ably a cyclopentane with two chlorine atoms attached to a ring carbon (141). Terrein, of Aspergillus terreus, not a chlorine compound, is the only other cyclopentane derivative known in fungi (32, 255). In a survey of 139 fungi, it was found that strains of Absidia spinosa, Alternaria tenuis, Aspergillus spp., Caldariomyces fumago, Penicillium spp., Syncephalastrum racemosum, and Trichoderma viride incor- porate 15 per cent or more of the available chlorine into organic combinations (141). Frequently a compound and its dechloro derivative occur together in one organism, e.g., griseofulvin and dechlorogriseofulvin in Penicil- lium spp. (362). The antibiotics tetracycline and chlortetracycline are produced by different species of Streptomyces, the related oxytetracy- cline by a third (61). The substitution of bromide for chloride in the medium has specific effects of two kinds: either the organism forms the bromo analogue of a chlorine compound (363), or chlorination is suppressed and the dechloro derivative is formed (468). The incorpo- ration of chlorine into tetracycline is also suppressed by copper-corn- ]84 CARBON METABOLISM III plexing agents, e.g., thiourea (468), which are known to interfere with iodination of tyrosine in the thyroid (551); presumably, the chlorina- tion step requires copper. 8. PENICILLIN The discovery by Fleming (197) of an organism producing penicil- lin initiated a new field of medical and biological investigation, the study of the formation and clinical use of antibiotics. The history of the exploitation of Fleming's discovery has been told elsewhere (1); in general, although extensive application of our knowledge of fungal physiology was essential to the development of methods of production, the physiological studies were less dramatic than the genetic, and few new principles of physiology or biochemistry have emerged from the work on penicillin and other antibiotics. The penicillins are produced by several different fungi, including Penicillium spp., Aspergillus spp., and a thermophilic organism tenta- tively identified as Malbranchea pulchella (441). Strains of the Peni- cillium chrysogenum series, including P. notatum, are the most active, and supply the penicillin of commerce. The penicillins are alike in their basic chemical structure, but differ in the side chain (R in the formula): S / \ /CH3 R— CO— NH— CH— CH C< I I I XCH3 CO— N CH— COOH The natural penicillins whose structures have been determined in- clude: 1. 2-Pentenylpenicillin (penicillin I, penicillin F) 2. Benzylpenicillin (penicillin II, penicillin G) 3. p-Hydroxybenzylpenicillin (penicillin III, penicillin X) 4. n-Amylpenicillin (dihydro penicillin F) 5. w-Heptylpenicillin (penicillin IV, penicillin K) 6. 4-Amino-4-carboxy-w-butylpenicillin (cephalosporin N) (393). It is likely that other penicillins formed only in small amounts have not been identified. Benzylpenicillin, now available in crystalline form, is the penicillin of medical practice. Extensive information on the metabolism of penicillin-producing fungi, reviewed by Chain (115) and Perlman (417), is available, but it must be said that there is no correlation of any other metabolic PENICILLIN 185 process with penicillin formation. It is possible to specify fairly exactly the optimal conditions for penicillin formation by a given strain, but not possible to define the way in which these conditions act. The metabolic changes which take place during penicillin produc- tion are conveniently divided into three phases: an initial phase of rapid growth and metabolic activity, with assimilation of the more available nutrients, a second phase of slower growth with carbohydrate utilization, and a third phase of autolysis and low metabolic activity. These are illustrated in Figure 9. Penicillin formation begins late in the first phase and is most vigorous during the second. Rates differ, but in broad outline this pattern is followed by different strains in both complex and synthetic media, with various carbon sources, and under diverse conditions of aeration (31, 209, 217, 239, 300, 324). Lactose or other slowly utilizable carbohydrates yield both a favorable rate of growth and a favorable pH for penicillin formation (385, 484). Penicillin production is also directly affected by aeration (483). The metal requirement for maximal penicillin formation by a strain of o E < 1 1 I Phase 1 1 ! 1 Phase 2 i i Phase 3 \ \ \ \ Residual lactose \ \ -^1 \/ 1 ^3r- Residual lactic \ \ \ \ / y v \ \ \. acid Penicillin Dry weight i i i 1 1 4 6 Time, days 10 Figure 9. Metabolic changes accompanying penicillin formation by Penicillium notatum in shaken flasks in a corn steep liquor-lactose medium. Redrawn from Koffler, Emerson, Perlman, and Burris (323), by permission of the Williams and Wilkins Company. 186 CARBON METABOLISM III P. chrysogenum is somewhat higher, particularly as to iron and phosphorus, than that for growth (301). Corn steep liquor, a complex mixture of organic substances derived by bacterial action on maize (339), probably has more than one role in penicillin production; its principal function, however, appears to be as a source of /?-phenylethylamine and related compounds (375), which are specific precursors of benzylpenicillin. In the biosynthesis of mixed penicillins, phenylacetic acid and its derivatives shift the balance of natural penicillins toward benzylpenicillin (273). Similarly, p-hydroxyphenylacetate increases the relative amount of p-hydroxy- benzylpenicillin. Although only a small fraction of the phenylacetate supplied is incorporated into the molecule (295), isotope studies have shown that it is incorporated without dilution, i.e., that the intact benzyl group enters as such into penicillin (38, 240, 467). Not only are close relatives of phenylacetic acid incorporated into benzylpenicillin, but many other substituted acetic acids can serve as precursors for new "biosynthetic" penicillins (37). Fatty acids, espe- cially those which are not rapidly broken down by the fungus, are incorporated into both natural and new penicillins (380, 516). The sulfur of penicillin can be supplied by inorganic sulfate; sulfur of methionine, cysteine, cystine, or glutathione is, however, utilized in preference to that of inorganic sulfate (490). The origin of the main part of the penicillin molecule has been clarified by Arnstein and Grant (22). Using C14-labeled valine and triply (N15, C14, S35) labeled cystine, they have shown that: (1) cystine — probably after conversion to cysteine — is incorporated directly into the penicillin molecule, and (2) the remaining five carbons are sup- plied by valine. This is consistent with earlier work on the incorpora- tion of small molecules into penicillin (368, 489, 517). The molecule of benzylpenicillin may be viewed, therefore, as made of three meta- bolic units, one formed directly from phenylacetic acid, one directly from cystine (probably via cysteine), and one supplied, directly or in- directly, by valine. The steps by which these structures are combined remain for future investigation. It is to be expected that energy is necessary for penicillin formation from these units, and the inhibitory effect of 2,4-dinitrophenol suggests that the energy is derived from oxidative phosphorylations (449). 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In this chapter, after touching on a few general problems, we shall consider primarily those reactions which are or may be part of organized respiratory systems. Many individual reactions described in the preceding chapter undoubtedly yield energy, but their relation to the major pathways of respiration is not yet known. Respiration may be aerobic or anaerobic. Aerobic respiration is defined by the fact that the ultimate hydrogen acceptor is molecular oxygen; in anaerobic respiration, or fermentation, hydrogen from substrate is transferred to compounds other than oxygen (107). Use of the term "fermentation" to describe an aerobic process is mislead- ing. The maximum energy available from a reaction or sequence of reactions is, of course, the free energy change; thus, complete aerobic oxidation of glucose to carbon dioxide and water involves a change in free energy of approximately —700,000 calories per mole under standard conditions. Some fraction of this energy is trapped by the cell and used for energy-requiring reactions. So far as we now know, the size of the fraction so trapped depends on the ability of the or- ganism to couple the oxidation to phosphorylation of adenosine mono- phosphate or adenosine diphosphate to yield adenosinetriphosphate (ATP). In turn, the hydrolysis of ATP is coupled to energy-requiring reactions. The common practice of describing ATP and similar com- 202 PROBLEMS OF METHOD 203 pounds as having "high energy" phosphate bonds is ambiguous (103). In general, most of the oxidative phosphorylations associated with glucose or other substrate oxidations are carried out at the cofactor level; electrons from substrate are passed along a chain of respiratory carriers to oxygen and in the process, in ways still unknown in detail, the energy released in electron transfer is used to form ATP. Sub- strate-level phosphorylations occur in the anaerobic conversion of glucose to ethanol and carbon dioxide — the Embden-Meyerhof path- way— and, possibly, in the oxidative decarboxylation of a-ketoglutaric acid. As described briefly in Chapter 9, fungi during growth transform inorganic into organic phosphate. Organic phosphorus compounds which have been identified in fungi and actinomycetes include sugar phosphates (38, 94, 134), adenosine phosphates (9, 162, 219), and various of the other known nucleotides (9). A mycelial extract of Rhizopus nigricans has myokinase activity (228), catalyzing the re- versible transphosphorylation: 2 adenosine diphosphate ^± adenosinetri phosphate + adenosinemonophosphate (1) The utilization of ATP to phosphorylate organic molecules is im- plicit in the two major respiratory pathways for glucose, both of which start with glucose-6-phosphate. There is very suggestive evidence that ATP generated by oxidative processes is involved in the streaming of the myxomycete Physarum polycephalum as it is in muscular con- traction (3, 177, 303, 304, 305). 1. PROBLEMS OF METHOD The respiration of whole cells and of extracts is usually measurable in terms of gas exchange — oxygen uptake or carbon dioxide formation. The basic instrument in most laboratories is the Warburg-Barcroft constant-volume manometer, described in the manuals of Dixon (77) and Umbreit et al. (308). Macrorespirometers with larger capacity have been designed (170, 188, 282, 328), and a few studies on fungi have been performed with more sensitive manometric apparatus (78, 214, 324). An amperometric procedure for measuring oxygen utiliza- tion has been applied successfully in studies on Penicillium chryso- genum (52). Dye reduction methods have a more limited application but are useful in some problems. The best known is that of Thunberg (298), 204 RESPIRATION which depends on the reduction of methylene blue coupled to oxida- tion of substrate: Substrate + oxidized dye — > oxidized substrate + reduced dye (2) The Thunberg method has been used to demonstrate the presence of respiratory enzymes in cell-free extracts from fungi (182, 226). Methyl- ene blue is often inhibitory (271) and other dyes, e.g., 2,6-dichloro- phenolindophenol, are in general to be preferred (30, 207, 216, 225). Spectrophotometric methods are both more powerful and more quantitative. The most generally useful of these relies upon the fact that the reduced forms of the coenzymes diphosphopyridine nucleotide (DPN) and triphosphopyridine nucleotide (TPN) absorb strongly at 340 m^; the change in density can be followed easily and rapidly after addition of substrate to a suitable system (312). Exam- ples of the use of this method in studies of fungi and actinomycetes include work on the enzymes of Neurospora crassa (81, 301), Aspergil- lus niger (148), Fusarium lini (62), and Streptomyces coelicolor (66). Spectrophotometric measurements can, of course, be used whenever a component of the system changes its light absorption as a conse- quence of an enzymatic reaction. Oxidation and reduction of cyto- chrome c can be followed at 550 imx (46, 47, 71, 280), or reducible dyes may be used (50). Like all other sensitive measurements, the spectrophotometric re- quires a certain amount of judgment. Too often, a crude enzyme preparation contains unsuspected activities which complicate a given determination. For example, an extract may reduce both DPN and TPN when fructose- 1,6-diphosphate is added. The DPN reduction is straightforward: aldolase forms triose phosphate, which is then oxidized with concurrent reduction of the coenzyme. The reduction of TPN is not, however, as might first be thought, the same process; instead, fructose-l,6-diphosphate is first converted to glucoses-phos- phate, which is oxidized by glucose-6-phosphate dehydrogenase and TPN to 6-phosphogluconate. The crude experiment falsely suggests that triose phosphate dehydrogenase can react with either DPN or TPN. These manometric and spectrophotometric methods are general in their application and therefore stand in contrast to the problem of preparing cells and extracts of fungi for respirometric study. In a certain sense, each organism is a problem in itself, but it may be useful here to mention some of the methods which have been used in particular instances. Discs of growing mycelium from a surface PROBLEMS OF METHOD 205 culture may be transferred to a respiromctcr flask (36, 78, 214, 313), but most workers have preferred to use when possible the more homo- geneous mycelium obtained from shaken or aerated cultures (65, 70, 102, 163). The mycelium from such cultures is often in the form of small pellets; if these are too large, above 0.4 mm. for Myrothecium verrucaria (70), the rate of diffusion of oxygen into the interior of the pellet may be limiting. Either surface or shake culture mycelium may be homogenized briefly, but there is a considerable risk that mechanical injury may reduce respiratory capacity (70, 80). The preparation of active cell-free extracts from fungi is an even more vexing problem. We may list here, in outline, some of the methods which have been successful: 1. Hand grinding with abrasives (7, 44, 50, 65, 71, 140, 182, 301). 2. Acetone drying (14, 129, 212, 288). 3. Physical drying, with or without a preliminary freezing (89, 155, 204, 218, 331). 4. Mechanical disruption in a ball mill (240) or in a motor-driven homogenizer (46, 274). 5. Freezing, followed by grinding with abrasives or by homogeniza- tion (190, 201, 258, 274). 6. Sonic oscillation (210, 264). 7. Disruption in the Hughes press (142) or in a small-orifice press (55, 200, 228). 8. Treatment of frozen mycelium with dilute ammonium hydroxide, followed by mild pressure (148). Of these, the most generally useful appear to be hand grinding with sand or powdered glass, sonic oscillation, and treatment in the Hughes or similar presses. Hugo (143) reviews the methods available for the preparation of extracts from bacteria. Suspending media for extraction may be water, phosphate buffer, or salt mixtures. Protective materials are often essential — both cys- teine (62, 209) and ethylenediaminetetraacetate (110) protect against inactivation of enzymes by heavy metals liberated during the extrac- tion process. During the preparation of cell-free extracts the terminal respiratory system is usually disrupted and must be replaced by an added hydro- gen carrier or hydrogen acceptor. Methylene blue, 2,6-dichlorophenol- indophenol, and other dyes have often been used; phenazine deriva- tives (76), ferricyanide (134, 235), and manganese dioxide (133) are of value for the same purpose. 206 RKSPIRATION 2. FACTORS IN RESPIRATION In general, it is probable that the most important factor in respira- tion is the species. Few comparative data are available, but we may assume that fungi which can grow on a given substrate can also oxidize it (291), so that the omnivorous saprophytes, e.g., Aspergillus spp., may be expected to oxidize a greater variety of compounds than do nutritionally more specialized forms. Within a given species, age and developmental status are the primary non-environmental factors; the permeability of the cell is also presumed to be important by the usual indirect evidence. The principal external factors are acidity, temperature, oxygen pressure, carbon dioxide, and accessory nutrients. Age and Developmental Status. As mentioned in Chapter 1, the highest respiratory activity is usually found during the period of most rapid growth. Subsequent to this period, respiration, especially endog- enous respiration, falls off with increasing age. This may, of course, reflect only the fact that more and more dead cells accumulate in the older mycelium; quite possibly, the respiration of the growing tips of the thallus is independent of the age of the culture so long as nutrients are available. The data of Figure 1 are typical, and many others of the same type have been reported in studies of fungi and aerobic actinomycetes (52, 70, 95, 96, 115, 134, 163, 249). Ustilago zeae appears unusual, in that once the maximum respiratory rate is reached it is maintained (260). Spore respiration is considered briefly in Chapter 12; there is as yet no reason to think that it differs fundamentally from that of mycelium, although it would not be surprising to find generally higher activity in germinating spores than in older cultures. The yeast phase of Blasto- myces brasiliensis exhibits an endogenous oxygen uptake greater than that of the mycelial form (215), and the same relation prevails between cell types of Mucor guillermondii (179). In Blastocladiella emersonii the two different types of mature thallus differ in their content of important respiratory enzymes and, presumably, in their respiratory pathways (44). Oxygen consumption of Dictyostelium discoideum is in part a function of developmental stage (116). Cell Permeability. It is difficult to distinguish between intrinsic inability of a cell to respire an exogenous substrate and failure of the compound to penetrate to the interior of the cell. As in the utiliza- tion of organic acids for growth (Chapter 3), it is often found that a low pH is required for or accelerates the oxidation of organic acids FACTORS IN RESPIRATION 207 Figure 1. The effect of culture age on the endogenous respiration of Fusarium solani mycelium. Curve 1, dry weight of culture; curve 2, respiratory rate, in ^1 oxygen per milligram dry cells per hour. 2 4 Age, days (93) or that provision of an ester of the acid increases its biological effect (13). Or, intact cells of a fungus may fail to oxidize a substrate while cell-free extracts do so rapidly (13, 65, 89, 258). All of these types of evidence suggest that the intact cell is impermeable to the substrate. In general, ionizable substances, e.g., phosphorylated com- pounds and organic acids, are the most likely to be excluded by a permeability barrier. However, failure of exogenous substrate to be utilized cannot al- ways be ascribed to membrane impermeability. Thus, in yeast, frozen cells are permeable to citrate but metabolize it only after further treatment with chloroform (90). It is possible for enzymes in the cell to be inaccessible by virtue of subcellular organization; in most cases we cannot distinguish between this situation and true membrane effects. Induction of Respiratory Enzymes. The enzymes of whatever funda- mental respiratory pathways an organism has are probably always present whatever the substrate. But it is often observed that some compounds are oxidized only after a long lag or only if the cells have previously been grown in contact with them. This suggests that cer- tain enzymes, probably those concerned in the preliminary steps of respiratory breakdown, are not constitutive but are induced by sub- strate. The oxidation of phenylacetic acid is an example (Chapter 6), and attack on other aromatic compounds by Hormodendrum sp. 208 RESPIRATION appears also to require the formation of inducible enzymes (127). In Streptomyces coelicolor gluconic acid and pentoses are oxidized only by specifically adapted cells (66). In none of these cases just cited, however, has the specific enzyme been isolated and studied as such. Acidity and Respiration. In most experiments reported, the pH of the suspending medium has little effect on endogenous metabolism over the range pH 5 to 8 (3, 70, 187), and the data of Figure 2 are typical of many. Exceptions to this general rule have, however, occa- sionally been found (93, 127, 214). Acidity is more apt to be important in substrate respiration, e.g., the oxidation of phenylacetic acid by Penicillium chrysogenum (135) and of glucose by Streptomyces griseus (134). Since the internal pH of the intact cell is probably protected against all but the most extreme changes in the medium, it seems likely that pronounced pH effects on respiration of undissociated compounds or of very weak acids signify that surface-located enzymes are involved in the total process of metabolism. Phosphate buffers are the most generally satisfactory for respiro- metric studies; others, particularly acetate, are often toxic (Chapter 1). Temperature. Most respirometric studies of saprophytic fungi and actinomycetes are performed at moderate temperature, 20-30°C; higher temperatures are more suitable for the fungi pathogenic to animals (215). Carbon dioxide evolution from growing mycelium of Aspergillus niger has a high Q10 at low temperature, e.g., 312 over the range 7-17°; the Q10 drops as the temperature is raised, and is only 1.7 over the range 27-37° (217). Oxygen and Carbon Dioxide Pressure. The response of aerobic respiration to oxygen pressure varies with the organism studied (3, 72, Figure 2. pH and the endo- genous respiration of Strepto- myces coelicolor (curve 1), My- rothecium verrucaria (curve 2) and Fusarium solani (curve 3) mycelium. FACTORS IN RESPIRATION 209 Figure 3. The effect of oxygen pressure on the rate of respiration of Aspergillus niger. Redrawn from P. Shu, Journal of Agricul- tural and Food Chemistry, Vol. 1, p. 1119, November 25, 1953. Copy- right 1953 by the American Chemical Society and reprinted by permission of the copyright owner. 400 Oxygen pressure, mm Hg 800 259, 260, 291). Respiration depends immediately upon dissolved oxy- gen, which in turn is proportional to the partial pressure of oxygen in the gas phase. Finn (83) defines Ccrit as the concentration of dis- solved oxygen above which cell respiration is independent of the oxygen concentration; data from several sources show that Ccrit for Penicillium chrysogenum is about 0.022 millimoles per liter (equiva- lent to an oxygen tension of 16 mm of mercury), that for yeast and Escherichia coli considerably lower. Amperometric measurements show that the rate of oxygen utilization is independent of oxygen pressure down to very low values (52). It follows, therefore, that the requirement of many fungi, e.g., Aspergillus niger (Figure 3), for rela- tively high oxygen pressure for maximal respiratory rate means that other factors must be operative. Studies on the respiration of my- celium of Myrothecium verrucaria indicate that the rate of oxygen con- sumption by these rather active cells is limited in part by the rate at which oxygen reaches the interior of the cell mass (70). The oxygen uptake of Micromonospora vulgaris is reduced by high oxygen pressure (315), but this effect is not usually encountered in the fungi. Little evidence is available on the influence of carbon dioxide pres- sure on fungus respiration. Most respirometric techniques involve removal of metabolic carbon dioxide during measurement of oxygen uptake. In many bacteria oxygen uptake is in fact depressed by such removal of carbon dioxide (119). Two methods are available to deter- mine if this occurs with any given organism: use of Warburg's "in- direct" method (308) or use of diethanolamine as a carbon dioxide "buffer" (166, 231). Nutrients. Potassium, phosphorus, and magnesium are usually sup- plied to respiring cells, although it is likely that endogenous reserves 210 RESPIRATION of these and other minerals are sufficient. Aerobic oxidation with exogenous substrate is generally markedly increased if a source of nitrogen is provided (199, 265, 275); this, of course, converts the preparation at least potentially to a growing culture carrying on amino acid or even protein synthesis with an attendant demand upon energy and upon metabolic intermediates of the citric acid cycle. The effect of such organic materials as yeast extract may be the same, but there is the added complication that respirable materials are also being supplied. 3. ENDOGENOUS RESPIRATION Endogenous respiration, or "autorespiration," defines the metabo- lism of the cell in the absence of an external substrate insofar as it is expressed by gas exchange. In yeast there is normally no anaerobic endogenous carbon dioxide liberation (270), although 2,4-dinitro- phenol initiates fermentation of carbohydrate reserves (251). Fusa- rium lini, on the other hand, has a vigorous endogenous fermentation (98). The substrate of endogenous respiration in fungi is not known; respiratory quotients are consistent with lipid oxidation (186, 230), but suitable analytical data to discriminate between this and other possi- bilities are not available. Cell-free extracts of Neurospora crassa are stimulated in endogenous respiration by triphosphopyridine nucleo- tide (301). Scopulariopsis brevicaulis cells are unable to assimilate nitrogen in the absence of exogenous substrate, i.e., the endogenous respiration cannot be coupled to amino acid or protein synthesis (186). This is in contrast to both Chlorella vulgaris (284) and Torulopsis utilis (332). The high endogenous rate characteristic of many fungi and actino- mycetes complicates experimental study; obviously, if some limiting terminal system is fully saturated by the endogenous system, then an addition of substrate will not cause any further increase in oxygen up- take. However, it cannot be assumed that under these circumstances the exogenous substrate is inert — conceivably some of it will be oxi- dized in competition with the oxidation of endogenous materials. Partial responses are also possible: if, for example, the terminal system were 50 per cent saturated by endogenous metabolism, addition of substrate could at most double the oxygen uptake rate. But — and this may be a real problem — one can imagine that in fact the substrate is competing on, say, equal terms with the endogenous metabolism for ENDOGENOUS RESPIRATION 211 the terminal system. Were this so, not 50 per cent but 75 per cent of the total metabolism would be substrate metabolism; put another way, the endogenous system would be suppressed 50 per cent. Barker (11) proposed a method to determine whether or not sub- strate utilization does suppress endogenous respiration. The method relies essentially on the assumption that the oxygen uptake per mole of substrate consumed is not affected by the concentration of substrate. Application of this technique to Penicillium chysogeniim indicates that glucose does not suppress endogenous respiration (272). In yeast, on the other hand, suppression of endogenous respiration by acetate occurs with agar-grown cells but not with cells grown in a liquid medium (322). A second approach to this problem is to use cells the endogenous reserves of which have been labeled by cultivation on a carbon- 14 substrate (37, 244). It is essential that all of the reserves be labeled by growth from a very early stage on the isotopic substrate; a short exposure to labeled acetate, for example, may not succeed in marking all of the reserves. This method has received only a limited trial to date (25, 63, 101, 128, 203), and it is, of course, open to the criticism that not enough is known of the composition and dynamics of the many metabolic pools involved. Experimentally, it is only necessary to determine wrhat the total activity of carbon dioxide from labeled cells is and whether or not this activity is affected by the simultaneous oxidation of substrate. Amino acids have been reported as affecting endogenous respiration both positively (17) and negatively (93). These findings cannot at present be interpreted, since the presence of amino nitrogen in the system and the possibility of amino acid toxicity unduly complicate the picture. Experimentally, it is often desirable or even essential to reduce the level of endogenous respiration. Starving the cells for a period of time prior to measuring their respiratory response to an exogenous substrate has been successful with some fungi and actinomycetes (34, 70, 316, 320). Others, however, lose substrate respiratory capacity during the starvation period (14, 134, 195). There is also the danger that the starvation period may result in developmental changes in the organism (145). Since endogenous respiration is affected by the com- position of the growth medium (254), the better approach would seem to be an attempt to find a medium in which the cells do not accumu- late reserve materials in large amounts (102). Many of the media com- monly used for fungi are so concentrated that accumulation of such reserves is almost forced upon the organism. 212 RESPIRATION 4. OXIDATIVE ASSIMILATION Oxidation of carbon compounds by many organisms is not accom- panied by the oxygen uptake theoretically necessary to account for the known disappearance of substrate. This has been explained as the result of the assimilation of part of the utilized carbon into reserves, presumably carbohydrate in nature, of the cell (59). Oxidative assimilation occurs in yeast (234, 322), actinomycetes (254), and fungi (70, 80, 253). Its extent may be rather high; for Penicillium chrysogenum (272), the data fit the equation: C6H1206 + 02 -> 5(CH20) + C02 +H20 (3) Thus, in this organism about 83 per cent of the glucose disappearing is assimilated. Fermentative, i.e., anaerobic, assimilation is known in yeast and bacteria (297). In what is presumably the same process, Aspergillus niger forms intracellular mannitol and carbohydrate anaerobically (255). Fermentative accumulation may account for the finding (154, 247) that A. niger forms less ethanol from glucose than the theoretical amount expected from a fermentation of the yeast type. 5. THE EMBDEN-MEYERHOF PATHWAY The anaerobic breakdown of glucose by yeast is diagrammed in part in Figure 4. This series of reactions is of very general, although probably not universal, occurrence in microorganisms (79, 118). In muscle the reactions, termed glycolysis, are similar, except that pyruvic acid is reduced to lactic acid in the final step and that the starting material is glycogen. Since the filamentous fungi require oxygen for growth (Chapter 1), there has been a tendency to discount the possible importance of the Embden-Meyerhof pathway. First, it should be realized that the products of glucose metabolism by other routes may feed into the pathway; thus the phosphogluconate oxidation pathway, as discussed later, forms triose phosphate. Second, the Embden-Meyerhof pathway is not restricted to anaerobic conditions; it is entirely possible for a portion of it to function simultaneously with aerobic processes or to be coupled to terminal aerobic reactions. Finally, some fungi seem to be able to ferment sugar anaerobically even though they require oxygen for growth. Although in the bacteria there are routes of ethanol formation other THE EMBDEN-MEYERHOF PATHWAY Glucose 213 © ^ ATP ADP Glucose-6-phosphate ©11 Fructose-6-phosphate © ^ ATP ADP Fructose-1, 6-diphosphate © D-Glyceraldehyde-3-phosphate ^ © +H3PO4 r DPN" © — Dihydroxyacetone phosphate DPNH + H' 1,3-Diphosphogly eerie acid © V ADP ATP 3-Phosphoglyceric acid ®\\r ©IN ®\\ Pyruvate Ethanol ADP ATP © ■DPN v^ DPNH + H' Acetaldehyde + CO2 Figure 4. Schematic outline of the Embden-Meyerhof pathway of yeast. Abbrevia- tions: ATP — adenosine triphosphate, ADP — adenosine diphosphate, DPN* — di- phosphopyridine nucleotide, DPNH — reduced diphosphopyridine nucleotide. En- zymes are numbered as follows: 1 . Hexokinase 2. Phosphohexoisomerase 3. Phosphofructokinase 4. Aldolase 5. Triosephosphate isomerase 6. Triosephosphate dehydrogenase 7. Phosphoglyccryl kinase 8. Phosphoglyceryl mutase 9. Enolase 10. Phosphoenolpyruvic transphosphorylase 1 1 . Pyruvic carboxylase 12. Alcohol dehydrogenase 9J4 RESPIRATION than the Embden-Meyerhof, wc may consider ethanol production as at least suggestive of the pathway. Formation of ethanol from glucose is rather common among the fungi. Early work, chiefly with species of Aspergillus, Fusarium, Mucor, and Penicillium, is reviewed by Wehmer (317), Czapek (69), and Foster (84). A number of the wood- destroying basidiomycetes form detectable amounts of ethanol in culture (178, 221, 232, 233, 319); other ethanol producers include Ashbya gossypii (194), Rhizopus spp. (121, 310, 314), Diplodia tubericola (121), Dematium pullulans (278), Mortierella spp. (233), Neurospora crassa (232), Stemphylium radicinum (178), and, probably, several of the Fungi Imperfecti (20). The most active producers are in the genus Fusarium, and, as will be seen later, a functional Embden- Meyerhof pathway is present in F. lini. Under the usual experimental conditions ethanol is oxidized con- currently with its formation, so that an accurate stoichiometry is im- possible (5, 194, 255). However, under anaerobic conditions not permitting either growth or ethanol oxidation it has been possible to show that the production of alcohol and carbon dioxide from glucose follows the quantitative predictions of the Embden-Meyerhof pathway (62, 290, 302). Species of a given genus vary considerably in their accumulation of alcohol under growth conditions; this has been studied especially in Aspergillus (21, 292, 333). Ethanol production by fungi is generally favored by restricted aeration (168, 285, 290), although even under conditions of rapid aeration some fungi form large amounts of alcohol (176, 314). The accumulation of ethanol, like that of other carbon-containing metab- olites, is greatest in media relatively high in carbohydrate (146) and low in nitrogen and minerals (243). Ethanol oxidation in Fusarium lini is a special case of a more general capacity to oxidize primary and secondary alcohols (111, 112); ethanol is oxidized to acetaldehyde, 2-butanol to 2-butanone, and 2,3- butylene glycol to acetoin. Acetaldehyde, the precursor of ethanol in the yeast fermentation, is formed, sometimes in appreciable amounts, by growing cultures or resting preformed mats of several fungi (16, 22, 27, 171, 176, 211, 221, 255, 257, 279, 286). Most of these forms are ethanol producers, and the acetaldehyde may be formed either as a precursor of ethanol or during oxidation of it (112). In the yeast fermentation small amounts of glycerol are formed; more is produced in the presence of sulfite or at alkaline pH. The same observations have been made with fungi (211, 287). Pyruvic acid is frequently produced in isolatable amounts by fungi THE EM BDEN-ME YERHOF PATHWAY 215 and actinomycetes (60, 131, 237, 238, 257); the presence of nitrite in- creases pyruvate formation by Fusarium spp. (220). Added thiamine decreases pyruvate accumulation (273, 323), presumably by reason of its function in diphosphothiamine, the coenzyme of pyruvic car- boxylase. The formation of ethanol, acetaldehyde, and pyruvic acid is con- sistent with the functioning of a pathway of the yeast type, as is the appearance of other minor products (53, 109, 237, 255, 281), but does not prove the occurrence of the pathway in any sense. The occurrence of lactic acid among fungi has been reviewed in the preceding chapter. Among the phycomycetes, the only true fungi to form large amounts of lactate from glucose, there appear to be two different types of metabolic pattern. The lower phycomycetes so far investigated anaerobically convert virtually all of the glucose con- sumed to lactic acid (40, 41, 114, 145). This immediately suggests comparison to the metabolism of muscle and of the homofermentative bacteria, in both of which glucose is converted anaerobically to two moles of lactic acid via the Embden-Meyerhof pathway. The com- parison must, of course, be only tentative in the absence of further data; the demonstration of a diphosphopyridine-linked lactic dehy- drogenase would be of especial interest. Members of the genus Rhizopus constitute the second group of lactate-forming fungi. Under anaerobiosis glucose is converted to lactic acid in 50 per cent yield; Waksman and Foster (310) suggest the over-all reaction: Glucose — » lactic acid + ethanol + C02 (4) The data of Table 1 show that R. oryzae ferments labeled glucose as expected from the Embden-Meyerhof sequence, that is, the path of glucose carbon is presumably, in outline: C3,4 C2,5 CI /Cl,6 02 C3 4 Lactic acid C3 -> C2,5 (5) C4 Cl,6 G5 \ C2,5 + C3,4 C6 Cl,6 Glucose Pyruvic acid Ethanol CO2 This implies that all products arise from a common pool of triose phosphate, leaving open the problem of how the 1:1 ratio of lactic acid to ethanol (Equation 4) is maintained. The conversion of pyruvate to 216 RESPIRATION lactate has been shown (84), and carbon- 14 data suggest that a direct reduction occurs (48). Leuconostoc mesenteroides yields the same products as Rhizopus spp., but the mechanism of this heterolactic fermentation is entirely different (75). Table 1. The Distribution of Glucose Carbon among the Fermentation Products of Rhizopus oryzae* Substrate co2 Ethanol Lactic Acid Exp. CH3 CH2OH COOH CHOH CH3 1 Glucose- 1-C14 — — — 0.5 0.5 16.0 2 Glucose-1-C14 0.5 14.1 0.9 0.2 0.2 3.8 3 Glucose-3,4-C14 13.0 0 0 13.2 0.35 0.5 4 Glucose-3,4-C14 — — — ■ 14 0.2 0.2 * Figures represent millimicrocuries of carbon-14 in each carbon atom. From M. Gibbs and R. Gastel, Arch. Biochem. Biophys. 43: 33-38 (1953), by permission of The Academic Press, Inc. Purely enzymatic studies can establish only the possibility of the Embden-Meyerhof pathway, since most of the enzymes of the sequence are also used for disposing of the triose phosphate formed in the phosphogluconate oxidation pathway and since the enzymes, even if present, are not necessarily functional. Pyruvic carboxylase has been detected in several fungi (62, 88, 148, 230, 274). Aspergillus niger extracts contain virtually the complete battery of enzymes necessary for the conversion of glucose to ethanol and carbon dioxide (148); this is consistent with the data cited earlier indicating that some species of Aspergillus produce ethanol. Several of the same enzymes appear in extracts of Penicillium chrysogenum (264) and P. notatum (182). Aldolase, one of the enzymes which is characteristic of the Embden- Meyerhof sequence, is found in Aspergillus spp. (7, 15a, 149, 150), Neurospora crassa (277), Fusarium lini (62), Penicillium spp. (182, 264), Streptomyces coelicolor (61), and Tilletia caries (212). Ethanol production in Fusarium spp. has already been mentioned. Carbon balance studies, enzymatic analyses, and isotope distribution experiments agree that anaerobically the Embden-Meyerhof pathway is functional and that no other anaerobic mechanism need be postu- lated (62, 125). Balance and fermentation data on Ashbya gossypii also implicate the yeast system (194). Streptomyces coelicolor is unable to form either carbon dioxide or acids anaerobically; it contains, however, the enzymes necessary to convert glucose to pyruvic acid by way of the Embden-Meyerhof pathway. The explanation for its failure to ferment seems to be that THE PHOSPHOGLUCON ATE OXIDATION PATHWAY 217 no enzymes are present which can anaerobically metabolize pyruvate and regenerate diphosphopyridine nucleotide from its reduced form (61). This biochemical lesion occurs also in certain of the bacteria (132). These data suggest that failure of certain of the true fungi, e.g., Myrothecium verrucaria, to ferment glucose may ultimately be traced to the same enzymatic deficiency. In summary, the cumulative evidence for the Embden-Meyerhof pathway in at least some of the filamentous fungi is impressive. There are, however, several different variations of the pattern. Fusarium spp. and, probably, Aspergillus spp. appear to resemble yeast most closely. The enzymatic basis of the two types of lactic acid formation in the phycomycetes remains unknown, and at present it is only on the basis of comparative biochemistry that we regard these as subtypes of the Embden-Meyerhof sequence. Finally, it is possible that non-fermenta- tive forms can utilize the pathway by coupling it to an aerobic process in which reduced coenzyme is regenerated by an oxidase system and pyruvate is metabolized via the citric acid cycle. 6. THE PHOSPHOGLUCONATE OXIDATION PATHWAY Studies carried out during the decade 1930-1940 by Dickens, Lipmann, and Warburg and more recently by Horecker, Racker, and many others have established the occurrence of a second sequence of respiratory reactions, variously known as the "hexosemonophosphate shunt," the "direct oxidative pathway," and the "pentose phosphate cycle." The system appears to be widespread in nature, occurring in animals, plants, and microorganisms (238). For convenience and to avoid unnecessary detail, we may, following Racker (236), divide the pathway into two phases. The first is an oxidative phase, in which glucose-6-phosphate is transformed to pentose phosphate via 6-phosphogluconic acid; carbon atom 1 of glucose, the aldehyde carbon, is converted to carbon dioxide in the process: Glucose-6-phosphate + TPN+ -» 6-phosphogluconic acid + TPNH + H+ (6) 6-Phosphogluconic acid + TPN+ — » pentose phosphate + TPNH + H+ + C02 (7) The key enzymes of this phase are glucose-6-phosphate dehydrogenase (Equation 6) and 6-phosphogluconic acid dehydrogenase (Equation 7); both are linked to triphosphopyridine nucleotide (TPN). Reac- 218 RESPIRATION tion 7 is probably tbe sum of two reactions. It is assumed that the aerobic reoxidation of reduced coenzyme (TPNH) occurs and that in the process the energy of the oxidation is utilized for synthesis of adenosine triphosphate or related compounds. The phosphoglu- conate pathway is an aerobic system; evidence for an analogous fermentative pathway is summarized by Gunsalus et al. (118). The first pentose phosphate formed, i.e., the product of Equation 7, is ribulose-5-phosphate; this rapidly comes into enzymatic equilib- rium with two other pentose phosphates, ribose-5-phosphate and xylulose-5-phosphate. The second phase of the sequence is a complex series of non-oxida- tive conversions of the pentose phosphates into other phosphorylated sugars. Direct intermediates in this phase include sedoheptulose-7- phosphate, glyceraldehyde-3-phosphate, a tetrose-4-phosphate (prob- ably erythrose-4-phosphate), and fructose-6-phosphate. Since glucose-6- phosphate is regenerated, this second phase may be summarized: 6 Pentose phosphate -> 5 glucose-6-phosphate (8) The key enzymes in this phase are transketolase and transaldolase. In the presence of enzymes of the Embden-Meyerhof sequence, still other sugar phosphates, fructose- 1,6-diphosphate and sedoheptulose- 1,7-diphosphate, appear; in addition a greater or less amount of the triose phosphate formed may be converted to pyruvic acid and thereby removed from the system under discussion. It was natural to investigate the fungi for an aerobic system of this type, but so far comparatively little progress has been made toward an organized and coherent picture of the role of this cycle in the oxidative metabolism of the fungi. The evidence at hand includes three types of study: demonstration of particular enzymes, demonstra- tion of one or more of the characteristic sugar phosphates, and study of the distribution of isotope from specifically labeled glucose. The two dehydrogenases of the oxidative phase of the pathway have been identified in Neurospora crassa (213), Tilletia caries (212), and Streptomyces coelicolor (66). Extracts of Aspergillus niger contain glucose-6-phosphate dehydrogenase (147). The conversion of pentose phosphate to hexose phosphate and the formation of sedoheptulose phosphate and other known intermediates are easily demonstrated in cell-free preparations of both fungi and actinomycetes (64, 212, 263). A variety of methods depending on the formation of metabolites from carbon-14 labeled glucose have been devised in an attempt to assay the quantitative importance of the phosphogluconate oxidation THE PHOSPHOGLUCONATE OXIDATION PATHWAY 219 pathway. One group of methods is based upon the fact that operation of the phosphogluconate oxidation pathway results in a preferential liberation of the aldehyde carbon of glucose as carbon dioxide. The limitation of this approach is that glucose goes through many more metabolic pools and is more diluted in the Embden-Meyerhof than in the oxidative pathway before liberation of carbon dioxide; this results in a bias which may exaggerate the contribution of the oxidative path- way. Application of various modifications of this method indicates that there is a substantial flow of carbon over some pathway prefer- entially liberating carbon- 1 of glucose as carbon dioxide in Penicillium chrysogenum (80, 124), Aspergillus oryzae (6), Streptomyces coelicolor (66), and Neurospora crassa (293). A modification of this method indicates that in growing mycelium of Penicillium chrysogenum about 60 per cent of the glucose carbon goes over the oxidative path- way (124), while in Fusarium lini a minimum of 17 per cent of the glucose catabolic mechanism involves preferential liberation of car- bon-1 (125). A more promising method involves determination of acetate, ethanol, pyruvate, or compounds derived therefrom during aerobic respiration of specifically labeled glucose (26, 318). Application of this technique to Penicillium chrysogenum suggests that the Embden- Meyerhof pathway is the principal route of glucose breakdown (172). Still other methods may be applicable but have not yet been tested in the fungi (18, 19, 23, 24, 105, 139, 156, 236, 268). Wood (327) discusses the methods critically and points out their limitations. In Aspergillus niger the distribution of isotope in citric acid in- dicates that the Embden-Meyerhof sequence accounts for 80 per cent of the total aerobic glucose catabolism (261). Similarly, in Ustilago zeae the labeling of the carbohydrate moiety of the glucolipid ustilagic acid is consistent with the resynthesis of glucose from triose phosphate and consequently with the operation of the Embden-Meyerhof path- way (29). The labeling of kojic acid formed by Aspergillus flavus from pentose is, on the other hand, suggestive of a preliminary con- version of pentose, through the non-oxidative phase of the phospho- gluconate pathway, to hexose (8). The various approaches which attempt to utilize labeled sugars each has its own difficulties and ambiguities, and we should regard the data collected so far as preliminary and tentative. Some attempt should be made to see whether two different methods agree with regard to the same material before reliance can be placed on any single method. There seems little doubt that the phosphogluconate oxidation pathway is utilized by many fungi and actinomycetes, and it is not unreasonable 220 RESPIRATION to anticipate that for some it may be a major pathway of respiration or of pentose biogenesis. It is also clear that in some organisms both the oxidative and the Embden-Meyerhof systems are present and presum- ably complete for glucose metabolism (64). It is to be expected that cells in different physiological states will strike different balances between the two systems. 7. THE NON-PHOSPHORYLATIVE OXIDATION OF GLUCOSE The single reaction of this type known is the oxidation of free glucose to gluconic acid (the first product is in fact glucono-8-lactone) by the enzyme usually described as glucose oxidase or notatin. It is debatable whether this reaction can be classed as respiratory, since we know neither the subsequent metabolic history of the gluconic acid nor the way in which energy from the oxidation can be used by the cell. However, it seems likely that further study will reveal a genuine respiratory pathway. The enzyme responsible for the oxidation of free glucose was dis- covered by D. Muller (205, 206) in Aspergillus Jiigcr and named glucose oxidase; earlier results of Maximov (193) probably involve this enzyme. Interest in it was revived by the later discovery that an antibiotic effect of Penicillium resticulosum and P. notatum is trace- able to the hydrogen peroxide generated by the reaction: Glucose + <32 + H20 -» gluconic acid + H202 (9) As an antibiotic, and before its identity with the enzyme was known, the material was variously named penicillin B, notatin, and penatin. The enzyme is a flavoprotein with two flavin adenine dinucleotide groups per molecule (158). It is highly specific for /3-D-glucose; purified preparations are virtually inactive toward other common sugars (160), although 2-deoxyglucose is oxidized (267). The specificity has been taken advantage of in a manometric assay for glucose (159) and in the demonstration of the mutarotase of Penicillium notatum (161). Studies with isotopic oxygen show that the enzyme transfers hy- drogen from glucose to oxygen (15). It is therefore a dehydrogenase rather than an oxidase, and the name glucose aerodehydrogenase is to be preferred. The enzyme differs from the glucose dehydrogenases of liver (120) and of Pseudomonas fluorescens (330), neither of which is a flavo- protein. Claims that other glucose dehydrogenases are present in fungi (207, 224, 225) have been disputed (91), and it appears that only one such enzyme is involved in the organisms studied. PENTOSE FERMENTATION AND OXIDATION 22 1 The importance of the oxidation of free glucose is difficult to assess. A relatively large number of fungi form gluconic acid (Chapter 6) and we may reasonably assume that the same reaction is involved in all. However, the known glucose aerodehydrogenase has been found only in the fungi mentioned above and in Penicillium glaucum, apparently not occurring in Aspergillus jumigalus, Mucor racemosus, Rhizopus nigricans, or Dematium pullulans (92). As mentioned earlier, the availability of the energy of the reaction to the cell is uncertain; the hydrogen peroxide formed could, in the presence of catalase, serve as an oxidizing agent for other cell metabolites (157, 294). The further metabolism of gluconic acid is similarly obscure; the likeliest possibili- ties are that the gluconate is phosphorylated and enters the hexose monophosphate pathway, or that there is a continued non-phosphoryla- tive metabolism after the pattern of the bacterial genera Pseudomonas and Acetobacter (297, 329); discovery of 2-keto-gluconic acid in small amounts in the culture fluid of Penicillium brevi-compactum (Chapter 6) suggests that the bacterial pathway may be worth investigation. 8. PENTOSE FERMENTATION AND OXIDATION The fermentation of pentoses by fungi has been studied primarily as it occurs in the genus Fusarium, although other fungi can carry out at least a slow fermentation of pentose (221). Early results and theories on the reaction are reviewed by Foster (84), and are of historical interest only. Resting cells of Fusarium lini ferment xylose in accordance with the equation: C5H10O, -> C2H5OH + CH3COOH + C02 (10) Xylose Ethanol Acetate With xylose- 1-C14 as substrate, virtually all of the label appears in the methyl carbon of acetate (98). This system resembles that of Lacto- bacillus spp. (169), with the difference that the lactic acid character- istic of the bacterial fermentation is replaced by ethanol and carbon dioxide. In Lactobacillus pentosus preparations the pentose chain is split between carbons 2 and 3, yielding triose phosphate and acetyl phosphate (35, 123). Presumably in F. lini the triose phosphate is then metabolized to ethanol and carbon dioxide — the enzymes neces- sary for this transformation are all present (62). The pentose fermentation by F. lini is striking in another connec- tion: there does not seem to be any conversion of pentose to hexose by the non-oxidative reactions of the phosphogluconate oxidation path- 222 RESPIRATION way, as described earlier for other fungi and actinomycetes and as is common in yeast and bacterial pentose metabolism (4, 99). The oxidative breakdown of pentose by fungi has not been inten- sively investigated. It has been mentioned in the preceding chapter that there is one report of the oxidation of pentoses to pentonic acids; the more recent and better documented report (175) that Pscudomonas spp. form pentonic acids from pentose suggests that a re-examination of the problem in fungi would be of interest. Streptomyces coelicolor extracts oxidize xylose and ribose only if the cells have been grown on the particular sugar; indirect evidence suggests that these pentoses must be phosphorylated and that the kinase responsible is an in- ducible enzyme not present in glucose-grown cells (66). 9. THE CITRIC ACID CYCLE In animal tissues the functioning of a respiratory cycle, the citric acid cycle (also called the tricarboxylic acid cycle or the Krebs cycle) is well established. The historical development of the concept has been reviewed by Krebs (165); other valuable general reviews are those of Wood (326) and Ochoa (222). In Figure 5 the principal reactions of the citric acid cycle are out- lined. It can be seen, first, that the net effect of one turn of the cycle is the complete oxidation of a 2-carbon fragment, CH.CO — , to carbon dioxide and water. This acetyl fragment is known to be attached to coenzyme A, forming acetyl-coenzyme A. The first reac- tion in the cycle is the transfer of the acetyl fragment to oxaloacetic acid, forming citric acid. In subsequent reactions, two molecules of carbon dioxide are split off and oxaloacetic acid is regenerated. In principle, therefore, only one molecule of oxaloacetate is required, to initiate the cycle, after which the reaction becomes self-sustaining. In fact, of course, carbon is drawn off, especially for the synthesis of amino acids, so that steady state operation of the cycle under normal condi- tions presumably requires that 4-carbon dicarboxylic acids be formed de novo continually. Mechanisms are known in bacteria by which pyruvic acid, phosphoenolpyruvic acid, and propionic acid can be carboxylated by carbon dioxide fixation (118); in addition, the pos- sibility of malate synthesis from acetate has been raised by work with bacteria (p. 227). Viewed strictly as a respiratory system, the net effect of the cycle shown in Figure 5 is the oxidation of pyruvic acid: CHa— CO— COOH + 2.5 O, -* 3 CO. + 2 HoO (11) the citric: acid cycle Pyruvate co2-VDPN + ' ^DPNH + H + Acetyl-coenzyme A Oxalacetate -^=» ^DPNH + H+V~/ © -*- Citrate ©1 v^DPN+ cis- Aconitate r Malate ©1 / |© if D-Isocitrate Fum. i 2H+ +2e^ irate © © ^TPN + ^TPNH + H + • Succi nate Oxalosuccinate 223 C02 + 2H+ +2e a-Ketoglutarate Figure 5. Schematic and simplified outline of the citric acid cycle. Abbreviations: DPN+ = diphosphopyridine nucleotide, DPNH = reduced diphosphopyridine nucleo- tide, TPN+ = triphosphopyridine nucleotide, TPNH — reduced triphosphopyridine nucleotide. Enzymes are numbered as follows: 1. Condensing enzyme 5. Complex system 2. Aconitase 6. Succinic dehydrogenase 3. Isocitric dehydrogenase 7. Fumarase 4. Oxalosuccinic decarboxylase (?) 8. Malic dehydrogenase Free acetate may enter the cycle and be oxidized, by 2 moles of oxygen. The occurrence of the cycle is well established in animal cells, and there is good evidence that it is at least potentially functional in yeast, bacteria, and higher plants. There is, however, a real question whether its major metabolic importance is in supplying the carbon skeleton of amino acids (167, 248) or whether it has an additional role in providing energy to the cell (74, 283). With the probable exception of the oxidative decarboxylation of a-ketoglutaric acid, the phos- phorylations associated with the citric acid cycle are at the cofactor level. The organic acids of the citric acid cycle which are formed in sub- stantial amounts by fungi include succinic, citric, fumaric, and acetic 224 RESPIRATION acids; malic acid appears usually in smaller quantities (Chapter 6). Although their occurrence is suggestive of the cycle, it is in no sense proof. Nor can it be argued from the mere fact that intact cells oxidize these organic acids that the cycle is present. Alpha-ketoglutaric acid is formed in detectable amounts by Penicil- Uwn chrysogenum from lactic acid (136), by Aspergillus oryzae from glucose (252), and by A. niger from endogenous reserves (311). Its role in glutamic acid synthesis is considered later (Chapter 8). Individual reactions of the cycle have been studied especially in seven organisms: Blastocladiella emersonii, Ashbya gossypii, Neuro- spora crassa, Aspergillus niger, Penicillium chrysogenum, Streptomyces coelicolor, and Zygorhynchus moelleri. For some of these more or less data is also available from isotope distribution experiments. Most of the known enzymes of the cycle have been demonstrated in Blastocladiella emersonii, as has also the formation of citrate (43, 46, 47). The cycle, however, appears to be at best weakly functional in this organism, and development of the resistant sporangia is accom- panied by loss of most of the enzyme activities of the cycle (43). Can- tino and Horenstein (45) have suggested that in B. emersonii a reduc- tive succinate-ketoglutarate-isocitrate (S.K.I.) cycle is operative; the most striking feature of this proposal is that light, which accelerates growth, is visualized as providing the energy for the carboxylation of succinate and a-ketoglutarate. In Ashbya gossypii acetate oxidation is accelerated by dicarboxylic acids, citrate is synthesized from acetate and oxalacetate, and citrate accumulation is increased by malonate (195). Several of the characteristic enzyme activities of the cycle have been demonstrated in extracts of Neurospora crassa (82, 213, 258, 275). Incorporation of carbon-14 from several substrates into aspartic and glutamic acids indicates that in this fungus the citric acid cycle plays a major role in the synthesis of these amino acids (248). Aspergillus niger is of particular interest in that some strains ac- cumulate citric acid (Chapter 6) and in that tracer studies have been made. Enzymatic analyses are the most complete for any fungus, and show that all of the known enzymes of the citric acid cycle are present in extracts made from mycelium (190, 239, 240, 241). The enzyme systems acting upon tricarboxylic acids are rather complex: there is evidence for an aconitase of the usual type, an aconitic hydrase con- verting aconitate only to citrate (73, 210), and two isocitric dehydro- genases (241). Excellent agreement between the enzymatic content of cells and the accumulation of citric acid affords further evidence for THE CITRIC ACID CYCLE 225 the role of an interrupted tricarboxylic acid cycle in the biogenesis of citric acid (242). Extensive studies of the incorporation of isotopic carbon into the citric acid molecule lend further and decisive support to the idea that bulk citric acid synthesis in Aspergillus niger occurs via the citric acid cycle. The labeling of citrate formed in the presence of C1402 is consistent with fixation of carbon dioxide into oxalacetate and con- densation of the oxalacetate with an acetyl fragment (191, 192, 198). Lewis and Weinhouse (174) showed experimentally that the sym- metrical citric acid molecule is asymmetrically synthesized, as first suggested by Ogston (223). Experiments with the metabolism of labeled acetic acid (28, 49, 173) similarly implicate the known initial reactions of the citric acid cycle in the synthesis of citrate by A. niger. A study of the incorporation of glucose-3,4-C14 into citrate provides evidence that pyruvate, arising from glucose, is converted to oxalacetate and an acetyl fragment, and that these condense to yield citrate with almost theoretical incorpora- tion of carbon dioxide and little or no randomization of the carboxyl carbons of oxalacetate (58). The last observation is important in showing that oxalacetate does not equilibrate with a symmetrical acid like fumaric or succinic. Although it has been suggested (85) that a condensation of 2-carbon fragments may yield the 4-carbon dicarboxylic acid skeleton for citrate synthesis, work with isotopic acetate indicates that this pathway is at best of minor significance (28, 173), and the studies of Cleland and Johnson (58) with labeled glucose afford no evidence at all for such a reaction. For Penicillium chrysogenum there is good evidence of the forma- tion of citrate from acetyl phosphate and oxalacetate and for the presence of enzyme systems oxidizing citrate, isocitrate, a-ketoglutarate, succinate, and malate (51, 126, 229). A partially purified succinic dehydrogenase is inhibited by malonate (110), and acetate oxidation is depressed by the same poison (13). The incorporation of acetate carbon into glutamic acid conforms to that expected if the citric acid cycle is responsible for the metabolism of acetate (113). Extracts of Streptomyces coelicolor carry out the major reactions of the cycle, including the formation of citrate, the oxidation of «- ketoglutarate and other acids, and the decarboxylation of oxalacetate (65). The incorporation of labeled carbon dioxide and acetate into amino acids by S. griseus is also consistent with the operation of the cycle (39, 104). 926 RESPIRATION Most of the enzymes of the cycle are demonstrable in extracts of Zygorhynchus moelleri (201), and isotope distribution experiments indicate that the cycle plays a major role in energy metabolism (202). Apart from these more intensively studied species, individual en- zyme activities have been determined in many other fungi and actinomycetes. These include oxalacetic decarboxylase (87, 306), a- ketoglutaric acid oxidase (46), "oxalosuccinic decarboxylase" (183), enzyme systems oxidizing succinic acid (46, 89, 137, 182, 216), and isocitric dehydrogenase and condensing enzyme (12). In conclusion, it is evident that the enzymes of the citric acid cycle are of quite general occurrence among fungi and actinomycetes and, indeed, that further enzyme surveys can add little to our understand- ing. The more important question is the physiological role of the citric acid cycle. Two such roles may be envisaged: provision of energy to the cell, and provision of carbon skeletons for amino acid synthesis. The second of these is reasonably certainly a real function of the cycle in Neurospora crassa, Penicillium chrysogenum , and Streptomyces griseus. A role in energy metabolism is more difficult to establish, but would be of great interest. Citric acid accumulation seems to result directly from an interruption of the cycle, under special nutritional conditions, at the citric acid stage. We must also recall from Chapter 6 that isocitritase, catalyzing the conversion of isocitrate to succinic and glyoxylic acids, is known to occur in fungi; its activity may contribute to some of the complexities of the metabolic data. 10. DICARBOXYLIC ACIDS IN RESPIRATION Apart from their role in the citric acid cycle, it has long been speculated that the 4-carbon dicarboxylic acids (succinic, fumaric, malic, and oxalacetic) may be components of another respiratory cycle in which acetate is oxidized to carbon dioxide. The initial reaction of such a cycle would be the reductive condensation of acetate, first suggested by Thunberg (298): 2 CH3COOH -» HOOC— CHo— CH2— COOH + 2 [H] (12) Further reactions can be imagined whereby the succinate so formed is converted to 1 mole of acetate and 2 moles of carbon dioxide, the acetate re-entering the cycle. The net effect would be the oxidation of one mole of acetate for each turn of the cycle. Possible dicarboxylic acid cycles are described by Ajl (1), Ochoa (222), and Umbreit (307); Siegel (262) discusses energy relations. DICARBOXYLIC ACIDS IN RESPIRATION 227 The formation of 4-carbon dicarboxylic acids has often been as- cribed to the Thunberg condensation solely on the basis that resting or growing cells given acetate form one or more of the dicarboxylic acids. By itself, this observation is of no significance and cannot be offered as evidence for the Thunberg reaction. The most effective evidence for some sort of Thunberg reaction in fungi comes from studies on the formation of fumaric acid by Rhizo- pns nigricans, and consists of the finding that the organism forms fumarate from ethanol in yields so high as to make it unlikely that the citric acid cycle is the pathway responsible (89). The reaction may be written: 2C*H3CH2OH + 02^ 2C*H3COOH -> HOOC— C*H=C*H— COOH (13) As indicated in the equation, the methyl carbons of ethanol appear as the methine carbons of fumarate. However, in this instance the dis- tribution of isotope is not of critical value, i.e., the labeling results do not require us to postulate the Thunberg reaction (2, 283, 309); the entrance of formic acid into the methyl group of pyruvate also com- plicates the interpretation of the isotope data (151, 152). It has been suggested that an "acetic dehydrogenase" of Aspergillus fumigatus carries out the Thunberg reaction (12), but the products of the reaction have not been isolated. Studies on bacteria offer a possible model for the results on Rhizopus nigricans. The enzyme malate synthetase of Escherichia coli carries out the reaction (325): Acetyl phosphate + glyoxylate —> malate (14) Kornberg and Madsen (164) find the same enzyme in Pseudomonas sp. and suggest the following sequence of reactions: Acetate + oxalacetate — » citrate O^a) Citrate —> isocitrate (15b) Isocitrate— > succinate + glyoxalate (15c) Acetate + coenzyme A—» acetyl-CoA (15d) Acetyl-CoA + glyoxylate-^ malate -f CoA (15e) Malate — > oxalacetate + 2[H] (15f) 2 Acetate-^ succinate + 2[H] (15) This complex system requires condensing enzyme (15a), aconitase (15b), isocitritase (15c), some mechanism generating acetyl-coenzyme A (15d), malate synthetase (15e), and malic dehydrogenase (15f). Al- 228 RESPIRATION though the accumulation of malate or of fumarate by such a system can be imagined, the cyclical character of the system as written would not be maintained. Most of the enzymes required for Equation 15 are known in fungi, and it will be of considerable interest to see if some such series of reactions participates in energy metabolism or in the synthesis of dicarboxylic acids. A comparable reaction system in Tetrahymena pyrifonnis converts succinate reversibly to acetyl phos- phate (256). 1 1 . CARBON DIOXIDE FIXATION Evidence for a physiological role of carbon dioxide in the growth of fungi appears first in relatively crude experiments on Fusarium spp. (180); retardation of early growth in the absence of carbon dioxide has been observed in many other fungi (245, 246, 250). The carbon dioxide requirement is enhanced by mineral deficiencies (269). An apparent effect of carbon dioxide on dehydrogenations (130) has been shown to be exerted in fact on endogenous methylene blue reduc- tion (97). Carbon dioxide stimulation of growth could, of course, be ascribed to other phenomena than fixation. However, in Penicillium chryso- genum there is good evidence that fixation is related to the essentiality of carbon dioxide (106). In this organism, fixation is negligible un- less both carbon and nitrogen sources are provided; this situation, coupled with the fact that carbon dioxide is not essential in a complex medium and that aspartic and glutamic acids are labeled very early by C14 from isotopic carbon dioxide, suggests strongly that the physio- logical role of carbon dioxide is in the synthesis of essential amino acids via the citric acid cycle. Carbon dioxide fixation in Neurospora crassa is similarly enhanced by provision of nitrogen (275) and carbon (128). It has been mentioned earlier in this chapter that under appropriate conditions there is extensive incorporation of carbon dioxide into citric acid. Labeled carbon dioxide is also incorporated into the carboxyl group of pyruvic acid by Rhizopus nigricans (86), in the carboxyl of lactate by R. oryzae (100), in oxalic acid by Aspergillus niger (174), in succinic acid by Streptomyces coelicolor (60), in strepto- mycin by 5. griseus (144), and in unidentified cell constituents by several fungi (181). In organisms generally there are several different pathways by which carbon dioxide is fixed (10, 118, 309). Indirect and not entirely conclusive evidence suggests that fixation occurs into oxalacetate in TERMINAL RESPIRATION 229 Rhizopus nigricans (86) and Neurospora crassa (276). We have already noted the proposal that the light-stimulated carbon dioxide fixation in Blastocladiella emersonii reflects the reductive carboxylation of suc- cinate and a-ketoglutarate (45). The pathway of fixation can presum- ably only be detected with finality in experiments with active cell-free preparations. 12. TERMINAL RESPIRATION In aerobic organisms the ultimate oxidizing agent is molecular oxygen; the reaction or sequence of reactions by which the electrons withdrawn from the substrate are transferred to oxygen is denoted terminal respiration. Conventionally the process is visualized as transfer of hydrogen from substrate to oxygen. The transfer is mediated by a system in which substances of increasingly positive oxidation-reduction potential form a chain of carriers. The simplest system involves only one carrier, the flavoprotein prosthetic group of an enzyme; examples are the amino acid oxidases and glucose aerodehydrogenase. More usually, the pyridine nucleo- tides— diphosphopyridine nucleotide or triphosphopyridine nucleotide — are the first hydrogen carriers of the sequence. Perhaps the most common terminal pathway involves a pyridine nucleotide, a flavo- protein, and the cytochrome system in succession, that is, the flow of hydrogen (electrons) is over the pathway: Substrate— > pyridine nucleotide —> flavoprotein — > cytochromes — > oxygen (16) The cytochrome system is complex and there is considerable doubt whether what is known of animal systems can be applied directly to microorganisms. The flavoprotein is probably always a metallo- enzyme (141). Under some conditions, e.g., a high partial pressure of oxygen, flavo- proteins may react directly with oxygen without the intervention of the cytochrome system (295). The cytochrome system appears to be general in the fungi, as judged in the first instance by spectroscopic observations (32, 33, 56, 117, 122, 197, 227, 291, 300). However, it should be noted that experience with bacterial cytochromes indicates that it is not possible from simple spectroscopic measurements to state with precision whether the com- ponents of a new system are the same as those of the more familiar yeast and animal systems (266, 321). Consequently, it is well to re- serve judgment for the time being as to whether the fungal cyto- 230 RESPIRATION chromes are the same as those of yeast, and to be content with the generalization that cytochromes are generally present in fungi. The cytochrome c of Ustilago sphaerogena, for example, has a spectrum like beef heart cytochrome c, but has a higher molecular weight and a lower isoelectric point (208). In this organism, too, the conditions of culture, especially the supply of zinc and of thiamine, markedly affect the concentrations of the cytochromes (117). Exogenously supplied cytochrome, from animal sources, functions as an electron carrier for the aerobic lactic dehydrogenase of Penicil- lium chrysogenum (50) and for the succinoxidase systems of all forms tested (13, 190, 258). Cytochrome oxidase may be assayed either manometrically or spectrophotometrically (Figure 6), usually with animal cytochrome c as substrate. The enzyme, operationally denned by these assays, seems to be of universal occurrence in the fungi; experiments have covered representatives of all of the major taxonomic groups (31, 32, 33, 46, 47, 57, 71, 117, 213, 230, 300). As in other organisms, cytochrome oxidase activity is associated with the insoluble and sedimentable fraction of homogenates (57, 71, 122, 258, 299). In bacteria, this fraction is apparently the cytoplasmic membrane or a similar structure (266). The flavin components of the terminal system which proceeds over the cytochromes (Equation 16) have not been systematically in- vestigated. 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The dependence upon carbon is illustrated in Figure 1; in this organism, Aspergillus niger, enough is known of other cultural factors so that a strict proportionality between carbo- hydrate supply and nitrogen demand can be demonstrated (496). No single pattern of nitrogen assimilation can be described to apply to all fungi. In general, however, inorganic or organic nitrogen is taken up rapidly during the phase of growth. Even in this phase, however, there may be a back movement of ammonia nitrogen from the cells to the medium (211), reflecting the permeability of the cell to ammonia. Certain enzymes also begin to appear in the medium during this period (115, 278), and in at least some organisms amino acids and other soluble nitrogen compounds are liberated (139, 181, 374, 413). The major liberation of proteins and other nitrogenous 241 242 NITROGEN NUTRITION AND METABOLISM 200 400 Nitrogen, mg per liter 600 800 Figure 1. The relation between growth of Aspergillus niger and nitrogen supply (ammonium nitrate) in media containing sucrose at 50 gm per liter (curve 1) and 25 gm per liter (curve 2). Drawn from data of Steinberg and Bowling (496). compounds occurs, of course, in the phase of autolysis (Chapter 1). Studies of Scopulariopsis brevicaulis indicate that upon exhaustion of the utilizable nitrogen of the medium, there is a mobilization and reutilization of at least part of the nitrogen from older hyphae to sup- port development of new mycelium (374). 1. INORGANIC SOURCES OF NITROGEN Primary attention must be devoted here to three inorganic forms of nitrogen: ammonium salts, nitrites, and nitrates. Other inorganic nitrogen compounds have been little studied but appear in general to be utilized only if easily or spontaneously converted to one of the types above-named (278, 494). Cyanide as a nutrient and metabolite is considered separately. The Utilization of Nitrate Nitrogen. Nitrates are excellent sources of nitrogen for many fungi (30, 221, 326, 459). Inability to utilize nitrate is especially common in some groups, e.g., the higher basidio- mycetes (221, 234, 275, 327, 397, 546), the Saprolegniaceae (55, 434, 562), and the Blastocladiales so far as known (90). Nitrate utilization, or at least nitrate reduction, is common in the actinomycetes, although INORGANIC SOURCES OF NITROGEN 243 one could ask for more quantitative data (15, 306, 419, 534, 569, 570), but some strains definitely cannot grow with nitrate as the sole source of nitrogen (138, 144, 393, 541). It is unlikely in the extreme that inability to use nitrate can serve as a phylogenetic criterion. Within a genus, individual species differ in nitrate utilization (150, 327, 397, 610). The capacity to use nitrate can be lost by mutation (250, 255, 420, 428, 455), and survival in nature of strains carrying this mutation is presumably governed by their ecological situation rather than their taxonomic position. Failure to utilize nitrate is usually presumed to be absolute, but there has been little exploration of the possibility that nitrate can be metabolized once growth is initiated on another more readily available nitrogen source. When spores of Streptomyces griseus are inoculated into a synthetic nitrate medium, no growth occurs, but a pregrown mycelium uses nitrate readily (105). As mentioned below, nitrite may be toxic in an acid medium. It follows that the kind and the concentration of the carbon source, insofar as these affect the pH, will decisively influence the response of some organisms, e.g., Streptomyces coelicolor (107), to nitrate. Ammonium nitrate is often used in media, and it is of interest to ask whether there is preferential utilization of either ion. The frequent experience that utilization of ammonium nitrate is accompanied by a pH drop (227, 256, 409, 415, 529) is presumptive evidence for prefer- ential utilization of the ammonium ion. More direct evidence is pro- vided, however, by analytical studies. In Scopulariopsis brevicaulis and other fungi, nitrate utilization does not begin until virtually all the ammonium has disappeared from the medium (Figure 2); this is Figure 2. The utilization of ammonium and nitrate by Scopulariopsis brevicaulis in a medium containing glucose, suc- cinate, and ammonium nitrate. Curve 1, nitrate concentration in the medium; curve 2, ammo- nium concentration in the me- dium. From tabular data of Morton and MacMillan (375). 2 4 6 8 Time, days 244 NITROGEN NUTRITION AND METABOLISM also true of Neurospora crassa (511). Detailed study of the phenome- non (345, 373, 375) brings out that nitrate utilization is completely stopped by the addition of ammonium salts, provided the ammonia is assimilated, i.e., that a carbon source is available. Since nitrite uti- lization is not blocked by ammonium, it must be assumed that in these fungi ammonium prevents the reduction of nitrate to nitrite; this is borne out by enzymatic studies, which show that the nitratase system declines in quantity soon after ammonia assimilation begins. Analytical data on utilization of ammonium nitrate by Helmintho- sporium gramineum show a different relation: ammonium ion is used much more rapidly than nitrate, but there is a slight assimilation of nitrate even in the early stages of growth (110). This relation is stated to prevail in some other fungi (375). The preferential utilization of ammonia from ammonium nitrate is influenced by pH. Foster (163) reviews several studies in Aspergillus niger, all of which agree that at very low pH ammonium utilization is reduced and at least some nitrate is assimilated. Attempts have been made to explain this phenomenon as one of permeability or as based on the adsorption capacity of cell colloids (86, 264); neither of these explanations is convincing, and it would appear desirable to reinvesti- gate the phenomenon itself before speculating on its theoretical im- plications. As shown later (Figure 3), the optimum pH for nitrate uptake in Scopulariopsis brevicaulis is about 6.0, far above the "critical" pH at which Aspergillus niger is claimed to assimilate nitrate (439). Nitrate reduction in the fungi is believed to be strictly assimilatory in nature, that is, to occur only in the incorporation of nitrate nitrogen into the cell. This process is in contrast to that of the bacteria, many of which reduce nitrate without assimilation of the nitrogen; nitrate, if it has an essential function, is acting as a hydrogen acceptor lor sub- strate oxidations (556). This "nitrate respiration" appears to be mediated by the cytochrome system in Micrococcus denitrificans and other bacteria (460, 557); whereas, as will be seen, nitrate reduction in the fungi is mediated, so far as we know, by flavin enzymes. It should be obvious, of course, that utilization of nitrate in fungi does depend upon substrate oxidations and affects, therefore, the respiratory quo- tient (521). The nitrate reductase of Neurospora crassa has been studied inten- sively; this work is reviewed by Nason (384). The enzyme is formed only on nitrate or nitrite media, i.e., it is inducible. Flavin adenine dinucleotide is the prosthetic group; sulfhydryl groups are essential to activity, and molybdenum is an essential factor in electron transport. INORGANIC SOURCES OF NITROGEN 245 The passage of electrons is visualized: Reduced triphosphopyridine nucleotide -> flavin adenine dinucleotide -> molybdenum -+ nitrate (1) That is, nitrate is reduced with concurrent oxidation of some other, presumably carbon, compound by a triphosphopyridine-linked dehy- drogenase. The enzyme of Hansenida anomala differs in that either di- or triphosphopyridine nucleotide is functional (483). Two other flavin enzymes of Nearospora crassa, nitrite reductase and hydroxylamine reductase, also inducible metal-dependent enzymes, have not as yet been separated. Together, and possibly with the aid of a third enzyme, they catalyze the reduction of nitrite to ammonia (384,385,451). From these enzymatic studies, and from the somewhat uncertain occurrence of hydroxylamine in nitrate cultures (322, 396, 494), an inorganic pathway of nitrogen reduction has been proposed (384): N03_ -» N02~ -> ? -> NH2OH -> NH3 (2) The unknown intermediate, possibly hyponitrite, is included on the assumption that the four-electron change from nitrite to hydroxyla- mine is unlikely to be a single reaction. Whether this inorganic pathway is the sole and obligatory sequence of nitrate reduction in fungi is not yet certain. Since molybdenum deficiency reduces both nitrate reductase content and the ability to grow on nitrate in N. crassa and Aspergillus niger, it seems probable that the first proposed step is essential. Genetic studies bear out this conclusion (484). However, we cannot be sure that succeeding steps are necessarily inorganic in nature, as implied in Equation 2; incor- poration into organic combination may occur at an oxidation level above that of ammonia (220, 338, 484). A nitroaryl reductase system in Neurospora crassa catalyzes the re- duction of m-dinitrobenzene by reduced pyridine nucleotides to ra-ni- troaniline (384). This system, although possibly not specific, is of interest as a model of the reduction of organic nitro compounds as an alternative to the inorganic pathway of Equation 2. The increased toxicity of thiourea to Aspergillus niger in nitrate media suggests that the poison interferes with assimilation (158); chlorate toxicity is also more evident in nitrate media (203). Fluoride causes the accumulation of nitrite in fungi, presumably acting spe- cifically on the nitrite reductase system (373). 246 NITROGEN NUTRITION AND METABOLISM The Utilization of Nitrite Nitrogen. Nitrite serves more or less well as the sole source of nitrogen for a number of fungi, e.g., Fusarium niveum (622), Coprinus spp. (165), Phymatotrichum omnivorum (520), Scopulariopsis brevicaulis (375), and Rhizophlyctis rosea (424). Dif- ferent species of Aspergillus use it well, poorly, or not at all (305, 457, 494). Failure to utilize nitrite, which is the more common situation in fungi, is explained on the basis of its known toxicity (233, 402, 623). The fact that growth in the presence of nitrite is best in alkaline media (107, 402, 454) indicates that it is the free unionized acid, rather than the nitrite ion, which is the toxic species. Nitrite toxicity to Fusarium lint is evidenced by accumulation of pyruvic acid (396); induction by nitrite of morphological variants in Aspergillus spp. also suggests toxicity (495, 497, 498). The Utilization of Ammonium Nitrogen. Studies on Scopulariop- sis brevicaulis (336, 338, 375) provide the best general view of the physiology of ammonium utilization. Both oxygen and an exogenous carbon source are required, i.e., assimilation depends on respiratory energy or glucose breakdown products or both. Ammonium utiliza- tion increases with pH, and, unlike nitrate absorption, has no definite pH optimum (Figure 3). Ammonia enters and leaves the cell by passive diffusion of the undissociated NH3 molecule; the requirement for respiration is not therefore an indication of accumulation against a gradient. In organisms generally (123, 558), in Torulopsis utilis in par- ticular (620), it is believed that the major and primary reaction of ammonium assimilation is the formation of glutamic acid. Other Figure 3. The rate of uptake of ammonia and nitrate by Scopu- lariopsis brevicaulis as a function of pH. Curve 1, ammonia (two buffers); curve 2, nitrate. From Morton and MacMillan (375), by permission of the Oxford University Press. 1NOR r, A NIC SOURCES OF NITROGE N 247 Day incubation at 30° C Figure 4. The effect of nitrogen assimilation on pH in Karlingia (Rhizophlyctis) rosea. Redrawn from Haskins and Weston (227), bv permission of the American Journal of Botany. amino acids can be formed from glutamate by transamination (p. 267). Secondary roles in ammonium assimilation may be filled by reactions which incorporate free ammonia into alanine, aspartic acid or gluta- mine. Respiratory breakdown of glucose is obviously necessary to supply the carbon skeleton. One of the best established physiological correlates of ammonium assimilation from such salts as the sulfate, nitrate, or chloride is the rapid and often quantitatively large drop in pH consequent upon preferential utilization of the cation. Typical data are shown in Figure 4. The generality of this reaction may be emphasized by listing a few of the fungi in which careful observation demonstrates the phenomenon beyond question: Alternaria solani (391), Aspergillus niger (496), Chaetomium funi- cola (477), Choanephora cucurbitarum (43), Chytridium sp. (113), Diplocarpon rosae (482), Entomophthora spp. (604), Myrothecium verrucaria (122), Mycosphaerella pinodes (45), Penicillium spp. (119, 254), Phymatotrichum omnivorum (520), Piricularia oryzae (406), Psalliota bispora (546), Pythium spp. (459), Polyporus spp. (221), Verticillium spp. (221, 256), Xylaria mali (221). In many, if not most, of the instances cited the decrease in pH was sufficient to reduce growth. Practically, several remedies exist: use 248 NITROGEN NUTRITION AND METABOLISM of ammonium carbonate or phosphate (177), increase in buffer con- centration (122), use of mixed nitrogen sources (254), or inclusion of utilizable organic acids in the medium. This last phenomenon has given rise to much confusion in the literature, the improvement of growth by use of organic acids in media relying on ammonium nitro- gen being taken for a specific nutritional or metabolic effect of the acid. Morton and MacMillan (375) correctly and with adequate evi- dence dispose of this argument; the organic acid effect is a pH effect. Illustrative evidence for the effect itself is abundant — work on the Mucorales (29), higher basidiomycetes (275), and miscellaneous fungi (221) may be considered typical. This point has been treated at some length because a requirement for "organic nitrogen," i.e., inability to grow with ammonium nitro- gen, may reflect only failure of the organism to grow at low pH. Allomyces javanicus var. macrogynus (340) and Chytridium sp. (113) illustrate this phenomenon. No claim of failure to utilize ammonium nitrogen can stand unless the pH effect is excluded experimentally. Other possibilities of error in claims for a non-specific organic nitro- gen requirement may be mentioned. The amino acids which appear to be essential may be acting to reduce, by complex formation, the inhibitory effect of heavy metals present in the basal medium; copper is especially likely to be removed by amino acids (16, 17), and histidine specifically antidotes cobalt toxicity (411, 462). Apparently essential sources of organic nitrogen may be effective by virtue of their content of vitamins (234,591). The apparent glutamic acid requirement of Allomyces javanicus var. macrogynus disappears when the concentrations of glucose, potassium, and minor elements are changed and sulfate is eliminated from the medium (339). It seems therefore that many reports of failure to utilize ammonium nitrogen (318, 343, 359, 506) must remain sub judice. There does appear, however, to be in some of the lower phycomycetes — Blastocla- diella emersonii (42), Sapromyces elongatus (204) and, probably, Leptomitus lacteus (463) — a genuine inability to grow with am- monium as the sole nitrogen source, but no specific amino acid re- quirement. Contradictory and only semi-quantitative data on Apo- dachlya brachynema (200, 461) do not permit of a decision. Finally, the relatively few naturally occurring fungi with specific amino acid requirements (p. 259) and the many known deficient mutants are, of course, exceptions to the rule that fungi as a group can, at least under specified conditions, grow with ammonium ion as the sole source of nitrogen. INORGANIC SOURCES OF NITROGEN 249 Ammonia in high concentrations is toxic (332) and may even find limited use as a fungicide (218, 388). Abnormalities in growth (458), spore germination (169), and development (108) have been traced to ammonia toxicity; especially in media with large amounts of organic nitrogen, ammonia may accumulate to an inhibitory level and con- tribute to the so-called staling effect (421). The toxicity of high am- monium ion concentration to Allomyces javanicus var. macrogynus is alleviated by phosphate (340). Nitrogen Fixation. From the earliest days of microbiology, claims have been made that fungi are able, like certain bacteria, to convert gaseous nitrogen to combined nitrogen. These studies have been re- viewed several times in the last generation (18, 137, 163, 202, 478). The evidence is overwhelmingly against the occurrence of nitrogen fixation in saprophytic fungi. Results with mycorrhizal fungi, par- ticularly Phoma spp., have been more often positive, but isotope re- sults fail to corroborate earlier claims (601). There is, therefore, at present no good evidence for fixation of nitrogen by the fungi. Nitro- gen fixation occurs in root nodules of Alnns glutinosa (426), but it is not yet known what microorganism is involved; the causal agent has been variously assigned to the bacteria, the actinomycetes, the fila- mentous fungi, and, most recently, the Plasmodiophorales (228). Un- til the organism can be cultivated, both its identity and its role in nitrogen fixation will remain uncertain. Oxidation of Inorganic Nitrogen Compounds. Heterotrophic oxi- dation of inorganic nitrogen compounds is effected by a few bacteria (126); this is to be distinguished from the better known autotrophic oxidation of ammonium and nitrite. Among the true fungi, Asper- gillus aureus and A. batatae are reported to form nitrate from nitrite during growth (457). Aspergillus flavus growing on a glucose-peptone- yeast extract medium produces small amounts of nitrite and rather larger amounts of nitrate (468). This finding, if confirmed, has im- portance to soil microbiology. Color tests suggest that A. niger oxi- dizes ammonia to hydroxylamine (494). Among the actinomycetes, Nocardia corallina oxidizes pyruvic oxime and hydroxylamine to nitrite (277, 321). An unusual species of Streptomyces, S. nitrificans, has been studied by Schatz and coworkers (257, 258, 464, 465). This organism utilizes urethan as the sole source of both carbon and nitrogen, and forms nitrite during growth on car- bamates and during metabolism of urea or ammonium carbonate. Activity of cell-free preparations suggests a pyridine nucleotide-linked ammonium dehydrogenase. Nitrate is not formed. 250 NITROGEN NUTRITION AND METABOLISM Cyanide as a Metabolite. Cyanide is, of course, an effective respira- tory poison, acting on iron enzymes. It has also, however, been re- ported to provide assimilable nitrogen to preformed mycelium of As- pergillus niger (271) and to cause an apparent increase in growth of Fusarium lini (395, 437). Data so far available, however, are not ade- quate for a final conclusion on the utilizability of cyanide as a nitrogen source; it is to be noted that Chughtai and Walker (104) found that the growth-promoting effect of cyanide is not duplicated by nitrate; they suggest a metabolic disturbance which diverts more carbon into dry matter. An unidentified soil organism, probably an aerobic acti- nomycete, makes visible growth with cyanide as the sole source of both carbon and nitrogen (578). The liberation of hydrogen cyanide from the fruit bodies of basidio- mycetes has been noted repeatedly; reports are reviewed by Bach (38), Miiller (379), Robbins et al. (444), and Zellner (629). An unidentified Mucor sp. was reported in 1916 to form both cyanide and benzalde- hyde (219). Cyanide is produced in culture, but apparently only dur- ing autolysis, not during growth (444). Fruit bodies of Pholiota aurea do not contain free cyanide when freshly collected, and circumstantial evidence indicates that it is released from another compound by an enzymatic reaction (37). Production of cyanide by an unidentified basidiomycete growing under snow cover is apparently responsible for crown rot in alfalfa; the fungus in culture produces cyanide at low temperature (319, 320). 2. PROTEINS AND PEPTIDES Nutritional Aspects. The fungi and actinomycetes as a group are active in the decomposition of proteins in soil and other materials (1, 568, 571, 572, 573). In pure culture it appears that proteins such as gelatin, casein, and egg albumin can serve as sources of nitrogen for at least the common saprophytic fungi and actinomycetes (278, 574); much more data are available on enzymatic destruction of common proteins than on their use for growth. Utilization of keratin is common only among dermatophytic fungi and, possibly, chytrids (Chapter 3). Whether fungi can attack highly purified proteins in the absence of any other nitrogen source is in doubt. Commercial peptone — a mixture of peptides of varying chain length — is generally utilizable, but in recent years has been replaced in culture media meant for physiological studies by materials of known composition. Dipeptides and tripeptides support the growth of Aspergillus spp. (2, 531) and Ustilago spp. (62, 470); the histidine PROTEINS AN D P E P T IDES 25 1 requirement of Trichophyton rosacea))) is met by carnosine and histi- dylhistidine (136). The widespread occurrence of enzymes hydrolyz- ing peptides to amino acids indicates that in general at least the simple linear peptides will be found to be adequate sources of nitrogen for most fungi. Tricholoma gambosum is unusual in that growth is tripled by glycylglycine, unaffected by glycine (398). The requirement of some Myxomycetes for protein has been men- tioned elsewhere (Chapter 10). Enzymatic Breakdown of Proteins and Peptides. Following current usage (487) we distinguish two general types of enzyme which attack peptide bonds: 1. Endopeptidases (proteinases), acting on peptide bonds either in simple peptides or in the interior of a protein chain. 2. Exopeptidases, acting only on peptide bonds which are adjacent to a free a-amino or a free carboxyl group and therefore restricted generally to hydrolysis of small peptides. Both these types are subsumed under the general term protease. Surveys of the activity of culture filtrates indicate than an enzyme or enzymes able to hydrolyze gelatin is extremely common among the Fungi Imperfecti (129, 130, 142, 351) and Streptomyces spp. (130, 276). Generalization as to other fungi is difficult; gelatin hydrolysis is, however, effected by some ascomycetes (130) and basidiomycetes (53, 629). Among the Mucorales, Rhizopus sp. grows upon and hydro- lyzes several proteins (574), and Mortierella renispora forms active proteases (587, 588). However, surveys limited to studies of the culture filtrate cannot be taken as final evidence of lack of a protease. The distribution of proteolytic enzymes between mycelium and culture fluid is conditioned both by age (185) and by the medium (208). This may explain some apparent disagreements (130, 142). The most extensive study of the utilization of proteins by fungi and actinomycetes is that of Waksman and Starkey (574). Streptomyces violaceus-ruber and Rhizopus sp. grow upon and decompose casein, zein, gliadin, fibrin, gelatin, and egg albumin; Trichoderma koningi utilizes edestin, zein, gliadin, and casein. In culture, proteins are used more rapidly if there is no other carbon source, but the liberation of ammonia under these conditions is large. Other less comprehensive studies show that fungi and actinomycetes generally hydrolyze fibrin (19, 185, 469, 567) and casein (262, 333, 485, 489, 524, 567). Keratin-hydrolyzing preparations have been ob- 252 NITROGEN NUTRITION AND METABOLISM tained from Streptomyces sp. (399), and it may be assumed that the destruction of keratin by dermatophyte fungi is carried out by an extra- cellular enzyme (491, 492, 555). A protease of Penicillium sp. converts trypsinogen to trypsin, but is distinct from the enterokinase which carries out this reaction in animal tissues (312). Fungal endopeptidases cannot be identified with known exocellular animal enzymes. However, it is reasonable to believe that studies of purified enzymes acting on synthetic substrates will eventually support a rational classification. For the present, a few general observations must suffice: 1. Crude and purified preparations from a fungus usually have their pH optimum somewhat on the alkaline side of neutrality (142, 185, 283, 335, 485), but particular activities may have an optimum as low as 4.5 (115). 2. Substrate and inhibition studies suggest that endopeptidases of the trypsin type occur in Streptomyces sp. (377), and in Trichophyton gypseum (185) and other fungi (335); a purified enzyme from Strepto- myces proteolyticus acts on pepsin substrates but at a pH very different from that of the animal enzyme (549). 3. Crude and highly purified preparations alike consist of mixtures of different enzymes (116, 279, 335, 588, 589, 621); enzymes of even closely related strains show differences in rate of attack on different proteins (142). Endopeptidases of Aspergillus oryzae have been studied thoroughly (115, 116, 198). Three different enzymes acting on proteins have been separated. The major component probably acts on most proteins, in- cluding those which lack aromatic acids, and has been obtained in a crystalline, although not completely pure, state (114, 198). Culture filtrates of Streptomyces spp. lyse bacterial cells. Lysis of heat- killed Gram-negative bacteria has been attributed to a protease (67, 285, 376, 519). Lysis of Gram-positive bacterial cells by different species of Streptomyces is effected, however, by an enzyme similar to lysozyme, i.e., a mucopolysaccharase (333, 334), or by the combined action of a ribonuclease and a protease (376, 378). The formation of exocellular protein-hydrolyzing enzymes by some fungi and actinomycetes proceeds in synthetic media with inorganic nitrogen sources (131, 185, 352); for other species a more complex medium may be required (131). There is as yet no good evidence of enzyme induction by substrate, although use of protein media may increase yields (53, 530). A study of cultural conditions as they affect PROTEINS AND PEPTIDES 253 endopeptidase formation by Aspergillus oryzae shows, as expected, optima for carbon source, salts, iron, and temperature (352). Culture nitrates or mycelial extracts of fungi commonly, perhaps uni- versally, hydrolyze peptides. However, it is hazardous at present to identify this activity with particular exopeptidases known from other sources or even to state categorically that endopeptidase activity has been excluded in many of the reports. Aspergillus parasiticus forms four exopeptidases, including an aminopeptidase which is activated by zinc and by reducing agents (284); these same activators affect the aminopeptidase of Penicillium spp. (52). A number of fungi — Asper- gillus spp., Penicillium spp., Cunninghamella sp., Coprinus radians, and Agaricus campestris — form a dipeptidase, an aminopeptidase, and a carboxypeptidase; many of these fungi also elaborate an aminotri- peptidase assayed with triglycine, and a glycylglycine dipeptidase (53). A similar range of activities is reported for Mortierella renispora, Chaetomium sp., and other fungi (336). Glycylglycine dipeptidase of Aspergillus oryzae is activated by co- balt (115), as is the same enzyme from other sources (487). Dipepti- dases also are produced by Physarum polycephalum (244), Piricularia oryzae (259), Penicillium notatum (263) and other fungi (336, 403). Hydrolysis of some amides may be effected by enzymes which also act on peptides or proteins; "hippuricase," for example, may be identical with a carboxypeptidase (182). It appears, in summary, that fungi produce a battery of enzymes attacking peptides of low molecular weight. Information is at present too fragmentary for a detailed classification of the fungal exopepti- dases; the availability of synthetic test substrates (487) makes the de- velopment of such a classification a possible enterprise. Peptides of Fungi and Actinomycetes. Following Bricas and Froma- geot (76), we may define peptides as compounds of low molecular weight which contain two or more amino acids and peptide or pepti- doid bonds. This excludes certain compounds such as pantothenic acid and biocytin (Chapter 10), in which one amino acid and a non- amino acid moiety are joined by a peptide linkage. The naturally- occurring peptides may be classified as homeomeric — consisting only of amino acids — and heteromeric — consisting of amino acids and a non- amino acid moiety. Glutathione perhaps deserves first place in this discussion because of its probable ubiquity in living cells, its vital role in triose phosphate dehydrogenase, and its probable role in peptide synthesis (76). Using 254 NITROGEN NUTRITION AND METABOLISM an iodometric method of possibly doubtful specificity, Miller and Stone (366) report the presence of glutathione in mycelium of Neuru- spora sitophila and other fungi. The common occurrence in fungi of triose phosphate dehydrogenase (Chapter 7), of which glutathione is the prosthetic group (308), indicates that the peptide is at least wide- spread among fungi and actinomycetes. A mutant of Glomerclla sp. requires glutathione, and nutritional evidence suggests that it is defi- cient in the ability to form the component dipcptide y-glutamylcys- teine (348). The actinomycins form a group of related antibiotics first discovered by Waksman and Woodruff (575) and produced by Streptomycrs spp. (79) and by Micromonospora sp. (157). Chemically they are hetero- meric peptides; all contain the same chromophore, or despeptido-acti- nomycin, of the structure (81): HO H3C I CH The peptide chain distinguishes the various actinomycins, very many of which have been described (78, 79); the identity of the individual actinomycins is much in doubt, since it appears that many of the iso- lated fractions are in fact different mixtures of the same components (452). A given strain may produce one type or mixture early, an- other later, in the culture cycle (210). Several other antibiotics of Streptomyces spp. are or contain peptide chains; examples include viomycin, streptothricin and streptolin (76), and the less completely characterized amphomycin (230) and levomycin (95). Polypeptide antibiotics are very common also among the true bacteria. Such compounds are not usual among the filamentous fungi, although penicillin (Chapter 6) may be considered as a heteromeric cyclic peptide, and the toxin of Helminthosporium victoriae is prob- ably a polypeptide (421a). Lycomarasmin, reviewed by Gaumann (190), is a peptide produced by Fusarium oxysporum f. lycopersici; although it is able to cause wilting of plants, its role in natural pathogenesis in the wilt diseases is in dispute. It appears in quantity in the medium fairly late in the culture cycle, after autolysis has begun (Figure 5). A heteromeric peptide, its structure has been determined (609) to be N-(a-[«-hydroxy- propionic acid])-glycyl-asparagine. PROTEINS AND P F. P T I I) E S 255 Figure 5. Growth and lycomaras- min formation (in the culture medium) in Fusarium oxysporum I. lycopersici. Redrawn from Dimond and Waggoner (127), bv permission of Pliytopathology. Dry weight 60 Time, days 100 Lycomarasmin forms a complex with iron, and it is the complex which is responsible for all or most of its toxicity to higher plants (190, 564). Both free lycomarasmin and the iron complex are rela- tively unstable in solution (128). Two heteromeric peptides, enniatin A and enniatin B, are also pro- duced by Fusarium spp. (190, 417, 418) and, like lycomarasmin, cause wilting of plants (193). Aspergillus fumigatus and Streptomyces spp. release into the culture medium a diketopiperazine, the anhydride of L-leucyl-L-proline, pre- viously known only from animal sources (280). Lastly, the toxins of Amanita spp. may be mentioned; they have been reviewed most recently by Block, Stephens, and Murrill (61). Origi- nally, the hemolytic nitrogenous glucoside phallin was separated from "amanitin" (8, 466); later, it was discovered that the second material is in fact three closely related peptides, now known as a-amanitine, /?-amanitine, and phalloidine. The European form of Amanita phal- loides contains all three of these compounds, amounting in total to about 0.005 per cent of its fresh weight (596). Other species of Amanita have only one or two of the peptides, and most species have none (61). A complete structure has been proposed only for phal- loidine (597), although the three toxins have been separated and purified (60, 594, 596). The suggested structure, that of a bicyclic hexapeptide, is unusual in that two amino acids arc joined by a sulfur bridge. The excretion of unidentified nitrogenous compounds, primarily peptides, into the medium during growth has been studied by Morton and Broadbent (374). This excretion occurs in Scopulariopsis brevi- caulis during the period of rapid growth, i.e., it is not an autolytic phe- 256 NITROGEN NUTRITION AND METABOLISM nomenon in the usual sense; about 25 per cent of the nitrogen supplied appears in the medium, and it is not reutilized. The enzymatic basis of peptide synthesis in fungi is at present un- known. Extracts of Neurospora crassa mycelium show y-glutamyl transferase activity; conceivably this system could function in the syn- thesis of peptides of glutamic acid (563). In Bacillus subtilis glutamyl di- and tripeptides are synthesized by transfer reactions of this general type (600). 3. UTILIZATION OF AMINO ACIDS AND AMIDES Amino Acids and Amides in Nutrition. The specificity of response to different amino acids indicates that in fungi as in other organisms amino acids are assimilated primarily as such, and that conversion to ammonia before assimilation of the nitrogen is at best a secondary pathway (44, 542). It can be shown by analysis that amino acids are assimilated at varying rates from a complex medium and that they exist in part, as mentioned earlier (Chapter 2), as free acids in the mycelium. Typically, the amino acid content of a medium drops as the acids are taken up, then rises again as they are liberated during autolysis (181, 413, 423). The value of individual amino acids for fungi is customarily as- sayed by growth on media containing the amino acid of interest as the sole nitrogen source. Studies of this type are numerous but generaliza- tion from them is difficult; we find that a given amino acid allows very good growth of one organism and only very little growth of another, but we are not able to say whether this reflects permeability, enzymatic capacities, or merely such secondary problems as acidity changes conse- quent upon utilization. In addition, amino acids and amides from natural sources may be contaminated with vitamins and there may also be metabolic consequences of the chelation of inorganic ions by amino acids. Most investigations more or less agree that glycine, asparagine, glu- tamic acid, and aspartic acid are the most likely to support good growth. However, it is relatively easy to find fungi which grow only poorly with asparagine (164, 252, 440, 625), aspartate (110, 278), or glutamate (275, 278). Conversely, leucine is generally not an adequate source of nitrogen (409), but for Trichophyton persicolor it is one of the most easily utilizable of the amino acids (506); tryptophan, usually not well utilized, supports normal growth of Streptomyces coelicolor (106). Examples of this type could be multiplied to establish the point that, although generalizations may be made which fit most UTILIZATION OF AMINO ACIDS AND AMIDES 257 No nitrogen DL-Tryptophan L-Cysteine L-Hydroxyproline L-Histidine L-Lysine DL-Methionine DL-Threonine DL-Leucine DL-Valine L-Asparagine DL-Aspartic acid L-Glutamic acid DL-Phenylalanine DL-lsoleucine L-Tyrosine DL-Alanine L-Arginine DL-Serine L-Proline Glycine Casein hydrolyxate 20 40 60 Relative growth 80 100 Figure 6. The growth of Venturia inaequalis in media with amino acids as the source of nitrogen. Each value is the mean of 27 observations (3 concentrations, 3 incubation periods, and 3 replicates). Growth on casein hydrolyzate has been given a relative value of 100. Drawn from tabular data of Pelletier and Keitt (409). fungi, they will not necessarily apply to any one fungus which happens to be of interest. The data of Figure 6 show the range of utilizability of the common amino acids for Venturia inaequalis. Statistical analysis of these and other data shows that the apparent relative value of different amino acids is affected by the period of incubation, the nature of the basal medium, and the concentrations of amino acids used (409). Figure 6 also brings out the point that growth is usually better with a natural 258 NITROGEN NUTRITION AND METABOLISM or artificial mixture of different amino acids than with any single amino acid (442). Asparagine deserves special attention, since it is so often one of the best sources of nitrogen for fungi and actinomycetes. The question is: does the amide have some particular value, or is asparagine merely a source of aspartic acid and extra nitrogen? Asparagine and aspartate are about equivalent for many fungi (25, 46, 275, 604), but the amide is superior to the acid for Tricholoma imbricatum (398), Piricularia oryzae (406), and Leptographium sp. (317), and, at least as sole nitro- gen source, for Phycomyces blakesleeanus (323). No decision can be reached at this time, nor can it be until possible pH effects of utiliza- tion of the two compounds are excluded; the role of pH is emphasized by the response of Coprinus hiascens to asparagine and aspartate (165). It is significant in this connection that aspartic acid does not replace the asparagine requirement of a mutant of Neurospora crassa (523). It is also known that asparagine is in some systems more active as an amino group donor to transamination reactions than is aspartate (354, 355). Piricularia oryzae utilizes the amide nitrogen of asparagine more rapidly than the amino nitrogen (260). Glutamine has been little studied in fungi; it is superior to glutamic acid as a nitrogen source for Tricholoma gambosum (398) and is gen- erally utilized by other basidiomycetes (275). Like asparagine, it is active in transamination (354, 355), and it may also have a role in transpeptidation. One specific and important function of glutamine has been pointed out (Chapter 5), the provision, by transamidation, of the amino group of glucosamine. As mentioned later (Chapter 12) spore germination of Neurospora crassa is inhibited by amino acids, e.g., glycine at 50 pg per milliliter (453). Hydroxyproline at about this level is markedly inhibitory to Trichophyton spp. and other dermatophyte fungi (443, 471). Hy- droxyproline is not toxic to other fungi; its action on Trichophyton mentagrophytes is partially reversed by L-proline (443). Methionine and phenylalanine are also inhibitory to Trichophyton spp. (506), methionine to other fungi (169). The possibility that toxicity is ex- erted at the cell surface deserves exploration, in view of studies on histidine mentioned below. The aliphatic amides, although possibly acted upon by fungal en- zymes (p. 265), are not generally utilizable; acetamide may be an excep- tion (39, 63, 120). Utilization of the D-isomers of the amino acids is specific; thus, Apodachlya brachynema utilizes D-leucine, but Leptomitus lacteus does not (463), and Ustilago scabiosae can grow with D-valine, D-isoleucine, UTILIZATION OF AMINO ACIUS AND AMIDES 259 or D-alanine, but not with D-leucine or D-phenylalanine as nitrogen source (62). It seems likely that utilization of the D-amino acids depends upon oxidative deamination by D-amino acid oxidase (p. 260); ammonia liberated by the oxidase system is then presumed to be incorporated into i.-acids (245). Specific racemases effecting direct interconversion of the d- and L-forms, are known in bacteria (22, 28, 286, 383, 608), not as yet in fungi. Presumably a second factor in utilization of the D-amino acids is their occasional toxicity (149). Specific amino acid requirements appear with reasonable frequency as induced mutations in fungi. They are, however, uncommon in nature and, when found, are more apt to be conditioned or partial deficiencies than absolute requirements. Thus, Mycena rubromargi- nata responds in growth to tyrosine or phenylalanine, but grows with- out either (169), and Cenococcum graniforme has a partial require- ment for histidine (358). Eremothecium ashbyii requires several amino acids only in acid media (619). An absolute requirement for histidine has been reported in Trichophyton spp. (135, 136. 196), as has a requirement lor arginine in T. tonsurans (515); these reports are based on responses in agar media. Labryinthula minuta var. at- lantica requires leucine (559), Lentinus omphalodes tryptophan (171). Requirements for methionine or cysteine are assumed to be related to sulfur rather than nitrogen metabolism, and are considered in Chapter 9; cysteine may also promote the growth of an organism re- quiring a low oxidation-reduction potential (275). Transport of Amino Acids into the Cell. In the present context, the main purpose of this section must be to call attention to a problem which has been only tentatively explored in the fungi, and for the solution of which promising experimental techniques are available. We know that a free amino acid pool exists in fungal cells, that trans- port across the membrane occurs in both directions, and that proteins and peptides are synthesized. In bacteria, yeast, and animal cells there is good evidence that some or all amino acids enter the cell by an active transport mechanism requiring an expenditure of metabolic energy, but different organisms cannot at present be fitted into any single pattern (100, 183, 188); lysine accumulation, for example, ap- pears to be independent of an energy source in Gram-positive bacteria, but glutamic acid enters only if glucose is being respired. Histidine enters the cells of Ncurospora crassa against a concentration gradient; the uptake is inhibited by arginine and methionine, which are, conse- quently, toxic to a histidine-requiring mutant (350). An analogous 260 NITROGEN NUTRITION AND METABOLISM interaction accounts for the toxicity to Lactobacillus arabinosus of D-alanine (293). Threonine uptake is possibly inhibited in N. crassa by other amino acids, but this is not yet certain (566). Catabolism of Amino Acids. The breakdown of amino acids and amides may be reviewed under two general headings, the processes which lead to removal of nitrogen and the processes which alter the carbon skeleton. These two categories of biochemical change are re- viewed by Cohen (109) and Greenberg (214), respectively. We must restrict this discussion largely to the fungi, and only a very incomplete picture of amino acid metabolism will emerge, inasmuch as many im- portant reactions known in other organisms have not yet been investi- gated in the fungi. One approach to amino acid catabolism is to determine what com- pounds are either oxidized or deaminated by resting cells, using mano- metric data or ammonia determinations as criteria. Penicillium chrysogenum, for example, oxidizes glutamic acid, proline, and alanine rapidly, other amino acids more slowly or not at all, as judged by oxygen uptake (603). Washed cells of Streptomyces venezuelae liber- ate ammonia from glutamate, arginine, proline, and other amino acids, not from tyrosine, trytophan, leucine or cysteine; only those amino acids which are attacked can serve as sole sources of nitrogen for growth (212). Leptomitus lacteus oxidizes and deaminates L-alanine, glycine, and L-leucine (463). A second avenue of investigation of amino acid catabolism is the study of particular enzymatic activities in cell-free preparations or as reflected in activities of intact cells. Most efforts of this type have been focused on amino acid oxidase (dehydrogenase) systems; scattered information on other reactions is available but difficult to systematize. The d- and L-amino acid oxidases are flavoproteins known and studied in animal systems; their general properties are reviewed by Cohen (109) and Meister (355). The usual manometric system, em- ploying crude extracts which contain catalase, may be formulated as follows: R— CH(NHo)— COOH + i/2 02 -> R— CO— COOH + NH3 (3) Amino acid a-Keto acid In the absence of catalase, the keto acid is decarboxylated and the over-all reaction then proceeds: R— CH(NHo)— COOH + 02 -* R— COOH + NH3 + C02 (4) The D-amino acid oxidase of Neurospora crassa acts at varying rates on a number of amino acids (Figure 7); detailed comparison with the UTILIZATION OF AMINO ACIDS AND AMIDES 261 Time, minutes Figure 7. The oxidation of some amino acids by the D-amino acid oxidase system of Neurospora crassa. Curves 1 to 6 represent, in sequence, the rate of oxidation of methionine, leucine, isoleucine, valine, lysine, and ornithine. The broken horizontal line is drawn at the theoretical value for the complete oxidation of the D-isomer. Redrawn from Horowitz (245), by permission of the Journal of Biological Chemistry. corresponding enzyme of sheep kidney reveals that the rates of attack on amino acids and the response to inhibitors of the two systems are somewhat different (47). The enzyme exhibits maximal activity at pH 8.0-8.5 (245). The same or a very similar enzyme is found in mycelial extracts of Aspergillus niger and three Penicillium spp., not in P. sanguineum (143). The different fungal preparations differ some- what in their substrate preferences (47, 143). Penicillium chryso- genum forms much more of the enzyme in a synthetic than in a com- plex medium (143). An oxidase system acting on L-amino acids has been shown in Neurospora spp. (47, 87, 536), both in cells and in the medium (48). The enzyme has a somewhat broader substrate specificity than the L-amino acid oxidase of cobra venom (47). Indirect evidence sup- ports the assumption that the prosthetic group is flavin adenine dinu- cleotide (87). Enzyme yield is increased by cultivation with casein hydrolyzate, but this presumed induction occurs only in a low-biotin medium (536). Endogenous amino acids interfere with detection of the enzyme (536). The L-amino acid oxidase of Neurospora crassa converts lysine to its 262 NITROGEN NUTRITION AND METABOLISM kcto analogue, which exists in a cyclic form as, probably, A'-dehydro- pipecolic acid; this compound may be cither reduced to pipecolic acid or reconverted to lysine (476). Although several other fungi show L-amino acid oxidase activity (51, 87, 99, 224, 299), it is not yet certain that the enzyme is identical in all forms. The formation of a-keto acids by growing or resting cells sup- plied with the corresponding L-amino acid is often observed, and may be taken as presumptive evidence for an L-amino acid oxidase (551, 552, 602). The formation of isoamyl alcohol from leucine (422) prob- ably reflects oxidative conversion of the amino acid to a keto acid, followed by decarboxylation and reduction. It should be stressed that chemical attack on an L-amino acid does not, by itself, prove that an L-amino acid oxidase is present. A com- bination of transaminase and glutamic dehydrogenase activities could — and probably does in mammalian tissues (355) — bring about oxi- dative deamination without the participation of an oxidase, and the over-all stoichiometry would be identical with Equation 3. Glutamic dehydrogenase has a wide distribution in nature, and has been found in extracts of Neurospora crassa mycelium (152). Unlike the oxidases, it is specific to L-glutamic acid and requires the partici- pation of a pyridine nucleotide. The reaction may be formulated: HOOC— (CHo)j- CH(NH2)COOH + TPN + L-Glutamic acid — HOOC— (CH2)2— CO— COOH a-Ketoglutaric acid + TPNH + H+ + NH3 (5) Glutamic dehydrogenase is probably the key to synthesis of amino acids (see below) and to the entrance of ammonia into organic com- bination; its possible role in deamination of other amino acids has just been mentioned. Non-oxidative deamination of L-serine and L-threonine is effected by serine dehydrase of Neurospora crassa (435, 618); the products are, respectively, pyruvate and a-ketobutyrate. Another pyridoxal phos- phate enzyme of N. crassa deaminates D-serine (617). Neither D-serine nor D-threonine is attacked by the JV. crassa D-amino acid oxidase (47). Aspartase, converting aspartate non-oxidatively to fumaric acid and ammonia, is also reported to be formed by Neurospora crassa (480) and Penicillium notatum (547). Transamination results, of course, in the deamination of the donor amino acid; the process is considered later, in connection with amino acid biosynthesis. UTILIZATION OF AMINO ACIDS AND AMIDES 263 Catabolism of the carbon chain of amino acids by fungi can be considered at present only on the basis of fragments of information, many of which, however, are suggestive of breakdown pathways known in other organisms. Tyrosine in at least some fungi and actinomycetes is attacked by tyrosinase, a polyphenol oxidase (Chapter 6). The sequence of reac- tions in Neurospora crassa (248) appears to be at least in general the same as in mammalian systems (324): tyrosine is oxidized via 2,4-di- hydroxyphenylalanine ("dopa") through a red hallochrome pigment to an insoluble black pigment, a melanin. The pigment of Strepto- myces scabies probably— although evidence is incomplete — is formed by this same pathway (15, 133, 242, 243), and it is usually assumed that black insoluble pigments as a group are products of tyrosinase action. In mammalian liver, tyrosine is oxidized to acetoacetic acid and fumaric acid by the following sequence of reactions (214): Tyrosine — > p-hydroxyphenylpyruvic acid -^ 2,5-dihydroxyphenylpyruvic acid — » homogentisic acid — » acetoacetate + fumarate (6) The last reaction — actually three reactions (300) — is not known in fungi. Homogentisic acid can be isolated as a product of the tyrosine metabolism of Aspergillus niger and Penicillium spp. (261, 298, 554); inhibition data implicate p-hydroxyphenylpyruvate in the tyrosine metabolism of Neurospora crassa (125). The isolation of tyramine from Aspergillus niger cultures (552) sug- gests another type of tyrosine breakdown, by decarboxylation, as in bacterial systems. Tryptophan catabolism in fungi has been studied principally in rela- tion to nicotinic acid biogenesis (Chapter 10). Other pathways are known in bacteria (14, 121, 214, 493) involving indole, catechol, kynurenic acid, or 5-hydroxytryptophan. A preparation from Neuro- spora crassa converts 3-hydroxykynurenine, an intermediate in nico- tinic acid biosynthesis, to xanthurenic acid (353). Indoleacetic acid (auxin), a growth regulator for higher plants, is produced by several fungi, including Aspergillus niger, Phy corny ces nitens, and Rhizopus suinus, and by soil actinomycetes and bacteria (72, 73, 236, 302, 394, 446, 448). Its obvious structural relation to tryptophan and the effect of the amino acid in stimulating its forma- tion by fungi indicate that the auxin is formed from tryptophan (537, 605). Production by Rhizopus suinus is increased by aeration (538). Detailed enzymatic studies of the synthesis have not appeared, but it 2(34 NITROGEN NUTRITION AND METABOLISM has been suggested that oxidative deamination, decarboxylation, and oxidation reactions occur as follows (353, 584): Tryptophan -+ indolepyruvic acid -> indoleacetaldehyde — > indoleacetic acid (7) Although from time to time it is reported that indoleacetic acid in- creases the growth of fungi, most studies have not shown this (367, 438, 584) or show it only under special circumstances (235). However, acceleration of the maturation of the meiosporangium of Allomyces arbuscula on agar media, perhaps by an effect on the sporangium wall, indubitably occurs (341). Growth inhibition by indoleacetate is com- mon (71, 235, 438, 449); in Nectria galligena inhibition is reduced by biotin (400, 401). It has been argued, on the basis of inhibition studies alone, that indoleacetate is an essential metabolite of Diplodia natalen- sis (195); the evidence, however, is subject to other interpretations. Destruction of indoleacetic acid occurs in the cultures in which it is produced (584), and cell-free enzyme systems of several basidiomycetes inactivate the auxin (77, 148, 479, 545). The enzyme of Omphalia flavida is highly specific, oxidizing indoleacetate with equimolar amounts of oxygen at pH 3.5; the first product of the reaction is not known (430, 431). Threonine is cleaved by Neurospora crassa to glycine and acetalde- hyde (565); this non-oxidative reaction occurs also in animal systems (214). The breakdown of histidine in bacterial and animal preparations proceeds via urocanic and glutamic acids (339, 516); this system has not been studied in fungi, although glutamate does accumulate during histidine breakdown by Streptomyces venezuelae (212). Histidine de- carboxylase could not be detected in Claviceps purpurea or Aspergillus spp. (585). The oxidation of cysteine sulfur is mentioned in Chapter 9, and the role of methionine as a methyl donor in Chapter 6. No other signifi- cant information on the breakdown of the carbon chains of these compounds by fungi is available. Cysteine desulfhydrase is not de- tectable in Penicillium chrysogenum; its occurrence in bacteria is re- viewed by Fromageot (180). Arginine is broken down in the urea cycle, discussed later. Strep- tomyces griseus extracts contain an inducible enzyme which converts arginine to guanidinobutyramide (539, 540). Glutamic acid is probably the principal donor of amino groups in transaminations (p. 267); in the process it is converted to a-ketoglu- taric acid. Hence its carbon chain may be assumed to be oxidized by BIOSYNTHESIS OF AMINO ACIDS 265 the tricarboxylic acid cycle. Glutamic decarboxylase, presumably yielding y-aminobutyric acid, has been detected in Penicillium chryso- genum (201) and Endomycopsis vernalis (290). It might be at first glance surprising that proline, a ring compound, is generally an excellent source of nitrogen for fungi (275, 278, 409, 412). The answer conceivably lies in a ready conversion of proline to glutamic acid, i.e., reversal of the steps in the synthesis of proline from glutamate (p. 268). Hydrolysis of Acyl Amino Acids and Amides. Nitrogen-acylated amino acids are hydrolyzed, liberating the free amino acid, by prepara- tions from Aspergillus spp. and Penicillium spp. (263, 364, 389, 405). Although these reactions may be referred to as acylase activity, it must be realized that there is in fungi no clear separation of acylases, ami- dases, and peptidases, all of which act on the — C — O — N< bond. Hippuricase (histozyme) splits hippuric acid (N-benzoylglycine) to benzoic acid and glycine. Its identity and relation to other acylases and to peptidases is still obscure; certain other N-benzoylated L-a-amino acids are attacked (325). It has most recently been studied in Peni- cillium spp. (263, 456); reports of its occurrence in other fungi are reviewed by Leuthardt (325). Asparaginase, hydrolyzing asparagine to aspartic acid and ammonia, has received some attention, although it has not been purified. Ac- tivity is found in Aspergillus niger (467), Penicillium spp. (134, 456, 535), Microsporum spp. (51), and Piricularia oryzae (260). The opti- mum pH is about 8 (32, 51, 467). The enzyme of A. niger is liberated into the medium only on autolysis (33), is not easily solubilized in sand-ground preparations (35), is unstable (36, 630), and is formed in cells grown on a synthetic medium (467). Amidase activity against glutamine is found in yeast, bacteria, and higher animals (630); it is probably common in the fungi, but has been reported so far only in Neurospora crassa (523). Several early reports, reviewed by Foster (163), suggest that fungal enzymes also act on aliphatic or aromatic amides, e.g., acetamide and benzamide. In view of the negative results of Schmalfuss and Mothes (467) it is perhaps best to suspend judgment, although aliphatic amides are hydrolyzed by Torula utilis (630). Penicillinase and penicillin amidase, both acting on amide linkages, have been mentioned briefly in Chapter 6. 4. BIOSYNTHESIS OF AMINO ACIDS The formation of amino acids by fungi may be considered for our purposes from three aspects: (1) the entrance of nitrogen into the 266 NITROGEN NUTRITION AND METABOLISM amino ac id pool, (2) transfer of the nitrogen from one amino acid to another, and (3) biosynthesis of the carbon chains of known amino acids either before or after incorporation of nitrogen. Most attention has been directed toward the synthesis of the pro- tein amino acids. Even a superficial survey reveals that many other amino acids are formed by fungi either free or in combined form. Gamma-aminobutyric acid, probably arising from glutamate by decar- boxylation, is rather common (374, 380, 486, 612). Djenkolic acid is found free in extracts and culture fluids of Tilletia caries (380). Beta- lysine occurs in several actinomycete antibiotics (56, 76, 382). Antibi- otics of fungi and actinomycetes yield on hydrolysis numerous N-methyl amino acids, for example, sarcosine, N-methylvaline, and N-methyliso- leucine (78, 417). Alpha-hydroxy amino acids occur combined in ly- comarasmin (609) and in ergot compounds (76), while D-a-methylserine is a constituent of the antibiotic amicetin (160). Entrance of Nitrogen into the Amino Acid Pool. We have observed that most fungi utilize ammonium nitrogen as the major or sole nitro- gen source; other inorganic forms are, we may assume with certain reservations, converted to ammonia before assimilation. The best- established reaction is the amination of «-ketoglutaric acid derived from carbohydrate via the tricarboxylic acid cycle, to form glutamic acid, i.e., by the reversal of Equation 5. As mentioned, the only fungus in which this enzyme has been unequivocally demonstrated is Nenro- spora crassa; distribution of carbon-14 in glutamate is consistent with the operation of this reaction in Streptomyces griseus (89, 199). Reversal of aspartase action occurs in bacteria (145, 183) and the enzyme is known in fungi (p. 262). It is not yet, however, known whether the reverse reaction: Fumaric acid + NH3 —> n-aspartic acid (8) makes a significant contribution to amino acid synthesis in fungi. This leaves us for the moment with only one mechanism for the entrance of nitrogen into amino acids. On grounds of comparative biochem- istry, however, it seems likely that future work will uncover other mechanisms, especially in the synthesis of amides and of tryptophan, histidine, serine, and other amino acids. BlastocladieUa emersonii, which will not grow with ammonia as sole nitrogen source, forms glu- tamate from labeled glucose, but isotopic equilibrium with a-ketoglu- tarate is not observed; this suggests that glutamic acid arises in some other way (91). BIOSYNTHESIS OF AMINO ACIDS 267 Transamination. In organisms generally, the amino group of glu- tamic acid can be transferred to various keto acids, yielding «-ketoglu- taric acid and a new a-amino acid, thus: HOOC— CH>— CH2— CH(NHo)- COOH + R— CO— COOH L-Glutnmic acid a-Keto acid — HOOC— CHo-CHo- CO— COOH a-Ketoglutaric acid + R— CH(NH2)COOH (9) a-amino acid Investigation of these enzymes in filamentous fungi has only be- gun, with the identification in Neurospora crassa of the transami- nase reactions in which aspartate and alanine participate as the R— CH(NH2)COOH of Equation 9 (151). Mutants which lack glu- tamic dehydrogenase exhibit a requirement for any of several amino acids; the fact that so many different amino acids support growth argues for an extensive transaminase system in Ar. crassa (151, 152, 154). Aspergillus fumigatus homogenates carry out the reaction (445): H2N— (CH2)3— COOH + HOOC— (CH2)2— CO— COOH — •y-Aminobutyric acid a-Ketoglutaric acid HOOC— (CH2)2— CH(NHo)— COOH (10) L-Glutamic acid This represents the transfer of an w-amino group, and is similar to the formation of glutamate from ornithine and a-keto-glutarate by extracts of Neurospora crassa (153, 155). Endomycopsis vernalis also forms glutamic acid by the reaction of Equation 10 (290). As in other organ- isms, transaminations in N. crassa are activated by pyridoxal phos- phate (151). A kynurenine transaminase, catalyzing the transfer of the a-amino group of kynurenine or 3-hydroxykynurenine to keto acids, has been partially purified from extracts of Neurospora crassa; again, pyridoxal phosphate is required (274). Asparagine and glutamine participate also in transaminations; the amino nitrogen is transferred to any of several keto acids (355). This reaction has not been explored in the fungi. Biosynthesis of Amino Acid Carbon Skeletons. Perhaps in no field of biochemistry have the techniques of biochemical genetics been more fruitfully employed than in that of amino acid synthesis; Neurospora crassa has been especially useful. In recent years, the application of isotype and enzymatic methods has provided additional valuable in- 268 NITROGEN NUTRITION AND METABOLISM formation. It is not always realized that there are serious pitfalls in all methods which seek to state unequivocally that this or that compound is an obligatory precursor of a given metabolite. Adelberg (11) has critically reviewed these problems with the conclusion that "present- day techniques are unable to establish metabolic intermediates with finality." In practice, one approaches certainty or conviction as more and more evidence from different types of experiment is accumulated (123). It seems appropriate to include here only a sketch of the more im- portant biosynthetic pathways which are known, or are likely, to be functional in one or more fungi. As will be seen, there is little evidence from fungi other than Neurospora crassa. Following Davis (123), we shall consider groups of amino acids which are known to be biosyn- thetically related. A family of amino acids is derived more or less directly from glutamic acid. Omitting details, the following over-all formulation may be sug- gested (123, 500): glutamine Glutamic acid * proline — > hydroxyproline (11) Evidence from fungi is, however, limited at present to the glutamate- proline interconversion (9, 64, 561, 624), and the appearance of A'- pyrroline-5-carboxylic acid, the immediate precursor of proline in Equation 11, in glutamate cultures of Aspergillus oryzae (486). Ornithine biosynthesis in Neurospora crassa starts from glutamic acid also; the pathways, however, are not at all clear. Vogel (560) sug- gests the following sequence in N. crassa and Torulopsis utilis: Glutamic acid -^ glutamic y-semialdehyde — > ornithine (12) This is rather different from the Escherichia coli system (123), which depends upon acetylated intermediates. There may be in N. crassa some other pathway of ornithine biosynthesis than that of Equation 12 (153, 560). Ornithine participates in the ornithine cycle, first worked out in animal systems (307) and later found to function in Neurospora crassa (156, 490). The basic elements of the cycle, leaving aside biochemical details, are schematized in Figure 8. N. crassa, although forming urease, does not liberate urea; we may suppose that the cycle here serves primarily to supply arginine for protein synthesis. Nutritional data suggest the same general pattern in Penicillium chrysogenum (64). Urea is a common product of fungi, but direct evidence implicating BIOSYNTHESIS OF AMINO ACIDS 269 L-Ornithine L-Citrulline L-Aspartic acid L-Argininosuccinic acid Urea L-Arginine If Fumaric acid Protein Figure 8. The ornithine cycle (429). the ornithine cycle in net formation of urea has not yet appeared. Ornithine appears in the medium during breakdown of arginine by Streptomyces venezuelae (212). Canavanine, a hydroxyguanidine derivative structurally related to arginine, inhibits the growth of some normal strains of Neurospora crassa (251). The inhibition has been traced (576) to the formation of a canavinino-succinate which blocks the splitting of arginino-succinate. Of the short-chain amino acids — glycine, alanine, serine, and cysteine — only the first named has received much attention in fungi. A glycine- requiring mutant of Neurospora crassa can grow normally if given either glycolic or glyoxylic acids (613). This is in agreement with animal studies (356, 582), and suggests the following sequence: HOOC— CH2OH Glycolic acid HOOC— CHO Glyoxylic acid HOOC— CH2NH2 (13) Glycine Drawing on experience with other organisms, we may suggest that the glycolate arises from pentose phosphate (583), and that the amino group is introduced by a transamination from glutamate or other amino donor. Glycine and serine are closely related in Penicillium notatum (26) and Eremothecium ashbyii (206, 207). Both participate in a variety of biochemical reactions and are clearly related in their biosynthesis; it cannot yet be said which is formed first. 270 Aspartic acid NITROGEN NUTRITION AND METABOLISM Homoserine Methionine ■*- — Cysteine Cystathionine Homocysteine Threonine Figure 9. The probable in- terrelations of aspartic acid, threonine, and the sulfur amino acids in Neurospora crassa. Several of the steps involve more than one re- action. All amino acids are of the L-configuration. Nutritional and metabolic studies on methionine-requiring mutants of Neurospora crassa established an over-all sequence leading from cysteine to methionine (159, 247, 533). A relation of threonine and methionine biosynthesis is suggested by both inhibition studies (132) and nutritional requirements of a single-gene mutant (533). These relations are schematized in Figure 9, to which has been added the postulate that aspartic acid is the precursor of both threonine and methionine, as it is in yeast and bacteria (59). The scheme is by no means complete; there is probably an intermediate between homocys- teine and methionine (92), and the source of the methyl group of methionine is not yet clear. An enzyme converting homoserine to threonine occurs in fungi (578a). Sulfur-methylcysteine is formed by Neurospora crassa and the iso- lated compound is utilizable as a source of sulfur (427). Canavanine has been mentioned as an inhibitor of arginine synthesis. It has also been suggested that canavanine-resistant strains convert canavanine to homoserine (329); Streptococcus faecalis carries out this reaction (292). Such a conversion would explain why canavanine re- places threonine for some N. crassa mutants (532). Isoleucine and valine are synthesized by Neurospora crassa from, respectively, a,/3-dihydroxy-/?-ethylbutyric acid and a./J-dihydroxy-/?- methylbutyric acid (13). An observed double requirement for both isoleucine and valine (65) has been traced to a deficiency in the de- hydrase — possibly dehydrases — which convert these dihydroxy acids to keto acids (381). There is some evidence that the precursors of valine and isoleucine ultimately arise from, respectively, pyruvic acid and threonine (12, 13, 565). The keto analogue of leucine, a-ketoisocaproic acid, replaces leucine for a mutant of Neurospora crassa, and is considered therefore to be a precursor of it (432). Earlier steps in leucine biosynthesis by fungi are not known. Studies on Neurospora crassa have shown that both a-aminoadipic BIOSYNTHESIS OF AMINO ACIDS 271 acid and a-amino-e-hydroxycaproic acid serve as precursors of lysine, probably both being converted to a semialdehyde (475, 611). Since Ophiostoma multiannulatiim lysineless mutants utilize a-ketoadipic acid, it is probable that this compound is the precursor of a-aminoadipic acid (54). A series of investigations of histidineless mutants of Neurospora crassa is reviewed by Ames (20); the following over-all pathway is sug- gested: Imidazole glycerol phosphate — > imidazole acetol phosphate — > L-histidinol phosphate -» L-histidine (14) Conventional nutritional techniques were of limited use in these studies, since the phosphorylated intermediates do not penetrate the cell. More reliance was placed on isolation of intermediates from blocked mutants and on direct enzymatic studies (20, 21). The known early steps of the conversion of glucose to aromatic com- pounds have been diagrammed in Chapter 6. Figure 10 takes up the story from shikimic acid on; although this presentation is based in large part on studies of Escherichia coli, all of the available evidence from Neurospora crassa is in agreement with it. In particular, a multiple requirement for p-aminobenzoic acid, phenylalanine, tyrosine, and tryptophan is met by shikimic acid (526). Additional evidence is found in the accumulation of prephenic acid by a strain deficient for the three aromatic amino acids (361), and in the replacement of tyrosine by phenylpyruvic acid (125). The suggestion that a-phenylglycine is a precursor of tyrosine and phenylalanine (222) has not been borne out by later work (124). Tryptophan synthesis from anthranilic acid and indole by Neuro- spora crassa is indicated by nutritional responses of deficient mutants (66, 171, 525) and by studies with isotopic nitrogen (408). The con- version of indole to tryptophan is catalyzed by tryptophan synthetase (indole-serine ligase); this enzyme has been isolated and partially puri- fied from N. crassa (616). The over-all reaction, which requires pyri- doxal phosphate, may be written: Indole + serine —> tryptophan + H20 (15) The mechanism of this reaction is considered by Tatum and Shemin (528). Tryptophan synthesis in Claviceps purpurea is probably effected by the same system (548). Presence or absence of the enzyme is controlled by a single gene in 272 NITROGEN NUTRITION AND METABOLISM COOH CH2— CH— COOH NH2 p-Aminobenzoic acid i . NH, Tryptophan COOH -* "X" HO ~OH OH Shikimic acid COOH I CH— NH2 I CH, COOH I CO I HOOC CH2 ^\^COOH ^^NH2 Anthranilic acid COOH i CO I CH2 Indole COOH I CH— NH2 I CH, OH Tyrosine Prephenic acid Phenylpyruvic acid '^ Phenylalanine Figure 10. The biosynthesis of tryptophan, phenylalanine, tyrosine, and p-amino- benzoic acid. This is a general scheme based on data from several organisms but consistent with knowledge of Neurospora crassa. Many of the reactions shown are multiple. Neurospora crassa (241). However, an antigenically related protein, possibly an enzyme precursor or possibly a complex of altered enzyme with an inhibitor, has been isolated from tryptophan-requiring mu- tants (514a). Cultivation of N. crassa in a medium deficient in zinc reduces the concentration of the synthesizing enzyme (386, 512). This summary of amino acid synthesis may give a false impression of finality. There are, in fact, many complexities and uncertainties which cannot be considered here, and it is to be expected that our pic- ture of these reactions will change as time goes on and more informa- tion becomes available. AMINES 273 5. AMINES Although only one specific decarboxylase activity — glutamic decar- boxylation, yielding y-aminobutyric acid — is known in fungi (p. 265), the number of amines which occur and which presumably originate by decarboxylation of known amino acids is large. Sclerotia of Clavi- ceps purpurea yield histamine, tyramine, isoamylamine, agmatine, pu- trescine and cadaverine (503, 508, 586). Occasional reports — not all certain — suggest that putrescine, phenylethylamine, isoamylamine, and cadaverine occur in fruit bodies of basidiomycetes (291, 310, 499, 580). A mutant of Aspergillus nidulans requires putrescine for growth, and the requirement is not met by L-ornithine or by other amines (488). Several fungi act on histamine (585). Amines are in general poor or unavailable sources of nitrogen (278). Muscarine, the toxin of Amanita muscaria, is an amine; Eugster (146) reviews earlier studies; a new structure has been confirmed by synthesis (301). Choline has been found as a constituent of several fungi (508, 581); whether, however, it occurs in the free state or only in phospholipids (Chapter 2) has not been determined. A single report (147) suggests that acetyl choline is a constituent of ergot. Cyclic choline sulfate has been mentioned (Chapter 2). Barley and other grains scabbed by Fusarium graminearum or other related Fusarium spp. contain a water-soluble material toxic to animals (101); identification of this substance with choline has been shown (473) to be in error. Choline is required by mutants of Neurospora crassa (246), and a mutant can be used for the bioassay of choline (249, 527). The synthe- sis of choline by successive methylations of aminoethanol has been described (Chapter 6). Trimethylamine has been isolated from numerous basidiomycetes, e.g., Tilletia levis, Phallus impudicus, and Russula aurata (225, 499, 580), and from Claviceps purpurea (508). In plants and in bacteria trimethylamine arises from choline (98), and the same origin may be proposed in fungi. Betaine, (CH3)3 — N+ — CH2 — COO-, has been isolated from fungi (581), but, as with all the amines found in gross studies, there is some doubt as to whether it occurs as such or combined. One such combined form of betaine, ergothioneine, has the structure of the trimethyl beta- ine of thiolhistidine: 274 NITROGEN NUTRITION AND METABOLISM HC N G— SH / C N— H I CH2 + I (CH3)3 - N— CH— COO- Ergothioneine occurs in mammalian blood and seminal fluid (182), in sclerotia and in cultivated mycelium of Claviceps purpurea (40, 508), and in the mycelium of several common fungi (360). Experiments with carbon- 14 indicate that ergothioneine and histidine are synthe- sized by the same pathway (229) and that histamine is not a precursor of either (599). Amanita mappa fruit bodies contain an amine first called mappin but now known to be identical with bufotenine; its structure, 5-hydroxy-N-dimethyl tryptamine (595), suggests that it is biosyntheti- cally derived from 5-hydroxytryptophan, an amino acid known in other organisms (353, 550) but not to date in fungi. 6. METABOLISM OF NUCLEIC ACIDS AND THEIR DERIVATIVES Nucleic acids, nucleotides, and the purine bases can probably all be used more or less well as sources of nitrogen by fungi (606, 607), but the important aspects of nucleic acid metabolism are more narrowly metabolic: the breakdown of nucleic acids and their biosynthesis. In- formation on nucleic acid breakdown is rather scanty and bears almost exclusively on the purine ribonucleotides. Biosynthetic studies are even less definitive, and are limited primarily to nutritional studies of deficient mutants. Catabolism of Nucleic Acids and Their Derivatives. That fungal enzymes attack nucleic acids has been known for some time. In early experiments liberation of phosphorus or of nitrogen bases, or utiliza- tion of nitrogen for growth were the criteria, so that it is not possible to specify what enzymes were involved (134, 265, 294, 357, 404). On comparative grounds, we may suppose that fungi, like bacteria, attack nucleic acids first by nucleodepolymerase action, yielding prima- rily mononucleotides. A few such activities have been shown in fungi (Table 1). The second step in the classical pathway of nucleic acid degradation is the dephosphorylation of nucleotides, yielding inorganic phosphate. NUCLEIC ACIDS 275 Table 1. Enzymes Acting on Nucleic Acids, Nucleotides, and Nucleosides* Substrate Type of Action Occurrence Ribonucleic acid Depolymerization Streptomyces spp. (67, 378), Aspergil- lus oryzae (311) Deoxyribonucleic acid Depolymerization Neurospora crassa (627), Streptomyces sp. (378) Nucleotides Deamination Aspergillus spp. (68, 69, 288, 311) Nucleosides Deamination Aspergillus oryzae (288, 311, 370), Neurospora crassa (350) Nucleosides Hydrolysis Aspergillus oryzae (31 1) * Dephosphorylation of adenosine phosphates is summarized in Table 1 of Chapter 9. This action is often, perhaps usually, carried out by non-specific phos- phatases (316). Hydrolysis of adenosine phosphates, summarized in Chapter 9, and the dephosphorylation of riboflavin monophosphate by extracts of Penicillium chrysogenum (96) are presumably, although not necessarily, effected by such phosphatases. A phosphatase of Strepto- myces sp. acts both on glycerophosphate and on nucleotides (378). Diphosphopyridine nucleotide is attacked by a specific enzyme, di- phosphopyridine nucleotidase, which splits off nicotinamide from the dinucleotide, leaving adenosine diphosphate ribose. The enzyme is formed by Neurospora crassa (289); its high concentration in the co- nidia suggests that it is formed only during sporulation (626). The con- centration of enzyme in the mycelium is increased by deficiencies of zinc, nitrogen, or, usually, growth factors (386, 387). The enzyme of N. crassa, unlike the corresponding animal enzyme, is not inhibited by nicotinamide, except at very high concentration, and does not catalyze an exchange reaction between the dinucleotide and free nicotinamide (628). Deamination of nucleotides and nucleosides is usually effected by specific enzymes, but an adenyl deaminase of Aspergillus oryzae acts on both types of compound, deaminating adenosine, adenosine-5'-phos- phate, adenosine-3-phosphate, adenosine diphosphate and diphospho- pyridine nucleotide (288). These scattered data do not, of course, give a complete or coherent picture of nucleic acid breakdown; they do indicate that catabolism of the purine ribonucleotides in fungi is similar to that in other organ- isms. It seems likely, therefore, that the free purines adenine, hypox- anthine, and guanine are converted to xanthine, which is then oxidized by xanthine oxidase to uric acid (518), the further metabolism of which is described below. In Nocardia corallina the pyrimidines thymine and uracil are metab- olized by way of barbituric acid (315); the over-all process, presumably -■ iMmrs cs Stttrztit N .: ■ : " :f iZiz izz - :■: - r .;_- - _ . re: :.z:I- : : z - : - : ' . 7 JDIJOB C : : -: .- . _ : ; :-• : : r_ :: :r- z :_. r : rn. ; :L~ zz i zz i> :!".:: m madl _4rvi". Ii ~zz± :: Lit* li: :.::i Le runber step cr step- added tc 1 :- i. - -- i . ..*..". - . -.m -. 11 . li.i : . ; n_; . 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Sadfc exj r -ill- -1 : "•• _i:i •"" — -" ~ i" ::i -"■- : ->rT ti:_ ; lit 1.. :n_-r: / : : mrc^i 278 NITROGEN NUTRITION AND METABOLISM Urease, the enzyme hydrolyzing urea, is well known from higher plants and bacteria (511). It is evidently of very general occurrence among fungi, including Aspergillus spp. (34, 35, 36, 84, 115, 161, 392, 181, 522), Penicilliurn spp. (134, 371, 547), Pythiacystis citrophthora (296), several dermatophytic fungi (524), and most of the fleshy basid- iomycetes which have been studied in this connection (70, 189, 209, 357). Both direct and indirect evidence indicate that urease is formed by at least some actinomycetes (10, 15, 240, 485). The urease of Aspergillus niger is most active at pH 7.6 (34), al- though its production is favored to a marked degree by low pH (371). It is formed by A. niger on various media and appears in the medium after autolysis (34). In basidiomycete sporophores the enzyme is most abundant in the hymenium (209). Urea is formed in the fruit bodies of the higher basidiomycetes (209, 268, 269, 579, 615), and by many saprophytic fungi and actinomycetes in pure culture (102, 194, 217, 266, 267, 579). Oxygen and exogenous ammonia increase urea formation by sporophores (272, 273). The function of urea — in some fungi very large amounts are reported (266, 268) — is presumably as a nitrogen reserve. The biochemical origin of urea in fungi has not been definitely proved, and there are several possible processes. First among these is the breakdown of arginine in the ornithine cycle (p. 269). Urea bio- genesis by this route is indicated by the finding that exogenous arginine increases urea accumulation in fungi (267, 522), and by the demon- stration of urea formation from arginine by cell-free preparations from basidiomycete sporophores (273, 615). Apart from the specific action of arginase on arginine, it appears that urea is also formed by one or more enzymes which act on guani- dine and derivatives of it to form urea and ammonia; this activity is found in Streptomyces griseus (540) and in several saprophytic fungi (103,270). Formation of urea from calcium cyanamide by cell-free preparations of Aspergillus niger has been reported (314). An origin of urea from purines is also possible in the fungi, by the general route: Xanthine -^ uric acid —> allantoin -» allantoic acid — > glyoxylic acid -f urea (19) Evidence for this pathway is still incomplete, but may be summarized briefly: • 1. Xanthine oxidase and uricase occur in several fungi (518). Uri- case is an inducible enzyme in Torulopsis utilis (450). E ROOT ALKALOIDS 27(J 2. Uric acid has been isolated, although possibly as a degradation product, from spores of Aspergillus oryzae (513). 3. Allantoinase, converting allantoin to allantoic acid, is found in cell-free extracts of basidiomycete sporophores (83) and Aspergillus niger (84). Allantoin serves as a nitrogen source for Fusarium oxy- sporum f. nicotianae (607). 4. Sporophores of basidiomycetes may contain significant amounts of allantoic acid (162). Proof of the role of this sequence in a single organism, and demonstra- tion of allantoicase and its products in a cell-free system are required for acceptance of the pathway as written in Equation 19. 8. ERGOT ALKALOIDS At least since the Middle Ages, it has been known that the black sclerotia of the ergot fungus cause disease and often death if eaten (41). The alkaloids of the fungus now also occupy a place in pharma- cology and have been intensively investigated as drugs (205, 436). Al- though the classical organism is Claviceps purpurea, there is reason to believe that sclerotia of other species of the genus, especially C. paspali, also contain the active materials (82). Chemically, the known ergot alkaloids comprise three basic types, all amides of lysergic acid, which has the structure: H COOH ' H2 N— CH3 H H Y/^N7 H In the ergotamine and ergotoxine alkaloids, lysergic acid is linked to a peptide; ergonovine (ergometrine, ergobasine) is made up of lysergic acid and L-2-amino-l-propanol (508). A number of compounds closely related to the pharmacologically active materials are produced in cul- ture by specific strains of Claviceps spp. (3, 509); the structure and pos- sible biosynthetic relationships of these are reviewed by Stickings and Raistrick (504). Agroclavine production is greater in still than in 280 NITROGEN NUTRITION AND METABOLISM shake culture (5, 6, 7). The ergot alkaloids, whether produced by the fungus or supplied exogenously, disappear in time from a culture (4, 363). 9. MISCELLANEOUS NITROGEN COMPOUNDS Although the antibiotics of actinomycetes form a large share of the known nitrogenous metabolites, a number of fungal products are also known. Table 2 lists some representative compounds in a classifica- tion based on chemical similarity. Almost nothing is known of bio- chemical relationships. Group I of Table 2 includes compounds more or less similar to amino acids. Azaserine is of some general interest by reason of its apparently specific blocking of a single step in the biogenesis of purine nucleotides (49, 543). Other serine derivatives include the anti- biotic cycloserine and the non-antibiotic O-carbamyl-D-serine (223), both actinomycete products. Mycelianamide, produced by Penicillium griseofulvum, is structurally related, as an acylhydroxylamine, to cy- closerine (57). The structure of the related antibiotics thiolutin and aureothricin has been determined (97), and their resemblance to cystine has been noted (14). Compounds more closely and obviously related to the amino acids include fumaryl-DL-alanine of Penicillium resticulosum (58), an uni- dentified ester of glutamine in Lepiota naucina (502), and the lichen product picroroccelin, a diketopiperazine (27). The organic nitro group occurs only rarely in biological material; chloramphenicol (Chapter 6) is one example, /3-nitropropionic acid another (Table 2). An antibiotic of Streptomyces thiolutens has the nitro group (238). Neurospora crassa forms an inducible enzyme acting on /?-nitropropionic acid and related compounds (328). Indigo is shown in Table 2 as a representative of Group III, com- pounds related to indole. There is good evidence that indole is the starting point for indigo formation by both soil bacteria (213) and in the mammalian gut (121); indole in turn, we may assume, arises from tryptophan. The indole nucleus is found also in lysergic acid (p. 279), and in echinulin, a product of Aspergillus glaucus (425). Gliotoxin, pro- duced by Aspergillus fumigatus, Penicillium spp., and Trichoderma viride (74), is believed to have a reduced indole nucleus (281). Peni- cillium terlikowski, earlier identified as P. obscurum, forms both glio- toxin and its monoacetate (282). Fusaric acid is the sole representative of Group IV, pyridine deriva- MISCELLANEOUS NITROGEN COMPOUNDS 281 Table 2. Representative Nitrogenous Compounds of Fungi and Actinomycetes Group* I Azaserine Compound Organisms Streptomyces sp. (186, 187, 372, 505) N+=N-=CH— CO— O— CH2— CH— COOH I NH2 Cycloserine (oxamycin) HoN— CH C=0 I I CHo NH \ / O II Beta-nitropropionic acid 02N— CH2— CH2— COOH III Indigo (indigotin) ^^ O O // X ^X^N ^^ N^V^ H IV Fusaric acid H HOOC- -(CH2)3— CH3 5N' OH "=::^Nvr;*\ OH * See p. 280. f Also known in higher plants (94). Streptomyces spp. (226, 237, 309) Aspergillus flavus\ (88) Schizophyllum commune (365) Fusarium spp., Gibber ella fujikuroi, Nectria cinna- barina (191, 192, 416, 507, 614) Penicillium spp. (75, 117) 282 NITROGEN NUTRITION AND METABOLISM Table 2 (continued) Group* Compound VI Aspergillic acid N / \ HC C— CH2— CH VIII IX Organisms Aspergillus flavus (141, 390, 591) CH3 CH3 Cordyceps mill tar is (50, 118) fN^ ^N"' CH2OH I I CH— CHOH— CH— CH2 I o 1 Cycloheximide CHi — CH — CH3 / \ CHj— CH C=0 \ / CH2— CH Streptomyces spp. (112, 304, 313, 590) CH2— C • O CHOH— CH«— CH N— H \ / CH2— C \ Actithiazic acid H O Streptomyces spp. (352) 0=C N CH2— S CH— (CH2)5— COOH MISCELLANEOUS NITROGEN COMPOUNDS 283 tives, although lysergic acid also has a reduced N-methyl pyridine structure. Fusaric acid is a potential wilt toxin in diseases caused by Fusarium spp. and related fungi. Its production by F. vasinfectum is narrowly controlled by the zinc content of the medium (287). Gib- berella fujikuroi also forms dehydrofusaric acid (510). Viridicatin (Group V, Table 2) is a derivative of quinoline; quino- linic acid is noted later (Chapter 10) as a possible intermediate in the biosynthesis of nicotinic acid from tryptophan. Some strains of Peni- cillium cyclopium form viridicatin, but in one it is replaced by the related cyclopenin (75). Aspergillic acid, one tautomeric form of which is displayed in Table 2, represents a sixth group, derivatives of pyrazine. Gliotoxin, men- tioned above as an indole derivative, also contains this grouping, as does pulcherriminic acid, the iron salt of which, pulcherrimin, is the red pigment of Candida pulcherrima (111, 297). Aspergillus flavus forms, in addition to aspergillic acid, the compound flavocol, a diiso- butyl derivative of deoxyaspergillic acid (140). The structure of the pyrazines strongly suggests a biosynthetic origin from leucine and isoleucine (74). Cordycepin yields adenine on hydrolysis. 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The known essential micronutrients are iron, zinc, copper, manganese, and molybdenum. The calcium requirement of fungi, if specific, is of the same order of magnitude as the micronutrient re- quirement, whereas calcium is a major nutrient for higher plants. Boron, required by green plants, is not known to be needed by fungi. The possibility of other micronutrient requirements in fungi must also be considered. Effects of sodium chloride have been assumed here to be non-specific, although it has been shown that sodium is required by Anabaena cylindrica (4). The history of investigations on the mineral nutrition of fungi is re- viewed by Foster (57). Early studies were primarily nutritional in nature and were severely hampered by methodological difficulties. More recent nutritional studies owe much to the pioneering work of R. A. Steinberg on both the techniques and the basic concepts of the field. Interest at the present time is shifting toward the problem of the metabolic function of the inorganic nutrients, with special atten- tion to enzymatic processes in which metals play a role. A return to the exploration of still unknown requirements should be possible as purer 300 PHOSPHORUS 301 chemicals and inert container materials come to the attention of the biologist. 1. PHOSPHORUS In culture media a level of potassium phosphate (monobasic or di- basic) of 0.004 M is usually provided; reported optimum concentrations — affected, of course, by the carbon and nitrogen level — are of the order of magnitude of 0.001-0.003 M (75, 100, 187, 241). These levels are, however, too low to provide adequate buffering against ap- preciable organic acid formation. Orthophosphate may be replaced, at least for some fungi, by other forms of inorganic phosphate (49), by phytic acid (220), by adenosine phosphates (223), or by casein (22). Aspergillus niger cannot utilize phosphite or hypophosphite (49); phosphites appear to be generally inert in biological systems (203). Phosphate deficiencies cause several metabolic disturbances. The most easily observed, as one would expect, is a lowered rate of glucose utilization (33, 123, 130); other metabolic symptoms of deficiency are presumably secondary to this direct effect. In the typical culture cycle, phosphorus is absorbed rapidly during Time, days Figure 1. Growth and phosphorus changes in Aspergillus niger. From data oi Mann (135). Seven-minute phosphorus is organic phosphorus mineralized by acid hydrolysis of 7 minutes duration. 302 INORGANIC NUTRITION AND METABOLISM the early stages of growth and is converted in the cells to organic com- pounds (69, 134). Absorption is dependent upon oxygen and a respir- able substrate and is inhibited if respiration is poisoned (79, 134, 205). The rate of absorption is retarded if available carbon or nitrogen is Table 1. Phosphatases of Fungi and Actinomycetes Substrate Beta-glycerophosphate, sugar phosphates, /;-nitrophenyl phos- phate, etc. Beta-glycerophosphate Glucosamine-6-phosphate Adenosine triphosphate Adenosine monophos- phate Monoallyl phosphate Colloidal polymetaphos- phate Low molecular weight metaphosphate Pyrophosphate Action Hydrolysis at acid pH Hydrolysis at alkaline pH Hydrolysis Hydrolysis Hydrolysis Hydrolysis Depolymeriza- tion Hydrolysis Hydrolysis Organisms Actinomyces Israeli (90), Aspergillus flavus* (251), A. niger (132, 134), A. oryzae (39, 41, 42, 179), Can- dida albicans, f Cryptococcus spp.,f Geotrichum candidum,\ and Torula cremoris\ (16), Streptomyces coeli- color (38), Torulopsis utilis (193) Allomyces javanicus\ (248), Aspergillus flavus* (251), A. oryzae* (7), Can- dida albicans^ (16), Microsporum canis (34), Penicillium chrysogenum (206,207), Torula cremoris\, (16). Trichophyton rubrum\ (172) Neurospora crassa (24) Aspergillus niger (112, 134), Penicil- lium chrysogenum (110, 111) Aspergillus niger (132, 134), Penicil- lium chrysogenum (206) Aspergillus niger (8) Aspergillus spp., Penicillium spp. J (95, 96, 97, 98, 111, 132) Aspergillus spp. (105, 106, 111, 112, 135) Aspergillus spp. (109, 132, 135, 159, 160, 164, 179), Penicillium chryso- genum (109, 206) Aspergillus niger (132), A. oryzae § (159, 160, 161) Aspergillus niger (111, 112, 113) Aspergillus niger (50, 134), Penicillium chrysogenum (206) Aspergillus spp. (56) Triphosphate Hydrolysis Polyphosphates Hydrolysis Phytic acid Hydrolysis Thionophosphate Hydrolysis * Two pH optima. f Histochemical method. t No depolymerization by extracts from Phycomyces blakesleeanus, Rhizopus nigri- cans, Collybia velutipes, Marasmius spp., Merulius domesticus, Polyporus betulinus, or Tricholoma equestre. § Two enzymes. POTASSIUM 303 made limiting (71, 72) and, of course, if the external phosphate con- centration is lowered (33, 70). When conidia appear, mycelial phos- phorus enters the spores (10). Autolysis is marked by the appearance of phosphate, primarily inorganic, in the medium (33, 42, 143, 217); most of this appears to be derived from mycelial organic phosphorus, inasmuch as the inorganic mycelial phosphorus remains relatively constant during autolysis. Most of our information is from studies on Aspergillus niger; the data of Figure 1 are typical. Combined forms of phosphate, organic and inorganic, are hydro- lyzed by fungal enzymes, the phosphatases, the occurrence of which is summarized in Table 1. It may be that the acid and alkaline phos- phatases of fungi are identical with phosphomonoesterases I and II of other organisms (204), but the specificity is very much in doubt. Indeed, there is no certainty in most of the examples of Table 1 that separate specific enzymes are involved. Probably most or all fungi and actinomycetes form a phosphatase active at low pH against organic phosphates, although particular prep- arations have been reported to lack this activity (34, 206, 207). 2. POTASSIUM Potassium is required by fungi; at the usual levels of carbohydrate, concentrations of 0.001-0.004 M are adequate (100, 187, 238). Sur- prisingly little information is available on the physiology of potassium in fungi; the most striking effect of potassium deficiency in Aspergillus niger is an increase in oxalic acid accumulation (29, 146, 196). A very low supply of the element is accompanied by poor sugar utiliza- tion (195), and it seems likely from work with organisms other than fungi that potassium has an essential function in carbohydrate metabo- lism (154, 176, 202). Certain enzymes, however, are more active in preparations from potassium-deficient cells (85). Sodium can partially replace potassium in the nutrition of Asper- gillus niger (73, 238), but the degree of replacement is small. Under some conditions, rubidium and cesium have slight effects (238), under others, none (149). In certain of the bacteria, rubidium and other alkali metals have a much more pronounced action, either as replace- ments for or competitors of potassium (55, 124, 130, 202, 247). It seems likely that studies of this problem in fungi other than Asper- gillus niger would be rewarding. Rubidium can replace potassium in certain cell-free enzyme systems (126). Figure 2 illustrates the typical growth response to the four major elements — potassium, magnesium, sulfur, and phosphorus. 304 INORGANIC NUTRITION AND METABOLISM Element added, mg per liter Figure 2. The response of Cephalosporium salmosynnematum to potassium, mag- nesium, phosphorus, and sulfur. Redrawn from Pisano et al. (187), by permission of the Williams and Wilkins Company. 3. SULFUR Media in common use incorporate excess sulfur as magnesium sul- fate; at moderate carbohydrate concentrations the sulfur requirement is about 0.0001-0.0006 M (13, 91, 228). Most fungi can supply all their needs for sulfur from inorganic sul- fate, i.e., they reduce the sulfate and incorporate it into organic mole- cules. The organic sulfur compounds known in fungi include amino acids (cysteine, cystine, and methionine), vitamins (thiamine and biotin), cyclic choline sulfate (Chapter 2), sulfur-containing anti- biotics, e.g., penicillin and gliotoxin, and such miscellaneous com- pounds as junipal (Chapter 6), thiourea (177), and methyl mercaptan (Chapter 6). Sulfur incorporation into organic combinations is often very large; whether or not sulfur is released on autolysis appears to be determined by the species studied (84, 192, 197). The fungi which as isolated from nature are unable to utilize sul- fate sulfur are all aquatic phycomycetes: most Saprolegniales and all known members of the Blastocladiales (30, 31). Other phycomycetes — Chytridiales, Leptomitales, Peronosporales, and the Zygomycetes — uti- lize sulfate. Whether dependence on reduced sulfur is biochemically or ecologically related to the lower oxygen requirement or to the fermen- tative capacity of the Blastocladiales is still uncertain. Mutants re- quiring reduced sulfur have been obtained from Neurospora crassa SULFUR 305 (89), Aspergillus nidulans (83, 188), Penicillium chrysogenum (82), Ophiostoma multiannulatum (64, 65), and Ustilago zeae (180). Some nutritional studies on sulfur are summarized in Table 2, from which it appears that most or all fungi utilize sulfide, thiosulfate, and Table 2. The Sulfur Nutrition of Some Fungi* Sapro- Brevi- Pesta- Asper- legnia legnia Pythium lotia gillus Penicillium Source of delica gracilis spp. malorum niger chrysogenum Sulfur (22) (22) (208) (244) (234) (82) Sulfate 0 + + + + + Persulfate 0 + + + + Sulfite 0 + + + Bisulfite 0 + + 0 + Dithionate 0 0 0 + 0 Hyposulfite + + + 0 + + Thiosulfate + + + + + + Sulfidef + + + + + + Disulfide f + + + Cysteine + + + + + + Cystine + + Methionine + Thiourea + + + + 0 + Thiocyanate + * + = growth, 0 = no growth. f See text. the more oxidized forms of inorganic sulfur (except dithionate). How- ever, the data on some of these compounds must be questioned in view of their known instability; thus, Steinberg (234) found that Aspergillus niger grows much better on old than on fresh samples of sodium sulfide and disulfide. It should be noted that the difference between Brevilegnia gracilis and Saprolegnia delica in their sulfur nutrition parallels that seen elsewhere (Chapter 3) in their carbon nutrition — the soil-inhabiting B. gracilis is more omnivorous than its aquatic relative. The details of sulfate reduction are not fully known. The available data suggest the following pathway (44): Sulfate (S04--)-» sulfite (SOs — )-> sulfide (S — ) or thio- sulfate (S203 — ) — » cysteine — > methionine (1) The obviously necessary intermediates are not known, nor is it possible to say at what point in Equation 1 the sulfur becomes attached to an 306 INORGANIC NUTRITION AND METABOLISM organic molecule, but the scheme is in agreement with nutritional data on genetically different mutants of Neurospora crassa (89), Asper- gillus nidulans (83, 188), and Venturia inaequalis (104). Shepherd (219) proposes a dual pathway of cysteine biosynthesis in A. nidulans. The naturally occurring fungi which require reduced sulfur are, we may suppose, unable to carry out one or more of the reactions of Equa- tion 1. Thus Blastocladiella emersonii grows with either cysteine or methionine (13) and is presumably blocked prior to cysteine synthesis; Allomyces arbuscula, on the other hand, appears unable to use any source of sulfur other than methionine (99). The same considerations apply to other fungi which require cysteine or methionine (131, 254). Bulk reduction of sulfate with evolution of hydrogen sulfide is not effected by fungi, as it is by some bacteria. Candida spp. reduce sulfite (173), many fungi enzymatically reduce elemental sulfur to H2S (125, 144, 216), and Neurospora crassa reduces selenite to selenium (259). Sulfur and sulfur compounds are oxidized by fungi: elemental sulfur by Penicillium luteum (1) sulfur-containing amino acids by Aspergillus niger (68, 148), cysteine by Microsporum gypseum (224). Cysteine-sul- finic acid has been proposed as an intermediate in cysteine oxidation (224). A few minor aspects of sulfur metabolism may be mentioned in conclusion. Commercial enzyme preparations from Aspergillus spp. contain a phenolsulfatase, hydrolyzing compounds of the type R — OS03K (2, 162, 163, 165); other sulfatases have not been reported from fungi (66). Penicillium notatum is able to split a carbon-sulfur bond (32). The toxicity of barium to Aspergillus niger results from the removal of sulfate, and barium is not toxic if sulfur is supplied in some other form (228, 234). Finally, the reduced sulfur requirement of the yeast phase of Histoplasma capsulatum (186, 209) is a require- ment for reducing conditions, not for reduced sulfur as such. Transfer of methyl groups to sulfur is summarized in Chapter 6; reductive steps occur in the over-all metabolism of sulfur, selenium, and tellurium. 4. MAGNESIUM Magnesium, customarily provided as the sulfate at about 0.001 M, probably has as its principal essential function the activation of en- zymes necessary to normal metabolism and growth. The optimum is dependent on the concentration of the carbon source (241), the production by the organism of hydroxy acids or other chelate-forming molecules which tend to render magnesium unavailable, and the con- centrations of other ions to which magnesium is antagonistic. Asper- iron 307 gilhis terreus in surface culture requires much more magnesium than it does in shaken culture (119, 120). The enzymes activated by magnesium in in vitro systems are often also activated by manganese or other divalent ions, but it is believed that magnesium is the physiologically active metal (115, 126). None of the physiological effects of magnesium deficiency has been conclu- sively related to enzyme malfunction, but it is reasonable to suppose that, for example, enhancement of riboflavin formation by magnesium deficiency in Aspergillus niger (114, 210) reflects an impairment of the normal oxidative system. In bacteria, magnesium accelerates the up- take of phosphate, possibly by reason of the role of the metal in transphosphorylations (3). The absorption of magnesium is somewhat slower at neutral than at acid reaction, resulting in a slightly higher optimum at neutrality (236). Magnesium is precipitated and rendered virtually unavailable at high pH in the presence of ammonium ion (62). As suggested above, the apparent magnesium requirement is prob- ably affected by other ions. This is deduced from the very great ac- tivity of magnesium in antagonizing the toxicity to fungi of such metals as aluminum, copper, and mercury (120, 122, 136); similar re- sults with other ions have been reported in studies on bacteria (3, 130). The most attractive theory explaining this antagonism is that of Mac- Leod and Snell (130), that other metals compete with magnesium for enzyme surfaces and that the resulting metal-enzyme complex is inactive or less active than the natural magnesium complex. Competition, how- ever, need not be limited to enzymatic reactions. Marsh (136) showed clearly that magnesium prevents the absorption of copper by conidia of Sclerotinia fructicola; this finding suggests a competition of the two elements at the cell membrane. Beryllium is said to replace magnesium partially and under special circumstances (238). 5. IRON Fungi and actinomycetes require approximately 0.1-0.3 ppm of iron, more in concentrated media (36, 37, 100, 169, 241). Aspergillus niger utilizes either ferrous or ferric iron (168). Iron is lost from solution at neutral or alkaline pH; inclusion in the medium of a chelating agent, e.g., citrate, reduces the loss (62, 88). The most obvious metabolic role of iron is in the formation of iron- containing metabolites. Those known in the fungi include catalase and the cytochromes (Chapter 7), coprogen and the ferrichromes 308 INORGANIC NUTRITION AND METABOLISM (Chapter 10), the heme pigment of Neurospora crassa and Penicillium notatum (103), and the pigment pulcherrimin of Torulopsis pulcher- rirna (40, 107). Preparations of aspergillin, the spore pigment of the black Aspergillus spp., contain 0.26 per cent iron (191). Aspergillus niger releases into the medium relatively large amounts of a substance or substances, not citrate, which bind ferric iron (67); the possible ecological value is obvious (158). A few of the metabolic effects of iron may be ascribed to direct use of the element; increases in catalase (257) and in pulcherrimin (201) are of this type. Other metabolic effects of iron concentration, e.g., on organic acid formation (45, 57, 184, 185), on penicillin production (108), on the balance of gentisyl alcohol and patulin (Chapter 6), and on sporulation and spore color (118, 211), are known, but the opera- tive mechanisms are obscure. Only one persistent pattern may be discerned: the maximum formation of certain metabolites — strepto- mycin (37), penicillin (100), and citrinin (9) — requires more iron than is needed for maximum growth. The data on organic acid accumula- tion as affected by iron are too much in conflict to allow any general- ization (36, 57, 183); as discussed elsewhere (Chapter 6), it is most probable that deficiencies or excesses of iron and other inorganic con- stituents act positively on acid formation, when they do, by virtue of interference with normal metabolism. 6. ZINC The essentiality of zinc for growth in fungi has been confirmed many times (35). Reported optima, determined, of course, under different conditions, range from 0.001 to 0.5 ppm; probably 0.5-1.0 ppm is adequate in routine work (5, 36, 37, 76, 241). Higher con- centrations of zinc may be toxic, especially at high pH (117, 174); the toxicity is reversed by certain other divalent ions (130). Inhibition of sporulation by zinc is reversed by iron (174, 200). High levels of zinc induce genetically stable variation in Fusarium sp. (46) and Helmin- thosporium sativum (145). Partial replacement of zinc by cadmium (101) probably results from contamination of the cadmium with zinc (227). The metabolic effects of zinc deficiency almost defy enumeration; several reviews consider them in detail (35, 57, 58, 183). The major types of metabolic effects are: 1. Accumulation of organic acids at levels of zinc too low for normal growth. Associated with this is a positive effect of the metal on the COPPER 309 economic coefficient and on carbon dioxide evolution. The effect of zinc on citric acid formation is, in almost all reported instances, to decrease the per cent conversion of carbohydrate to citrate (35). This zinc effect is not specific; citric acid accumulation is favored by other mineral deficiencies (36). 2. Lowering of specific enzyme concentrations in cells grown in low-zinc media. Enzymes affected include pyruvic carboxylase (59, 242) and ethanol dehydrogenase and the tryptophan-synthesizing en- zyme (156). Diphosphopyridine nucleotidase, on the other hand, is increased by moderate zinc deficiency (156, 157). It should not be assumed, however, that these effects are specific to zinc. 3. Decrease by zinc of the accumulation of certain antibiotics (141, 245). In Aspergillus ochraceus, the specific 6/?-hydroxylation of steroids is much retarded by zinc deficiency (51). 4. Increase in cytochrome synthesis in Ustilago sphaerogena at high zinc (76), with an accompanying decrease in ferrichrome accumula- tion (158). 5. Changes in cell composition (127, 189, 214). 6. Stimulation by zinc of the in vitro formation of vesicles by Puc- cinia coronata (218). 7. A shift in the proportions of related ring compounds produced by Penicillium urticae (52). 8. Effects of zinc supply on sporulation, reviewed by Foster (57). Zinc is known to activate a few enzymes specifically (126) and others non-specifically (207); ethanol dehydrogenase and glutamic de- hydrogenase are zinc proteins (249, 250). The diverse effects of zinc on metabolism suggest in addition that it is concerned with the syn- thesis of enzyme proteins; this hypothesis applies especially to the second, third, fourth, and seventh effects just listed. 7. COPPER Copper is required by fungi for normal growth and sporulation at about 0.01-0.1 ppm (36, 37, 100, 241). Higher concentrations are of course toxic, although two fungi make visible growth even in a satu- rated solution of copper sulfate (226). The uptake of copper is some- what greater at moderate pH than at low pH (215). Perhaps the most striking physiological effect of copper deficiency is the reduction in pigmentation of the colored spores of Aspergillus spp. (26, 102, 142, 150) and Trichoderma viride (27). Although other metal 310 INORGANIC NUTRITION A N D M E T A B O L I S M deficiencies also affect spore pigmentation (58), copper is probably the most important, at least for the dark spore pigments. It has been mentioned earlier (Chapter 6) that copper may play a role in chlorine metabolism of fungi. The possibility that, as in ani- mals (134), copper is essential for normal iron metabolism has not been explored. 8. MANGANESE The essentiality of manganese in small amounts, about 0.005-0.01 ppm, has been demonstrated for a number of fungi (23, 36, 53, 57, 86, 137, 190, 199, 227, 241); we may assume that fungi in general require it, although the effects of deficiency are not apparent for all. The re- quirement is more easily shown at low pH than at high (121). Manganese deficiency has several physiological effects (57, 183), in- cluding a pronounced decrease in sporulation. The frequent role of the metal in activation of enzymes, especially those of the citric acid cycle (136), suggests a role for it in metabolism at this point; manga- nese concentration also affects the concentration of other enzymes in the cell (86). The biological oxidation of manganese in soil can be demonstrated perhaps best in soil percolation experiments (129). It is probable that fungi as well as other organisms carry out this reaction; the limited evidence is reviewed by Mulder and Gerretsen (153) and by Starkey (225). 9. MOLYBDENUM The essentiality of molybdenum for Aspergillus niger was discovered by Steinberg (229). The requirement is small — estimates range from 0.1 parts per billon (152, 168) to 10 ppb (241) — and A. niger responds to as little as 0.02 ppb (152). Vanadium does not replace molybdenum (168). It has been observed repeatedly that the molybdenum requirement of both Aspergillus niger and Neurospora crassa is higher if nitrate is used as the source of nitrogen than if ammonium nitrogen is provided (152, 170, 230). The explanation for this phenomenon is the first un- equivocal demonstration of a specific enzymatic basis for a deficiency symptom in fungi. Molybdenum participates as electron carrier in the reduction of nitrate by enzymes of Neurospora crassa, Escherichia coli, and soybean; the evidence for this conclusion is reviewed by Nason CALCIUM 311 (155). The valence states of molybdenum which function in electron transport are the +5 and the +6 (171). Molybdenum must, however, have other functions, inasmuch as a molybdenum deficiency can be demonstrated in media not containing nitrate (152, 168, 170). 10. CALCIUM The effect of calcium on the growth of fungi has only recently been recognized, although the first report of such an effect was in 1922 (258). Species in 19 genera, including representatives of all the major groups of fungi and of the genus Streptomyces, have been found to respond to added calcium by an increase in dry weight (62, 63, 73, 99, 113a, 117, 128, 166, 175, 178, 198, 239, 246, 253). Reported optima range from 0.5 to 20 ppm. Some fungi, it should be noted, cannot be shown to require calcium (198, 239). Figure 3 illustrates the response to cal- cium. Strontium partially replaces calcium for Allomyces arbuscula (99) and several other fungi (117, 198, 239). Strontium is firmly bound by mycelium of Rhizopus sp. (116). One may still ask, however, whether the so-called calcium require- 7 9 Time, days Figure 3. The growth of Coprinus ephemeras at different levels of calcium (milligrams per liter). Redrawn from Fries (63), by permission of the Svensk Botanish T.idskrift. 312 INORGANIC NUTRITION AND METABOLISM ment of fungi is specific. Calcium in many organisms, including fungi (117), protects against the injurious effects of certain common mono- valent cations, especially hydrogen, sodium, and potassium. In bac- teria, calcium and related ions also protect against zinc toxicity (127). These non-nutritional effects, the wide range of optima reported, the extent of replacement by strontium, and the fact that some fungi do not respond at all to calcium all argue for some caution in interpreta- tion of the data as reflecting an absolute and specific requirement. It is noticeable that in a casamino acids medium, with high sodium con- centration, calcium affects the growth of Aspergillus oryzae (222), but the related A. niger in the usual low-sodium media does not respond to calcium (239). The crucial experiment would be, of course, to de- termine whether an apparent calcium requirement can be eliminated by adjusting the concentration of other ions. It has been found that calcium affects the time required for perithe- cial formation in Chaetomium spp. (14, 15) and the intensity of conidi- ation in Trichoderma viride (27). Calcium, like magnesium, may be made less available if chelating agents, e.g., citrate, are present in the medium, and is less available, because of precipitation, at high pH than at low. 11. COBALT In spite of early claims of the essentiality of cobalt for fungi, re- viewed by Marston (138), there is at present no conclusive evidence of a cobalt requirement; some more recent data are suggestive, however (11). Cobalt is required by animals but not, apparently, by higher plants (138). As mentioned elsewhere (Chapter 10), vitamin B12 is made by Streptomyces spp. — and for the synthesis cobalt must be re- quired— but whether the vitamin is produced by true fungi is uncer- tain. Furthermore, neither the fungi nor the actinomycetes are known to require the vitamin. Although cobalt activates some enzymes, e.g., the dipeptidase of Aspergillus oryzae (42), its only known essential function in organisms generally is as a constituent of vitamin B12. In view of the very great technical difficulty in establishing a cobalt re- quirement if the metal is only needed for B12 synthesis (94), it may be more profitable to explore the B12 metabolism of fungi than to attack the cobalt problem directly; preliminary results (168) suggest that nei- ther B12 nor cobalt is essential to Aspergillus niger. Cobalt is accumulated by Neurospora crassa (12), the degree of ac- cumulation— up to 23 times the external concentration — being a func- tion of the amount of cobalt in the medium. Up to 40 per cent of the BIOASSAY OF MINERALS 313 cobalt so accumulated is bound to a protein. Cobalt accumulation in N. crassa is affected indirectly by the iron concentration (11). Cobalt at appreciable concentrations is toxic to fungi and other or- ganisms* Toxicity is reduced by histidine (181, 182, 212), which pre- sumably forms a complex with the cobalt ion. The uptake of cobalt by Aspergillus niger is sharply reduced by the addition of relatively small amounts of magnesium (3). A third complication in cobalt tox- icity is the observation that, although iron and cobalt do not simply compete, the enzymatic symptoms of cobalt toxicity in Neurospora crassa resemble closely the symptoms of iron deficiency (80). 12. OTHER INORGANIC NUTRIENTS Aspergillus niger responds slightly to vanadium (17, 18, 19) and fleshy fungi take up vanadium from the soil (20), but satisfactory proof of an essential role is not at hand. Vanadium is essential, however, for the alga Scenedesmus obliquus (6). Steinberg has reported growth increases from addition of gallium (231) and — only with glycerol as carbon source — of scandium (233). Results on gallium have not, however, proved reproducible (237). Claims of an essential or growth-promoting role of boron (256) and organic compounds of silicon (87) have not as yet been confirmed; Winfield (255) reports negative results with boron. Chlorine appears to have no necessary role in the nutrition of fungi (60) although chlorin- ated metabolites are not uncommon (Chapter 6), and chlorine is es- sential to higher plants (28). Other metals, e.g., aluminum and chro- mium, often exert physiological effects and may even under peculiar circumstances improve growth, but it has not been suggested that they are essential. 13. BIOASSAY OF MINERALS The response of fungi to nutrients is sensitive enough so that a num- ber of bioassays for inorganic elements in soil have been developed to the point of practical utility. Almost all the work has been with Aspergillus niger, although a radial growth assay for phosphorus with Cunninghamella spp. was devised in 1934 (139, 140). Bioassays for minerals are more laborious than chemical determina- tions and are intrinsically less accurate; precision is especially low in those assays based on visual estimates of growth or spore color (151). The principal advantage of the bioassay method, and the reason for its popularity, is that it may more nearly approximate a determination of 3H INORGANIC NUTRITION AND METABOLISM Zinc ^* / / / / ^^ 1 Molybdenum^^---^7'^ (^"" jS / ^^Copper 4-» 00 ID /s" Q ''? r'/ 7/ Figure 4. The form of assay curves, employing Aspergillus niger, for zinc, molybdenum, and copper. Redrawn from D. J. D. Nicholas, The Analyst, Vol. 77, p. 629-642 (1952), by permission of the Society for Analytical Chem- istry. Concentration of elemeat the minerals actually available to a crop plant. The assumption made, therefore, is that the fraction of a given mineral which the plant cannot absorb is equally unavailable to the fungus. Consequently, the major criterion for the practicability of a bioassay must be the degree to which the assay results predict the behavior of crop plants in a given soil; correlation with chemical determinations is of secondary impor- tance. Correlative studies have usually shown at least fair agreement between growth of Aspergillus niger and growth of higher plants in soils deficient in phosphorus, potassium, zinc, copper, and molybdenum (47, 73, 147, 150, 168). The fungus is less suitable for manganese deter- mination because metabolic acids make more manganese available to it than is available for higher plants (153). Typical assay curves are shown, in simplified form, in Figure 4; it is to be noted that the response is not rectilinear. Detailed methods are described by Donald et al. (47), Gerretsen (73), Mulder (151), and Nicholas (168). 14. PROBLEMS IN MINERAL NUTRITION Two general approaches may be used in determining the essentiality of a given element. One may design a medium free of the element in question and containing all other materials necessary for growth. Es- sentiality is shown by a quantitatively determined growth response to graded amounts of some soluble compound containing the element. Obviously, the medium must be purified rigorously for the study of PROBLEMS IN MINERAL NUTRITION 315 micronutrients, and, equally important, the compound added must not contain any growth-promoting substances. The second approach, little used so far, is to determine that some metabolite which contains the element is essential to growth. For ex- ample, if the vitamins B12 prove to be essential, then cobalt must be essential. Details of the methods for purification of media for studies of micro- nutrient elements lie outside the scope of this work. There is no one best method for all materials, and the choice depends on the particular element under study. The most useful over-all methods involve adsorp- tion on calcium carbonate (240) or aluminum compounds (48, 221). Special methods have been described for copper (151, 167, 168), man- ganese (168), and molybdenum (81, 168). It appears that the chelating agents (8-hydroxyquinoline, diphenylthiocarbazone, etc.) are not only less efficient for most micronutrient elements but are likely to be toxic to the organism. Detailed comparisons of different methods have been published by Steinberg (240), Donald et al. (48), and Nicholas (168). Methods of obtaining inorganic salts of maximum purity are dis- cussed by Hutner et al. (94). The sources of contamination in micronutrient work are well known by now: the inoculum, glassware, distilled water from a metal still, su- gars and polyatomic alcohols, hydroxy acids, inorganic salts, and com- plex extracts of biological origin (92, 168, 229, 230, 232). Amino acids are often so heavily contaminated that they cannot be employed at all (235). Chelating agents may be used to exaggerate a requirement and make it more readily detectable. Thus, Hutner (93) added citrate to hold calcium and other elements in solution; the calcium requirement in the absence of the complexing anion is estimated by extrapolation. Work with the major inorganic nutrients is, of course, somewhat simpler inasmuch as deficiencies are not made up by minor amounts of contamination. Methodological problems do, however, arise even here. Thus, the usual practice in working at low or zero potassium is to supply phosphate as the sodium salt; this, of course, ignores the pos- sibility that sodium partially replaces potassium. In this particular instance, the solution is to use an amine in place of the alkali metal to control pH (126). In reporting results, the concentration permitting half maximal growth is a more useful datum than that at which maximum growth occurs. Although an optimum amount of a given element may be defined for rigidly specified conditions (236, 237), it must be realized that there 316 INORGANIC NUTRITION A N D M E T A B O L I S M is no absolute optimum concentration of an element for even one spe- cies. Some of the complicating factors may be mentioned. First, the need for inorganic nutrients is proportional to the carbohydrate sup- ply (241). Second, the need for an element may be increased or de- creased by the presence of a second element, the important nutritional factor becoming then the ratio of the two (60, 129); in practice, this means that equally good growth is observed over a range of total salt concentration (43, 77, 128). Third, the nutrients and metabolites which form chelate complexes with minerals are present in continu- ally changing amounts; although a chelating agent may on the one hand solubilize a mineral and so make it more available, on the other hand the cell must compete with the chelate complex for the element. Fourth, the pH of the medium — again a factor not usually constant for the entire life cycle — affects the solubility of most inorganic salts (62, 78, 88, 230), the state of oxidation of manganese (225), and the stability of chelated complexes (25). Finally, changes in the conditions of culture may change the apparent requirement for a mineral, as in the case of the magnesium requirement of Aspergillus terreus (119, 120). The use of metabolically inert chelating agents, particularly ethylene- diaminetetraacetic acid (EDTA, versene) solves some of the practical problems of supplying a fungus with minerals; the compound has been used successfully, at 2-5 ppm, for several fungi (194, 209a, 252) and for Streptomyces nitrificans (213). However, this and similar agents may prove to be toxic (63, 128, 243). Added citrate or malate, unless uti- lized by the organism, can be used to keep minerals soluble (92). The fungi accumulate minerals against a concentration gradient; the data are particularly clear for cobalt (11, 12), silver and cesium (144), and potassium (54). These examples, and the accumulation of rubid- ium by fleshy basidiomycetes (21), make it clear in addition that accumulation of non-essential elements is common. The energy for accumulation must come, of course, from respiration. The very strik- ing effect of magnesium on the uptake of cobalt by Aspergillus niger (3) and of copper by Sclerotinia fructicola (132) suggests that reactions at a semi-permeable membrane are of importance in mineral nutrition. In yeast, there is good evidence for carrier systems in the transport of cations across the membrane (61). The functions of the minerals required by fungi deserve further study. Phosphorus and sulfur occur in known essential compounds, and their major role appears to be constitutive. The other essential mineral elements are today more or less conventionally assigned a "catalytic" function as activators or active centers of enzymes. Reasons exist, of course, for this general belief: the function of molybdenum in BIBLIOGRAPHY 317 nitrate reductase and zinc in ethanol dehydrogenase, the occurrence of copper and iron in purified enzymes, and the known activating effect of magnesium, manganese, and calcium on isolated enzymes. 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We may, for example, include as growth substances all compounds required in small amounts and not used for energy (260). This broader definition includes amino acids, purines, and choline as growth factors. A nar- rower concept, the one used here, excludes compounds that function as structural materials, even though they are needed in small amounts. Most of the known vitamins have a catalytic function in the cell as coenzymes or constituent parts of coenzymes. This function is stressed in the definition of vitamins employed in this chapter: organic mole- cules required in small amounts and not, so far as we know, used as sources of either energy or structural materials of protoplasm. This definition is arbitrary and provisional. Arbitrary, because it separates constituents of coenzymes (e.g., riboflavin) from constituents of en- zymes (e.g., amino acids) and cannot separate, for example, the role of adenine in adenosine triphosphate from its role in the structure of nucleic acids. Provisional, because the cofactor role of some of the vitamins, especially inositol, is uncertain. 324 VITAMIN REQUIREMENTS This chapter, then, includes substances, primarily the water-soluble vitamins, which are thought to be catalytic in function; the amino acids and nitrogen bases are considered in Chapter 8, as is the plant growth factor indoleacetic acid. The history of our knowledge of the vitamin requirements of fungi is reviewed by Janke (117), Robbins and V. Kavanagh (213), Schopfer (239), and Knight (122). Although the work on yeasts and yeastlike fungi really began in the nineteenth century, the vitamin needs of the filamentous fungi have been studied intensively only since 1934, i.e., since the papers of Schopfer (236, 237) on thiamine and the growth of Phycomyces blakesleeanus. Most work has been devoted to the require- ments of naturally occurring fungi and, more recently, to the require- ments of induced mutants; both types of material have provided signif- icant information on the biosynthesis and the function of vitamins in living things generally. 1. PROBLEMS OF METHOD The central methodological problem in vitamin studies is that of the degree to which the investigator knows the composition of the medium. Almost any natural product, e.g., sucrose or asparagine, is contaminated with significant amounts of some vitamins; procedures for purification will be found in the literature on particular growth factors. For very limited purposes, e.g., in formulating a medium for routine use, it may be sufficient to use the approximate methods so common in the literature on vitamin nutrition. However, a serious study of vita- min nutrition requires at the least that three methodological criteria be met: 1. The measurement of growth should be objective and quantitative. Although growth on agar has been used fairly successfully in particular problems, it cannot be relied upon, and dry weight or turbidity is to be preferred (Chapter 1). Pyridine-extracted agar (214) may, however, be used for some problems. 2. Over some range of concentration of the vitamin, it should be shown that growth is a function of dose (concentration or total amount of the vitamin). 3. If growth occurs on the basal medium lacking added vitamin, an independent assay of the medium for the vitamin should be made. One very simple test consists of growing on the basal medium an or- ganism known to require the factor absolutely. Other problems of method are summarized by Robbins and V. Kavanagh (213). THE NATURE OF VITAMIN REQUIREMENTS 325 Especially with negative results, i.e., the finding that an organism does not need a vitamin, still further precautions may be mentioned. First, the basal medium must be free of the vitamin; even cotton plugs may contribute growth factors (248, 251). Second, it is essential that the organism be serially subcultured in the vitamin-free medium for at least three transfers. In some investigations (131) the inoculum has no effect, but this cannot be assumed. The criterion of serial subcul- ture is the one most frequently disregarded, and in consequence most reports of a lack of need for a vitamin — especially those, e.g., biotin, for which the requirement is always small — are not wholly reliable. The use of crude extracts of natural products in the preliminary search for vitamin requirements is, of course, helpful and, within lim- its, valuable. However, the simple observation that such an extract increases growth has no necessary bearing on vitamin nutrition, since so many other materials, known and unknown, are present in these ex- tracts. Thus, the effect of agar on growth of several fungi (132) and the effect of yeast extract on cytochrome synthesis by Ustilago sphaerogena (89) are both attributable to the zinc content of the natural product, not at all to organic materials. The vitamin content of many common constituents of culture media is known (34, 44, 95, 206, 217, 224, 264); occurrence in other natural products is reviewed in general works on the vitamins (225, 249). 2. THE NATURE OF VITAMIN REQUIREMENTS It is generally assumed that a fungus or other organism which is in- dependent of an externally supplied vitamin is so by virtue of its ability to synthesize the compound: a thiamine-independent fungus synthe- sizes thiamine, etc. The validity of this assumption is borne out by experimental evidence in all cases so far investigated, and is logically deduced from the premise that the known vitamins have essential functions in all cells. This premise, it should be noted, extends only to the water-soluble or B vitamins. Further evidence, if such is needed, for the essentiality of vitamins in those fungi which do not require an external supply is found in the fact that deficient mutants can usually be obtained by appropriate tech- niques; examples, in addition to the classical Neurospora crassa, in- clude species of Penicillium (17) and Aspergillus (115, 195, 199), gen- era in which naturally occurring vitamin deficiencies are uncommon. The simplest situation in vitamin studies is that of complete inabil- ity of a given fungus to synthesize one or more vitamins. In this cir- cumstance, the cell depends absolutely on an external supply and, at 326 VITAMIN REQUIREMENTS least over a certain range, growth will be proportional to the supply of the required factor or factors. Many natural and induced deficiencies are or appear to be of this type, which is the most easily detectable. A somewhat more complicated situation arises if a fungus is able to synthesize a vitamin, but so slowly that under the usual conditions of culture the rate of all other processes is potentially faster than vitamin synthesis. Here we find the organism growing slowly in the absence of exogenous vitamin but responding to an external supply by a faster rate of growth. Such partial deficiencies are also common, perhaps more common than complete deficiencies. Examples include the require- ments of particular fungi for thiamine (16, 65, 183, 186), biotin (144, 216), pyridoxine (66, 67, 216, 217), nicotinic acid (291), and inositol (238). ft also follows that any growth-regulating process — including vita- min synthesis — may be limiting under some conditions and not under others. A vitamin requirement that is manifest only under certain circumstances is termed a conditioned requirement. The known types of conditioned deficiency in fungi may be classified as follows: 1. The deficiency is apparent or more severe at particular tempera- tures, pH levels, or salt concentrations. 2. The requirement for a vitamin is reduced or, more rarely, elimi- nated by provision in the medium of a precursor or of a metabolite for the synthesis of which the vitamin is essential. 3. The deficiency is limited to or more acute at a particular stage of development. Environmental factors conditioning vitamin deficiencies include tem- perature, acidity, and salt concentration. A mutant of Nenrospora crassa, for example, requires riboflavin only at relatively high tempera- tures (172). Sclerotinia camelliae requires more inositol at 26° C than at 18° but is inhibited by inositol at 27° (8); a temperature-dependent inositolless mutant of N. crassa is also known (113). Sordaria fimicola requires thiamine only if the initial pH of the culture is less than 4.0, possibly because thiamine synthesis is inhibited by hydrogen ion (139). A similar phenomenon in mutants of Neurospora spp. which require pyridoxine appears, however, to reflect a requirement for free ammonia rather than a direct effect of acid (268). The influence of pH on the p-aminobenzoic acid requirement seems, finally, to be exerted through permeability effects (325). Pythium butleri requires exogenous thia- mine only in a high-salt medium (211). The second type of conditioned requirement is more easily ex- plained: a precursor of the vitamin or a metabolite in the synthesis of THE NATURE OF VITAMIN REQUIREMENTS 327 which the vitamin is essential will obviously reduce the demand for the exogenous vitamin. The replacement of nicotinic acid by large amounts of tryptophan represents replacement by a precursor (20). The replacement of p-aminobenzoic acid by methionine (269) may be interpreted as evidence that at least one function of the vitamin is in the biosynthesis of methionine, and a similar mechanism is invoked to explain the partial replacement of biotin by aspartic acid (190). The third type of conditioned requirement is the most interesting and the least studied: the possibility that fungi change in their syn- thetic capacity and in their vitamin needs during development. It must be realized that the typical vitamin experiment performed with fungi actually measures in the first instance the capacity of the spores to germinate in the absence of the vitamin in question; if the spore germination process requires the growth factor, the organism as such is recorded as having the requirement. It is entirely possible that spore germination may require factors which mature mycelium can synthe- size for itself. Thus, the growth of Memnoniella echinata from a spore inoculum requires biotin, which is not replaceable by desthiobiotin. However, if desthiobiotin is added to a growing culture supplied with limiting amounts of biotin, the desthiobiotin elicits a significant growth response (191). Evidently, the mycelium is able to convert desthiobiotin to biotin but the spore cannot. Successful use of the dif- ferential germination method for the detection of vitamin-deficient mutants (69, 308) shows that spore germination is affected by growth factor deficiencies. Several fungi — Alternaria solani (138), Fusarium solani (285), and Penicillium digitatum (61) — are reported to be accelerated in early growth by vitamins the requirements for which are not evident if total growth over a long period is measured. In Myrothecium verrucaria a requirement for biotin is detectable only at a stage shortly after spore germination (157). Among the dimorphic fungi (Chapter 1), a single study (229) sug- gests that the yeast and the mycelial phases of some forms differ in their biotin requirement. The onset of reproduction as a phase of growth may, although the data are far from decisive, involve changes in vitamin needs and re- sponses. There is, however, no evidence of a singular association of one vitamin with sporulation per se, independent of growth. This problem is considered in Chapter 1 1 . Multiple requirements are especially common in the yeasts; five or six vitamins may be needed (27, 29, 39). Among the filamentous fungi, there are several instances of a requirement for three different vitamins, 328 VITAMIN REQUIREMENTS e.g., in Ascoidea rubescens (67), Dlastocladia pringsheimii (35), Cera- tostomella microspora (216), and Trichophyton discoides strains (152, 222). Inhibitory effects of vitamins have been reported occasionally. Thi- amine depresses the growth of Fusarium spp. (55, 285, 304) and Rhizo- pns spp. (142, 210, 238); biotin may reduce growth or enzyme forma- tion (11, 228, 253, 316). Large amounts of pyridoxine do not, how- ever, appear to depress growth (67). The mechanism of inhibition is not always known; thiamine inhibition has been attributed to ammonia accumulation in Trichoderma viride (16) and to ethanol accumulation in Rhizopus suinus (247). 3. THE METABOLISM OF VITAMINS Apart from their catalytic role in metabolism, vitamins are them- selves metabolites and are both synthesized and destroyed in the cell. More or less comprehensive surveys of vitamin synthesis have been reported for Penicillium spp. (37, 100, 178, 274), and a good deal of information is available on vitamin synthesis by Aspergillus spp. (37, 60, 85, 127, 181, 233, 262) and Streptomyces spp. (47, 100, 103, 105, 106, 151). The vitamin content of microorganisms is reviewed comprehen- sively by Van Lanen and Tanner (288). As mentioned earlier, we may assume that a fungus which does not require one of the water-soluble vitamins known to be required by other fungi is able to synthesize it. Vitamins synthesized by a fungus usually appear in the medium and are detected there by bioassay; however, in Penicillium chrysogenum the greater part of the thiamine, riboflavin, and nicotinic acid pro- duced remains in the cell (100), and the same is true of thiamine in some other fungi (133) and of riboflavin in Aspergillus niger (60). The destruction of vitamins by fungi has been less thoroughly stud- ied. Phycomyces blakesleeanus and Sclerotium rolfsii destroy the thia- zole moiety of thiamine (21, 120). A mutant of Neurospora crassa destroys pyridoxine, and this destruction is inhibited competitively by thiamine (96). 4. FUNGI IN THE BIOASSAY OF VITAMINS Analysis for individual vitamins by a biological assay is often, for one reason or another, superior to chemical analysis. This is especially true if a biologically active vitamin exists in more than one chemical form. Bioassays employing filamentous fungi have been devised for THIAMINE 329 thiamine, biotin, pyridoxine, /?-aminobenzoic acid, pantothenic acid, and inositol (239, 259, 281). Usually, dry weight is the criterion, al- though pyruvic acid formation (93), pigment production (289), and rate of growth on agar (10, 277, 282) have all been proposed. Assays based on the dry weight of vitamin-requiring fungi have in general the advantages and disadvantages of any biological assay (171, 259). However, the relatively more tedious procedures necessary with the fungi have persuaded most investigators to use, whenever possible, bioassays based on yeast or bacterial growth or metabolism. In special cases, fungi may prove more valuable; thus, different fungi able to use either the pyrimidine or the thiazole moiety of thiamine were used by Krampitz and Wool ley (124) in a study of the thiaminase of carp. Bioassays using fungi are critically reviewed by Snell (259); others have been described for biotin (224), pantothenate (290), and thia- mine (93, 289). The Phycomyces assay for thiamine is reviewed by 0degard (185). A survey of the response of many fungi to graded doses of water-soluble vitamins (136) brings out some of the difficulties in- herent in biological assay procedures. 5. THIAMINE Thiamine (aneurin, vitamin Bx) is the first vitamin to have been studied as a known entity in the nutrition of fungi, and is the vitamin most frequently required by fungi. Thiamine deficiencies oc- cur in all major taxonomic groups — although the ascomycetes have been less thoroughly investigated than others — and in fungi from many dif- ferent natural environments. Reported requirements vary widely (65, 226, 300); time of harvest, temperature, and the composition of the medium can be expected to affect thiamine demand. It seems at least possible that those fungi with very small requirements are able to synthesize thiamine once growth has been initiated. A typical response curve is shown in Fig- ure 1. The metabolically active form of thiamine is the pyrophosphate, long known as cocarboxylase because of its coenzyme function in the decarboxylation of pyruvic acid. Thiamine pyrophosphate is also the coenzyme for transketolase and for a variety of enzymatic reactions of a-keto acids (118). In the fungi, its role in the metabolism of pyruvic acid is clear from its acceleration of ethanol formation (42, 247) and the accumulation of pyruvate in thiamine-deficient cultures (64, 93, 304). Acceleration of glucose utilization (101) and decrease in oxalate accumulation (175) may operate through the same mechanism. 330 VITAMIN REQUIRKM I NTS 1 — i — r 0 5 10 Time, days Figure I. The response of Phycomyces blakesleeanus to thiamine. Thiamine at zero (curve 1), 0.2 (curve 2), and 1.0 (curve 3) m^M per 50 ml. From data of Dr. Frederick Kavanagh. Cocarboxylase itself has been reported to replace thiamine (142, 256), possibly entering the cell without dephosphorylation (231). Other metabolic functions of thiamine in fungi are suggested by the sparing effect of oxaloacetate (13, 82) and the effect of thiamine in promoting cytochrome synthesis by Ustilago sphaerogena (89). Free thiamine chloride hydrochloride has the structure: [~ NH2HC1 H3C / HI II GH 2 XCH3 + ci- Thiamine can be synthesized chemically by coupling the two moie- ties, 2-methyl-5-bromomethyl-6-aminopyridine and 4-methyl-5-/?-hy- droxyethylthiazole, commonly referred to as pyrimidine and thiazole, respectively. Some microorganisms can use one or the other moiety by itself to fill their thiamine requirement. The different types of fungi, and some examples, are shown in Table 1 ; the most common require- ment is for the pyrimidine moiety, i.e., most thiamine-deficient fungi can synthesize thiazole and couple the two moieties to make the com- plete molecule. Several studies have established that an organism able THIAMINE 331 Table 1. Response of Fungi to Thiamine and Its Moieties* Minimum Requirement Examples Intact thiamine Ceratostomella fimbriala, Chalaropsis thielavioides (220), Cortinarius glaucopus (167); Phytophthora spp. (188, 204), Tilletia tritici (46), Trichophyton discoides (222) Both moieties Boletus variegatus (167); Ceratostomella spp. (220), Marasmius perforans (145), Phycomyces spp. (130, 205, 210), Piricularia oryzae (284), Polyporus versicolor (220), Tilletia horrida (245), Ustilago spp. (15) Thiazole Collybia velutipes\ (1 56), Endomyces magnusii (165), Mucor raman- nianus (173, 174), Stereum frustulosum (182) Pyrimidine Allomyces kniepii (196), Blakeslea tiispora (130), Blastocladiella emersonii (7), Ceratostomella spp. (220), Cercosporella herpotri- choides (45), Collybia tuber osa, Coprinus lagopus (130), Fomes spp. (332), Hypholoma fascicular e (245), Marasmius fulvobul- billosus (145), Nocardia spp. (157), Polyporus spp. (332), Sclerotium spp. (205), Sphaerulina trifolii (210), Stereum murraii (208), Trichophyton tonsurans (273) * A more inclusive list is given by Robbins and V. Kavanagh (213). f Not definitely established. to live with either moiety alone does in fact make the other and syn- thesize thiamine (14, 21, 133, 220, 239); neither pyrimidine nor thiazole by itself has a vitamin function. A great deal of attention has been devoted to the specificity of the structures of thiazole and pyrimidine for fungi. In general, the require- ments are quite specific (213, 225, 239); most utilizable modifications, e.g., the substitution of an acetyl for a hydroxyl group or of an ethyl for a methyl group, are either minor or are very likely transformed biologically into the natural product. Allithiamine fully replaces thi- amine in the nutrition of Endoconidiophora funbriota, Mucor raman- nianus, and other fungi and can be converted into thiamine in the cell (141). It might be assumed a priori that biosynthesis of thiamine proceeds via a coupling of the two moieties. However, there are some difficul- ties with this simple concept as applied to Neurospora crassa, and Harris (97, 98), on the basis of indirect evidence from nutritional stud- ies, has proposed that thiamine biosynthesis proceeds in this fashion: Pyrimidine + unidentified thiazole precursor —> thiaminelike intermediate -» thiamine (1) Direct coupling of pyrimidine and thiazole may occur as a minor path- way. 332 VITAMIN REQUIREMENTS We may assume that synthesis of the pyrimidine moiety follows that of other pyrimidines (Chapter 8). The origin of thiazole in Neuro- spora crassa is apparently related to amino acid metabolism (50). Two fungi, Phy corny ces blakesleeanus and Sclerotium rolfsii, which can use the two moieties in place of thiamine, also destroy or convert to an inactive form part of the thiazole, whether added as thiamine or as free thiazole. Phytophthora cinnamomi, which requires intact thi- amine, does not destroy free or combined thiazole. Thus the destruc- tion of thiazole seems to be unrelated to the use of thiamine in the growth processes of the fungi (120). The effect of temperature on the enzyme which destroys thiazole may explain the observation that the efficiency of thiamine for growth increases with decreasing temperature (212). The compound "pyrithiamine" is inhibitory to those fungi, e.g., Phytophthora cinnamomi, which require intact thiamine, and does not inhibit those, e.g., Neurospora crassa, which synthesize thiamine; organisms requiring only one moiety of thiamine are intermediate in susceptibility (312, 314). However, the problem needs further study, in view of the fact that thiamineless mutants of Neurospora crassa do not respond according to expectations (279). Further, it is believed that "pyrithiamine" is a mixture of different compounds; neopyrithi- amine has been synthesized (303) and has the structure, of a pyridine analogue of thiamine, originally assigned to "pyrithiamine." Neo- pyrithiamine competitively inhibits thiamine metabolism in animals (162). "Pyrithiamine" can be used by pyrimidine-deficient fungi as a source of the pyrimidine ring (207). Figure 2. The response of Stachy- botrys sp. (SN 1) to biotin. From data of Marsh and Bollenbacher (161). 0.004 0.04 0.4 Biotin, rriMg per ml 4.0 BIOTIN 333 6. BIOTIN Biotin (vitamin H, coenzyme R) is required by many yeasts and fungi; all the major taxonomic groups of the fungi are represented by biotin-requiring types (5, 35, 67, 128, 161, 214, 219, 240, 241). The absolute requirement is usually less than 5 txg per liter (Figure 2). The role of biotin is not certain; in bacteria, it or a derivative of it is probably involved in several enzymatic systems (126). Evidence from studies of several fungi (111, 155, 190) implicates biotin in the synthe- sis of aspartic acid, inasmuch as aspartate partially replaces biotin in nutrition. It seems likely that the reaction affected by biotin is either the carboxylation of pyruvic acid to yield oxalacetic acid or the conver- sion of oxalacetate to aspartate (92): Pyruvic acid + C02 — » oxalacetic acid — > aspartic acid (2) A second probable function of biotin in fungi is in the synthesis of essential fatty acids; oleic acid partially replaces biotin for a Neuro- spora crassa mutant (111) and for OpJiiostoma pini (163). It must be said that evidence for the essentiality of fatty acids for fungi is incom- plete; the possibility is raised by (a) the stimulating effect of oleic acid on growth (12, 84, 332) and (b) the occurrence in Neurospora crassa of mutants requiring fatty acids (129). Less well-defined and probably indirect effects of biotin on fungi include inhibition of growth of a pantothenicless mutant (228), inhibi- tion of enzyme synthesis in one strain of Neurospora crassa (11), and change in the growth pattern of the dimorphic Sporotrichum schencki (160). Mitrula paludosa requires biotin only if ammonium nitrogen is used in the medium (67). In relation to biosynthesis, the structures of three compounds are of interest: O O II II /°\ /°\ HN NH HN NH II II HC CH HC CH II II CH3 GH2— (CH2)4— COOH H2G CH2— (CH2)4— COOH V Desthiobiotin Biotin 334 VITAMIN RF.QUIRK M K NTS O I! • /c\ HN NH I I HC CH I i H2C CH— (CH2)4— COOH " V i o Biotin sulfoxide Tatum (276) studied the nutrition of biotinless mutants of Penicil- lium chrysogenum. One mutant can use desthiobiotin as well as bi- otin; a second mutant accumulates desthiobiotin but cannot use it. Production of desthiobiotin in this strain is increased by provision of pimelic acid [HOOC— (CH2)5— COOH]. Equation 3 therefore summa- rizes the known steps in biosynthesis: Pimelic acid — » desthiobiotin — » biotin (3) Biotin-deficient fungi isolated from nature fall into two groups. The first, exemplified by Ceratostomella ips, Neurospora crassa, Blastomy- ces derma titidis, and yeast (49, 95, 143), can grow, at least to a degree, with desthiobiotin in place of biotin; these fungi are presumably blocked in a step prior to desthiobiotin synthesis. The second group, which includes Ceratostomella pint and Sordaria fimicola, cannot use desthiobiotin (135, 143) and is therefore blocked in the conversion of desthiobiotin to biotin. Those fungi known only to be unable to use pimelic acid in place of biotin (219) cannot yet be placed in either group. We have noted earlier the different responses of spores and pregrown cultures of Memnoniella echinata to desthiobiotin. Biotin sulfoxide is formed by Aspergillus niger in synthetic medium; since pimelic acid and desthiobiotin increase the yield, and since added biotin is quantitatively converted to the sulfoxide, it is concluded that A. niger synthesizes biotin as in Equation 3 and then transforms it to biotin sulfoxide (54, 317, 320, 321, 322). Biotin sulfoxide is as active as biotin for Neurospora crassa but not for yeast or the bacteria so far tested (321). Aspergillus niger utilizes azelaic acid also in biotin syn- thesis (318). Biocytin, e-N-biotinyl-L-lysine, a naturally occurring form of biotin, is as active as biotin for Neurospora crassa and for a biotin-requiring mutant of Penicillium chrysogenum (319). The oxygen analogue of PYRIDOX1NE 335 biotin, S - >N a n 'o 1 ! 1 1 . . . 1 32 Inositol, pig per 25 ml 64 Figure 5. The response of an inositolless mutant of Neuro- spora crassa to inositol. From Leonian and Lilly (136), cour- tesy of the West Virginia Uni- versity Agricultural Experiment Station. ashbyii (170), but neither phytin nor lipositol is utilizable by an inosi- tolless mutant of Neurospora crassa (9). As mentioned earlier, no general function of inositol is known in fungi. Inositol overcomes the inhibition caused by high levels of some other vitamins (238, 253), and has been stated to cause a conidial type of growth in Ophiostoma multiannulatum (72). The response of Scle- rotinia camelliae to inositol is conditioned by temperature (8). The insecticide y-hexachlorocyclohexane (gammexane) has been in- vestigated as a possible inhibitory analogue of inositol; it is not, how- ever, structurally an analogue of meso-inositol but of mz/co-inositol (125). Gammexane inhibition of Neurospora crassa appears to be based on two mechanisms: an interference with the metabolism of exog- enous inositol in an inositol-requiring mutant and a second mechanism which is independent of inositol metabolism (93). 11. PARA-AMINOBENZOIC ACID Only one fungus, Blastocladia ramosa, deficient for p-aminobenzoic acid (PABA) is known to occur naturally (39a); a yeast, Rliodotorula aurantiaca, is reported to show an absolute requirement (221), and several bacteria require the compound (323). Mutants of several fila- mentous fungi require PABA (17, 68, 83, 115, 195), so it is presumably a normal metabolite. The deficient mutant of Neurospora crassa grows maximally with PABA at about 5 /xg per liter (281); the requirement is lower at acid pH than at neutral pH, presumably by reason of a faster penetration of the undissociated acid into the cell (325). The require- ment of the N. crassa mutant is highly specific: of closely related com- pounds only 2-fluoro-p-aminobenzoic acid has appreciable activity (323). PANTOTHEMIC ACID 341 Although the folic acid molecule contains the structure of p-amino- benzoic acid, it seems doubtful that the major function of PABA is in the formation of folic acid; for example, folic acid does not replace PABA for Neurospora crassa (1, 333). Several metabolic roles of PABA have been suggested, especially a function in the introduction of 1 -car- bon intermediates into other compounds (254). The fact that methio- nine replaces PABA for a mutant of Neurospora crassa supports this possibility; the vitamin here probably functions in the methylation of homocysteine (269). Other functions no doubt exist (266), but they have not yet been shown in the fungi. The biosynthesis of PABA by Neurospora crassa is a special case of aromatic synthesis. A single gene mutant is deficient for PABA, trypto- phan, phenylalanine, and tyrosine; shikimic acid replaces all these re- quirements (280). The probable pathway of the synthesis is consid- ered elsewhere (Chapter 8). PABA in high concentrations is toxic to fungi (38), but in Neuro- spora crassa it is converted to a pigment of unknown structure and to N-acetyl-p-aminobenzoic acid (200). The acetylation of PABA occurs also in animal tissues (323). The metabolic importance of PABA was first recognized by Woods (307), who found that the bacteriostatic effect of sulfanilamide is com- petitively antagonized by PABA. The same antagonism has been ob- served in fungi (24, 48, 99), and we may assume that the same mech- anism prevails. Emerson and Cushing (58) isolated a sulfonamide-resistant mutant of Neurospora crassa and a second mutant requiring sulfonamides for growth. The requirement is absolute at 35 °C, partial at lower incuba- tion temperatures (56). Later studies by Zalokar (333) suggest that the sulfa-requiring strain is abnormally sensitive to the PABA which is formed in its own cells; the beneficial effect of sulfonamides is, in this view, based on its antagonism of the toxic effect of PABA. Some of the difficulties of this hypothesis are discussed by Mitchell (171); a rela- tionship to amino acid metabolism is apparently involved (57, 334). The present uncertain status of the sulfonamide requirement reflects the limitations of growth methods used by themselves for the solution of essentially biochemical problems. 12. PANTOTHENIC ACID Requirements for pantothenic acid, although fairly common in yeasts (27, 29, 134), are rare or unknown in the filamentous fungi. Penicillium digitatum is stimulated in the early stages of growth by 342 VITAMIN REQUIREMENTS pantothenate (61, 315), and it has been reported that Polyporus texa- )ius requires pantothenate (332). Pantothenicless mutants have been obtained frequently in the fungi (83, 121, 195, 278). Pantothenic acid functions in metabolism as a constituent of the larger molecule coenzyme A, an essential cof actor in a number of acyl transfer and related reactions (147, 184). The effect of pantothenate on carotenogenesis in Mucor hiemalis presumably results from an in- crease in the rate of acetyl transfer (90). Presumably all fungi except those needing exogenous pantothenate synthesize the vitamin; this is borne out by analysis of, for example, Boletus edulis (40), Neurospora crassa (297), and a few other fungi (181). The ultimate step in pantothenate synthesis is known to be the coupling of pantoic acid and /^-alanine. This coupling is carried out by growing cells, resting cells, and a cell-free preparation (acetone powder) of Neurospora crassa (296). Surprisingly, the enzyme is found also in a pantothenicless mutant, the cells of which do not under the usual conditions of still culture form pantothenate from the two pre- cursors (295). However, it has been found that the coupling reaction above does proceed in "pantothenicless" cells grown under aeration (297). Either we have here in still culture a gene-controlled suppres- sion of an enzyme, or the mutant strain is unable under limited aera- tion to provide the energy necessary for the formation of the peptide bond of pantothenic acid. The origin of the pantoic acid moiety in fungi is not known; in bac- teria, p-aminobenzoic acid is involved, and there is some evidence that a-ketoisovaleric acid is a precursor of pantoic acid (266). 13. PTEROYLGLUTAMIC ACID Pteroylglutamic acid (vitamin Be, Lactobacillus casei factor, folic acid, folacin) and its relatives are required by microorganisms and higher animals; probably the several active compounds are all con- verted to the same form (265). Venturia inaequalis is reported to be stimulated in its growth by pteroylglutamic acid (63); no fungi or actinomycetes are known to require the factor. A substance active for Lactobacillus casei is produced by several fungi, including species of Absidia, Aspergillus, Ghaetomium, Circi- nella, Fusarium, Monascus, Mucor, Neurospora, Pencillium, and Rhi- zopus (30, 164, 178). The identity of this product is uncertain. Citro- vorum factor, another member of this group of compounds, is formed by Streptomyces griseus (59). Rhizopterin, N-formylpteroic acid, is formed by Rhizopus nigricans THE VITAMINS B12 343 (306) and replaces pteroylglutamic acid for strains of Streptococcus lac- tis (265). Presumably it and the factor active for L. casei are both utilized by the fungi producing them in the same biological reactions for which the coenzyme form of pteroylglutamic acid is required in bacteria and animals. 14. THE VITAMINS B12 The vitamins B12 comprise a family of compounds, the chemical and biological relationships of which are not fully understood. Vita- min B12 itself, or cyanocobalamin, was the first discovered, and its structure is now known (22, 110); other cobalamins, e.g., hydroxoco- balamin, can be converted to it by cyanide treatment. A second group of compounds differs from the cobalamins in the nucleotide moiety; these are active for some microorganisms only (25, 62). Vitamin BI2 is believed to function in the cell in the synthesis of 1 -carbon fragments, especially the labile methyl group (25). Vitamin B12 and related compounds are required by several bacteria and algal flagellates (62), not, so far as is now known, by actinomycetes or fungi. Higher animals require the vitamin, green plants do not. The synthesis of cobalamins occurs in certain of the true bacteria and especially in species of Streptomyces and Nocardia, of the Actino- mycetales (32, 43, 53, 94, 1 16, 203, 209, 232, 267, 305). Commercial sup- plies of the vitamin can in fact be obtained as byproducts of antibiotic production by Streptomyces spp. (324). Cobalt added to the culture medium increases the yield of cyanocobalamin from Streptomyces griseus (104). In addition to cyanocobalamin, species of Streptomyces form hy- droxocobalamin (vitamin B12a, B12b), in which the cyanide of cyanoco- balamin is replaced by hydroxyl, and vitamin B12c, containing a nitrite radical in place of cyanide; all three forms have been isolated from one species, 5. griseus (43). Synthesis of vitamin B12 in the filamentous fungi has not been demon- strated conclusively. Substances active for Lactobacillus leichmanii are formed by Ashbya gossypii (258) and Aspergillus niger (179). How- ever, the L. leichmanii assay is not specific enough to support a final identification (62). It has been mentioned earlier that inhibition studies point to 1,2- dimethyl-4,5-diaminobenzene as a precursor of riboflavin. These in- vestigations (310, 311) implicate this compound also as a precursor of the cobalamins; the structure is known to be part of the larger co- balamin molecule. Other evidence bears out the suggestion that the 344 VITAMIN REQUIREMENTS biosyntheses of riboflavin and of cobalamins are related (62, 266). Streptomyces olivaceus incorporates labeled 5,6-dimethylbenzimidazole without dilution into the cobalamin molecule (299); more recent and still preliminary evidence is in favor of ^-aminolevulinic acid as a precursor (250). 15. VITAMINS K Vitamins Kx and K2 are required by higher animals. Both are de- rivatives of 2-methyl-l,4-napthoquinone, and it appears that any con- pound containing this nucleus is active in animals (3). Many bacteria produce vitamin K2 (2), and the phthiocol of Myco- bacterium tuberculosis — chemically 2-methyl-3-hydroxy-l,4-naphtho- quinone — has K activity (3). The six fungi that have been tested (2) do not have detectable vitamin K activity. Other 1,4-naphthoquinones are known to be produced by fungi (Chapter 6), but none of these is a 2-methyl derivative. No requirement for the K vitamins is known in the fungi; Almquist (3) reviews the conflicting evidence for a K requirement in Mycobac- terium paratuberculosis (313). Some compounds structurally related to vitamin K, specifically 2- chloro-l,4-naphthoquinone (246) and 2,3-dichloro-l,4-benzoquinone, are fungitoxic (Chapter 14). Menadione (2-methyl- 1,4-naphthoqui- none) competitively inhibits the action of both of these compounds in certain biological systems (246, 309). However, the fungitoxicity and the reversal by menadione are not adequate evidence for a role of the K vitamins in fungal metabolism; one may equally well consider the antagonists as foreign toxicants competing for the same site of action (309). 16. OTHER GROWTH FACTORS The only well-characterized growth factor for fungi that has not so far been mentioned is coprogen, required by species of Pilobolus and first isolated from manure (107). Bioassays indicate that this factor is manufactured by some other fungi and several actinomycetes (108), and crystalline coprogen has been isolated from Penicillium sp. (193). Coprogen is related chemically and biologically to two other ma- terials, ferrichrome and the Arthrobacter terregens factor. Ferri- chrome and ferrichrome A are nitrogenous iron-containing pigments of Ustilago sphaerogena (76, 176); the effect of zinc on synthesis sug- gests that ferrichrome is a precursor of cytochrome c (177). The OCCURRENCE OF VITAMIN DEFICIENCIES 345 growth factor for Arthrobacter terregens, although not identical with either coprogen or ferrichrome, is evidently related to them; thus both ferrichrome and coprogen are active for A. terregens (33, 148, 149) and Pilobohis spp. (177a). Sterols are produced by many fungi (Chapter 6); so far only Laby- rinthula vitellina — of uncertain taxonomic position but possibly a myxomycete — has been shown to have a steroid requirement (293, 294). It has been claimed (4) that anaerobic growth of Saccharomyces cerevisiae in a synthetic medium is dependent on ergosterol or other sterols. Many still unknown factors have been postulated for particular fungi. Mycorrhizal fungi are stimulated by materials from plant roots and forest litter, and the effect cannot be traced to known vitamins or amino acids (166, 167, 168). The myxomycetes apparently require for their growth one or more bacterial proteins (112, 272), not vitamins as we have here used the term. As mentioned earlier, growth-stimulating effects of crude natural materials do not necessarily implicate a vita- min. Other vitamins — ascorbic acid and vitamins A, D, and E — required by animals do not appear to play any essential role in the fungi (28, 86); ascorbic acid may, however, affect growth through its effect on the oxidation-reduction potential. 17. THE OCCURRENCE OF VITAMIN DEFICIENCIES IN THE FUNGI It will be clear from the preceding discussion of individual vitamins that some deficiencies occur frequently, others more rarely. The vita- min most often required by fungi from nature is thiamine; usually the deficiency is for the pyrimidine moiety, and we have noted only a few instances of a deficiency for thiazole (Table 1). Biotin is very often required, and deficiencies in pyridoxine or its derivatives are fre- quently encountered. Pantothenic acid and nicotinic acid require- ments are rare in nature, and we have at present only single reports of requirements for riboflavin and p-aminobenzoic acid among filamen- tous fungi. This lop-sided distribution of vitamin requirements demands an attempt at an explanation. Assuming that spontaneous mutations in nature are like those induced by mutagens, forms deficient for ribo- flavin of p-aminobenzoic acid must arise from time to time. Their failure to survive — if it is real and not an artifact of screening methods — may result from either or both of two conditions, viz.: 3 I (', VITAMIN RF.QUIR I. Ml NTS 1. The required vitamin is not available in the environment. 2. The derangement of metabolism associated with the deficiency is so great that even with the missing factor available in the environment the mutant is at a selective disadvantage and cannot propagate itself. The second of these hypotheses is the more attractive, inasmuch as even the plant pathogens, living in close contact with higher plant cells, do not seem to develop strains deficient for certain vitamins. 18. VITAMINS, TAXONOMY, AND ECOLOGY Comparative physiology often affords clues to taxonomic and phylo- genetic relationships. Vitamin deficiencies do not, so far as can be determined, offer such clues. From the frequency of induced muta- tions, one would expect that vitamin deficiencies occur at random within species, genera, and higher categories; this appears to be true. Marked differences between the vitamin demands of strains of a single species are often encountered, e.g., Trichophyton spp. and other animal pathogens (5, 80, 81, 152), Lenzites trabea (HO), Boletus granulatus (169), and Glomerella cingulata (270). In culture, both Fusarium avenaceum (214) and Ustilago major (15) give rise to clones which differ in requirements from the parent strain. Within a genus, again, species differ widely in vitamin demands; examples include the genus Ophiostoma and its asexual form Cera- tostomella (66, 67, 215, 217, 224), Trichophyton (31, 80, 222, 223), Ustilago (245), Candida (241), Boletus (169), Marasmius (145), and Absidia (213). In the light of this intra- and interspecific variation, it is difficult to imagine that vitamin needs per se can separate taxons or can yield valid evidence of phylogeny. The theory of Lwoff (150) and Knight (122), that metabolic defi- ciencies have arisen in the evolution of microorganisms by loss of functions originally present, has been generally accepted. If we grant, in view of the argument just made, that this loss of function has not followed phylogenetic or taxonomic divisions, we may ask if there is any relation of vitamin needs to ecology. First, it appears that some genera as isolated from nature rarely have vitamin deficiencies. Only a tentative listing can be made at present, in view of the methodological difficulty in establishing the absence of a requirement: Aspergillus, Chaetomium, Fusarium, Mucor, Penicil- lium, Rhizopus, Streptomyces, and ZygorhyncJius (213). Exceptions occur in most of these genera, i.e., some isolates are deficient or par- BIBLIOGRAPHY 347 tially deficient for a given vitamin (61, 195, 214), and deficient mu- tants may be obtained in both Aspergillus and Penicillium (17, 115, 195). These genera are all vigorous saprophytes, widespread in na- ture, and frequently isolated from soil. They seem to form, therefore, an ecological type independent of taxonomic boundaries; at present, however, it is fruitless to speculate on the biochemical, genetic, or environmental determinants involved. Soil-inhabiting fungi are not, however, as a group independent of vitamins, many soil fungi requiring them for maximal growth (6). Soil, especially soil of high organic matter, contains several vitamins (234, 235), probably formed by higher plants (298) or algae (78); it is not surprising, therefore, to find deficient soil fungi. Certain major ecological groups show no general pattern of vitamin dependence; these include animal pathogens (5, 52, 223), plant patho- gens (45, 46, 55, 215, 217, 244, 336), and the aquatic phycomycetes as a group (36). Among the insect pathogens, however, limited data suggest a tendency for species of wide host range, e.g., Isaria farinosa, to be independent of growth factors, and more specialized pathogens, e.g., Hirsutella spp., to require vitamins and organic nitrogen (154). The nearest approach to a correlation of a taxonomic group with a vitamin requirement is in the Agaricales; almost all the fungi in this group that have been investigated require thiamine, replaceable for most by the pyrimidine moiety (71, 119, 145, 146, 166, 183, 213, 332). 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This com- plexity is particularly apparent in ecological studies of the higher fungi (115). Although some tentative conclusions on fungi in general have been proposed, it should be emphasized that any organism must be studied individually; there is no one "formula" for sporulation. It is fair to say that most of our knowledge of reproduction in the fungi comes from essentially qualitative data. Sporulation occurs under some conditions, but not under others, and data may be re- ported in simple "yes" or "no" categories. No objection can be made to this procedure in general; however, it is becoming clear that some problems require quantitative data on the intensity of sporulation. This is particularly true of nutritional studies, in which the response is likely to be quantitative rather than all-or-none. It has not been possible, with present information, to consider the physiology of resting structures, e.g., chlamydospores and sclerotia, separately or in detail. Chlamydospore formation is probably a re- sponse to unfavorable environmental conditions (214, 215, 231, 266). 356 LIGHT 357 Sclerotium development is, of course, often a prelude to sexual repro- duction and may be expected to be influenced by the same factors, allowing for a higher nutritional level entailed by the larger mass (1, 249, 252, 290). The "sclerotia" of myxomycetes are purely resting structures and appear to develop primarily as a response to desicca- tion (110, 154, 166). The geotropism of large sporophores is an established fact of ob- servation (27, 49, 50, 278), but its physiological correlates have not yet been uncovered. Finally, in the list of omissions, purely genetic problems are not here considered. These are, of course, most important to a thorough under- standing of reproduction, but they lie outside the field of physiology. 1. LIGHT The reactions of fungi to visible and ultraviolet light are of three principal types: inductive effects, inhibition, and trophic responses. Inductive effects include both absolute light requirements for initiation or maturation of reproductive structures and quantitative responses, i.e., an increase in the number of reproductive structures upon illumi- nation. Light is absolutely required for the formation of various types of reproductive organs in certain species. As examples we may cite the requirement of light for the formation of sporophores in the Agari- cales (226, 253), of perithecia (264), of apothecia (248, 283, 319), of basidiocarps in the Gasteromycetes (298), of pycnidia (105, 183, 185), of sporangia in the Mucorales (221), of conidia in the Mucorales (18) and Fungi Imperfecti (97, 159), and of Myxomycete sporangia (117). Smith (263) reviews still other examples. As might be expected, the effect of light is not always exerted at the same point in these diverse fungi: to take two extremes, light is re- quired for the first steps in sexual reproduction in Pyronema con- fluens (248) and for all stages in Polyporus arcularius (110a), but only for pileus development in some Agaricales (27, 106). Whether all developmental stages of the Coprinus lagopus sporophore require light is in dispute (196, 294). Light of the visible range often has the effect of increasing the num- ber of reproductive structures formed (82, 89, 120, 124, 130, 261). More striking effects of this type are induced by ultraviolet light in certain ascomycetes and Fungi Imperfecti; both sexual and asexual structures are affected (11, 66, 160, 198, 232, 262, 271, 272, 273, 271). Ultraviolet effects are exerted in the cell, not in the medium (74, 271), 358 REPRODUCTION and cannot be ascribed to temperature effects (262). The effective wavelength is still in doubt (88, 232), but it is probably longer than the well-known "germicidal" wavelength of 254 m^. Ultraviolet light, of course, in high doses represses both sporulation and growth. Most of the work on induction and stimulation by visible light has been at best semi-quantitative. The available data on the action spectrum indicate fairly consistently that the effective wavelengths are in the blue region of the spectrum (37, 83, 117, 198, 248, 250a, 263, 277, 319). Morphogenetic action on Coprinus lagopus is maximal at 440-460 m^ (255), close to the peak of the action spectrum of photo- tropism. The amount of light needed for inductive response is similarly un- certain; it is much less than normal daylight exposure (105, 168). The maximal effect of light on fruiting in Coprinus lagopus is exerted by exposure for 5 seconds at 0.1 foot-candles intensity (196). So much attention has been focused on the positive effects of light that the possibly more important inhibitory action may be overlooked. Induction of conidiation in Choanephora cucubitarum requires a dark as well as a light period (18), and light of wavelength below 580 m/A appears to be inhibitory (80). Dark periods are required for maximum production of ascospores by Diaporthe phaseolorum (286) and of coni- diophores by Alternaria brassicae (312). For the Myxomycete Didym- ium eunigripes, Leith (189, 190) presents evidence for both induc- tion and inhibition of sporangial formation by different wavelengths. Finally, the type of zonation in which spore production is greater dur- ing dark periods (see below) is most easily interpreted as resulting from a light inhibition of sporulation. The diurnal cycle of Pseudoperonospora humuli and other downy mildew fungi in nature involves both light inhibition and light stimu- lation, although effects on the host metabolism cannot be excluded (315). Other diurnal cycles in nature (314, 316) and the frequently observed nocturnal production of conidia (161, 162) may also result from light inhibition, although the dominant factor in noctural forma- tion of spores by Sclerospora spp. has been thought to be water (306, 307). Zonation — the formation of fruiting structures in concentric rings on an agar surface or on the host plant — is a common phenomenon and may best be considered at this point, since light is usually the primary environmental factor. The occurrence of light-induced zona- tion is reviewed by Hall (214) and by Hafiz (123). A simple explana- tion is rendered difficult by the observation that two extreme types of response to an alternation of light and dark periods are known, viz.: LIGHT 359 Type 1. The fungus speculates well in darkness, much less or not at all during the light period. Examples are Cephalothecium roseum (145) and Sclerotinia fructicola (124). Type 2. The organism, e.g., Fusarium spp. (29, 124, 320), sporulates poorly in the dark and forms a zone of spores after even a brief ex- posure to light. It seems necessary to explain Type 1 reactions as the result of light inhibition of sporulation. Type 2 responses are more difficult; we may propose, however, that light inhibits mycelial growth — reports of such inhibition are reviewed in Chapter 13 — and that stimulation to sporulation results from the check upon vegetative growth. The light effect in these fungi is exerted only upon actively growing hyphal tips (29, 45, 264). Temperature alternations also induce the formation of zones of reproductive structures (94, 123, 131, 250a, 320); it appears, especially from the data of Ellis (94) and Hafiz (123), that the immediate stimu- lus for sporulation is a check in growth. Zonation of reproductive structures in some fungi is dependent neither upon light nor upon temperature fluctuations (40, 75, 146, 206); presumably nutritional factors are involved here, but they are not understood. The pH of the medium has an effect (212, 240). Any unitary hypothesis to explain the action of light on sporulation will have to take account of several facts, viz.: 1. The indifference of many fungi to light stimuli. 2. The high specificity of the response. Thus, the formation of conidia of Choanephora encurbitarum is governed by light, but the development of the sporangia is independent of light (18), and the formation of zygospores is inhibited by light at low temperature (20). 3. The fact that light may either stimulate or inhibit sporulation. 4. The partial replacement of the light effect by high temperature (29, 37, 185, 212), by periodic exposure to air (206), by treatment with oxidizing agents (82), or by mechanical agitation (37, 275). 5. The observation of Houston and Oswald (159) that Helmintho- sporium gramineum forms spores in the dark if growing on host tissue but requires light for sporulation on agar media. 6. The extreme sensitivity of the response (29, 168), which suggests that an enzymatic mechanism is triggered by the stimulus. 7. The frequent efficacy of ultraviolet radiation, known to be in- jurious to all organisms in sufficient dose. Sporulation of Alternaria solani is induced by either ultraviolet or mechanical damage (74). 360 REPRODUCTION A unified theory should take as its principal postulate that light is inhibitory to one or more regulatory systems which in the normal culture prevent or retard reproduction and favor continued vegetative growth. That is to say, there is in truth no such thing as stimulation by light in the induction of reproductive activity; instead, growth is checked and the chain of events so initiated leads to reproduction il other factors are not limiting. Alternative explanations are possible, and several have held specifically that stimuli for reproduction do not check growth; nevertheless, the weight of the evidence favors the hy- pothesis presented. Confirmation must wait upon studies which deter- mine accurately and quantitatively the effects of light on both repro- duction and growth simultaneously. Phototropic responses are common in fungi; examples include the orientation of basidiomycete sporophores (37, 278, 298) and of asci or ascocarps (8, 41, 53). Gametic phototaxis has been reported (180). The most dramatic and the most thoroughly investigated phototropic reaction is that of the sporangiophores of Phycomyces spp. and Pilobo- his spp., observed also in others of the Mucorales (96). Consideration of phototropism in Phycomyces spp. should begin with the observation of Blaauw (32), confirmed by Castle (68), that a sporangiophore illuminated equally from two sides exhibits a measur- able temporary acceleration in vertical growth, the "light growth reac- tion." A more recent study of this phenomenon (86) brings out sev- eral important aspects: (1) the response probably involves no net increase in total growth; (2) the effect of the light stimulus depends in a predictable fashion on the level of illumination prior to the appli- cation of the stimulus; (3) there is a latent period of about 2.5 seconds; and (4) the magnitude of the increased growth is proportional to the strength of the stimulus. This last observation is important in show- ing that the light growth reaction is not an all-or-none, or threshold, reaction. Granted that illumination can increase growth, the fact of photo- tropism— unequal growth — requires some mechanism whereby the sporangiophore wall distal to the light grows more rapidly than the proximal wall. Castle (71, 72) argues that the size, shape, and refrac- tive index of the sporangiophore combine in the "lens effect" to con- centrate unilateral illumination in such a way that the distal region of the protoplasm receives more light than the proximal. This is con- sistent with the observation that sporangiophores immersed in paraf- fin oil become negatively phototropic (14, 48), with the effect of ultra- violet light (66a), and with the demonstration by Banbury (14) that LIGHT 3G1 illumination of only one edge of the sporangiopliore causes localized extension of that side and consequent curvature. This theory, and others of the same general type (32), fail, as Dcl- bruck and Reichardt (86) point out, to account for the extreme local- ization of the receptor which is implied by the unidirectionality of the phototropic curvature; if the entire growing zone were affected, uni- lateral illumination and the known spiral habit of growth would com- bine to generate a helical shape of the sporangiopliore. The action spectrum of phototropism in Phy corny ces blakesleeanus (Figure 1) and Pilobolus kleinii has a maximum at about 430-500 m^ (54, 55, 69, 99). This has often been taken as evidence for a carotenoid receptor pigment, carotenoids being present in these fungi and absorb- ing maximally in the same region of the spectrum (297). However, as Galston (107) points out, riboflavin or a derivative of it could with equal probability be the receptor. Carotenogenesis in Phycomyces sp. can be suppressed, at least below detectable levels, without impairment of the phototropic response (241). Nor are there any solid data on chemical mediators between the light absorbing system and the growth (or wall extension) response. In higher plants it has been suggested that auxin (indoleacetic acid) is involved, but its role is not yet entirely certain (107). Fungi do produce auxin (Chapter 8), and it is found in fact in the sporangio- phore of Phycomyces nitens (151), but there is at present no reason to accept — and there is considerable evidence against — an auxin theory of phototropism in the fungi (14). Phototropism in Pilobus kleinii has been investigated especially by Buller (53), who reviews the earlier literature. In this species a pig- Figure 1. The action spec- trum of phototropism in Phycomyces blakesleeanus. Redrawn from Castle (69), hy permission of The Rocke- feller Institute for Medical Research. 400 500 Wavelength, m/n 600 362 REPRODUCTION men ted "ocellus" at the base of the subsporangium is believed to be the photosensitive area, but it should be noted that photosensitivity is apparent before the formation of the subsporangium (222) and that other phototropic forms do not have an "ocellus." Other light responses of Pilobolus spp. may be briefly noted. Light induces the formation of the trophocyst, or sporangial primordium, of some isolates; Page (221) raises the possibility of a flavin receptor in this reaction. Second, alternations of light and dark, or of tempera- ture, set up a periodic rhythm in the development of sporangia (175, 196). This rhythm, once established, persists for two or three 24-hour cycles after the removal of the stimulus (254, 291). Finally, the forcible discharge of the sporangium is hastened by light (55). Light has other effects on the reproductive apparatus, notably a positive effect on the size and septation of macrospores of Fusarium spp. (130, 264). Pigment production is also enhanced by light (189, 264), but attempts to connect this phenomenon with induction of sporangia in Didymium eunigripes were unsuccessful (189). Exposure to light, unless appropriate filters and cooling devices are used, means an increase in temperature. Thus, the apparent attrac- tion of myxomycete pseudoplasmodia to light is a temperature effect; the plasmodium moves toward a source of heat along a temperature gradient as small as 0.05°C per centimeter (35). Similarly, a negative phototropic response of Phycomyces sp. to intense light was shown by Castle (70) to be a heat effect. Many reports on light effects have not included quantitative determinations of the ambient temperature; possibly some of the apparent complexities may disappear when such data become available. 2. TEMPERATURE Only two generalizations can be formulated with respect to tempera- ture and sporulation. First, the temperature range permitting repro- duction is usually narrower than that permitting growth. Second, each spore form has its own temperature optimum, which may or may not coincide with that of vegetative growth or of other spore forms of the same species. The relative narrowness of the temperature limits of reproduction is virtually universal in the fungi. Examples include both sexual and asexual spores in the phycomycetes (174) and ascomycetes (148, 194, 281), sporophores of basidiomycetes (5, 182), and spores of the Fungi Imperfecti (2, 204, 287). Usually the differences in range are not TEMPERATURE 863 great; Phoma apiicola is an extreme with mycelial growth on agar at 5-28°C but pycnidium formation only at 13-26° (25). The optimum temperature for sporulation of a given species may be either close to the growth optimum, as in Piricularia oryzae (149) and Rhizopus spp. (303), or rather different, lower in Fusarium con- glutinans (287) and higher in Sphaerotheca pannosa (194). Different spore forms of a given species often differ in their tempera- ture requirements. Thus, the zygotes of Sporodinia grandis and of Saprolegnia mixta form at lower temperatures than do the correspond- ing asexual sporangia (172, 173). Perithecia of Aspergillus spp. (171, 256) and of Ceratostomella (Ophiostoma) fimbriata (17) are produced in abundance at relatively high temperature and are replaced by co- nidia at lower. Pseudopeziza ribis forms conidia at 20-24°C, micro- conidia at lower temperatures (33). It must be borne in mind that most of these data are only semi- quantitative, and that the same uncertainties are involved in the defi- nition of an optimum for reproduction as in the definition of a growth optimum (Chapter 1). One such complication is illustrated by the findings of Robbins and Schmitt (245) on Phycomyces blakesleeanus: the failure of progametes to form at 26 °C is the result of the accumula- tion of acid in the medium before the two strains meet. Presumably, progametes would form at 26° if the pH were controlled. One may speculate that sexual stages adapted to survive a cold win- ter are formed prefentially at low temperature, and that fungi which must live through a hot and dry season will tend to form their dormant spores at higher temperature. However, there is as yet no ecological or physiological data bearing on this possibility, and the failure of sexual reproduction in Neurospora crassa at high temperature (305) speaks against the idea. The asci of Erysiphe graminis mature some- what more rapidly if chilled briefly, but they do mature without cold treatment (114). The apothecia of Claviceps purpurea appear, from limited data, to develop only if the sclerotia bearing them are exposed to low temperature (170). Zygospore formation in Thamnidium spp. is also best at low temperature (150). Temperature-induced zonation has been considered elsewhere with the conclusion that the positive effect of high or low tem- perature on spore formation is probably effected by retardation of mycelial growth. Morphological effects of temperature are easily observed; as exam- ples we may cite the finding that the conidia of Cercospora sesami are larger at an intermediate temperature than at higher or lower tem- 364 REPRODUCTION peratures (79), and the effect of temperature on the morphology of the conidial fructification in Aspergillus janas (239). 3. WATER. RELATIONS Satisfactorily quantitative determinations of the moisture require- ments of sporulation are rare. Indications are, however, that specific fungi have specific requirements. At one extreme, Colletotrichum lindemuthianum and other anthracnose fungi sporulate much more profusely at high than at low humidity (318). Conidiation in Sclero- spora spp. (306, 307) and in Peronospora destructor (317) is also fa- vored by high humidity. The formation of sexual structures in asco- mycetes and basidiomycetes (10, 111, 283) and sporangium formation in Sporodinia grandis (147) are believed to proceed more rapidly at high than at low moisture levels. Somewhat lower humidities are tolerated, but are not necessarily re- quired, by some of the rust fungi (318), by Botrytis cinerea (140), and by Bremia lactucae (228). Species of Alternaria in culture sporulate rather better if the culture is allowed to dry out (90, 233). Finally, the powdery mildew fungi appear to be relatively independent of hu- midity or possibly favored in both sexual and asexual reproduction by lower atmospheric moisture (47, 125, 318). Asci of Erysiphe graminis are reported to form only if the perithecium is alternately wetted and dried (76), but it seems doubtful that this is a general requirement among the powdery mildew fungi. The weight of evidence favors the generalization that reproductive processes, in most fungi at least, are favored by moderate rather than high atmospheric humidity. However, one may see from even a super- ficial glance at the original work that accurate control of the environ- ment and quantitative determination of the intensity of sporulation have not usually been achieved. Different spore forms of a single species occasionally show different humidity responses; examples include the two asexual spore forms of Choanephora cucurbitarum (19) and the zygote and sporangium of Sporodinia grandis (13, 172, 247). Miscellaneous reported effects of atmospheric humidity include negative hydrotropism of sporangiophores (172, 300), transitory ac- celeration of the growth of sporangiophores of Phycomyces nitens at high humidity (300), and changes in the size or morphology of co- nidia (98, 119) and sporophores (39). Spore discharge in fungi has been studied in some detail; the sum- OXYGEN, CARBON DIOXIDE, ET AL. 365 mary of Ingokl (162) has been supplemented more recently by investi- gations of spore discharge in rust fungi (220) and Nigrospora sphaerica (302). With the possible exception of "drop-excretion" discharge in basidiomycetes (50, 51, 52, 229), all these discharge mechanisms rely in some way on either substrate or atmospheric water. We infer that osmotic forces are at work. In immature asci (162) and in the imma- ture active layer of Sphaerobolus spp. (299) glycogen is abundant; its disappearance at maturity suggests that the uptake of water is medi- ated by enzymatic conversion of the osmotically inactive polysaccharide to osmotically active sugars. Such a mechanism, if suitably quantified, might be extended to cover all discharge mechanisms which depend on increasing turgor of a cell or cell layer. The elevation of the receptaculum in the Phallales is accompanied by disappearance of glycogen, but in this instance the increase in si/.e may be true growth (58, 95). Elongation of the Agaricus campestris sporophore has been shown to be a growth process rather than simple water uptake (36). 4. OXYGEN, CARBON DIOXIDE, AND VOLATILE SUBSTANCES It is a common observation that sporulation is reduced in closed containers (82, 126, 185) and that submerged mycelium fails to develop spores (84, 201, 321). Although this is usually ascribed to an oxygen deficit, it seems more likely that the accumulation of carbon dioxide is the critical factor. Pyronema conftuens, for example, does not fruit in a closed tube, but if alkali is present to absorb carbon dioxide, peri- thecia develop (248). Carbon dioxide accumulation inhibits the asexual reproduction of Choanephora cucurbitarum (19) and sporo- phore development in Agaricus campestris (181) and Collybia vehitipes (227). In large culture containers the sporulation of Piricularia oryzae is dependent upon aeration of the vessel; Henry and Anderson (149) suggest that metabolically produced ammonia is the inhibitory factor. An unidentified volatile substance, probably of microbial origin, in- hibits sporophore development in Psalliota campestris (199). The best available data on the oxygen requirement for sporulation are those of Denny (87). Any reduction of oxygen below atmospheric pressure delays the appearance of perithecia. Further, visually esti- mated mycelial growth appeared to be normal at oxygen pressures too low for reproduction. The same phenomenon — a higher oxygen re- quirement for fruiting than for growth — is reported by Ternetz (283). 366 REPRODUCTION Plant pathogenic fungi usually, and with an obvious ecological ad- vantage, form their reproductive structures on the surface of the host. It is reasonable to speculate that either the higher oxygen requirement or the lower carbon dioxide tolerance of sporulation is the mechanism that ensures extramatrical sporulation; it is significant that active anaerobic respiratory mechanisms are common in the lower phycomy- cetes and the genus Fusarium, forms in which resting spores or conidia often occur within host tissues (93). Several Penicillium spp. sporulate in shaken cultures, but the de- terminants are not known (102, 122). In our laboratory we have ob- served that Fusarium solani forms abundant macrospores in shake culture, particularly in certain media. Conidia of Streptomyces griseus appear in aerated cultures at the time of autolysis (67). In a very brief note, Richards (242) reported that ozone appears to stimulate sporulation in Alternaria spp.; Mycosphaerella citrullina forms more pyenidia if exposed to ozone, but the spores are non-viable. The striking effect of carbon dioxide and bicarbonate on sporangial development in Blastoclaclia and Blastocladiella spp. is considered later in this chapter. 5. ACIDITY Although pH has definite effects on reproduction, no unitary hy- pothesis or generalization is possible; as mentioned elsewhere (Chapter 1), pH effects are exerted in a variety of ways. Furthermore, the com- mon failure to determine the final pH casts doubt on much of the available data. In general, it appears that the pH range for sporula- tion is somewhat narrower than that for growth (5, 20, 23, 192, 193, 276). Within the physiological range, an acid reaction is often less favorable than a neutral or mildly alkaline reaction (170, 245, 276), but exceptions to this rule have been reported (5, 156). Lockwood (192) showed that in several fungi perithecia form over a wide range of pH values, but asci mature only at neutral or slightly alkaline reaction. This finding is of particular importance because often the only data submitted in pH studies concern macroscopically visible fruit bodies. In Mycosphaerella pinocles pyenidia, pseudothecia, and chlamydo- spores are reported to have different pH optima (266), but other data on the same species are not easily reconciled with this claim (23). The pH range for sporangium formation by Physarum polycephalum is narrowed by high temperature cultivation (116). CARBON NUTRITION 367 6. CARBON NUTRITION The classical work on the influence of nutrition, and for that matter of all environmental factors, on reproduction in fungi is that of Klebs (172, 173, 174). In relation to nutrition, his major points may be formulated briefly: 1. Sexual and asexual reproduction are most likely to occur when a vigorous mycelium exhausts its nutrients or is transferred to a medium low in nutrients. 2. Formation of fruiting structures therefore occurs over a narrower range of nutrient concentrations than does vegetative growth. 3. Fruiting structures of different fungi, or different spore forms of the same fungus, have specific requirements for nutrient concentration. In Saprolegnia mixta and Sporodinia grandis the sexual spore stage requires a higher nutrient level than the asexual stages. With minor modifications — most importantly that the concentration of the carbon source is usually more decisive than that of other nu- trients— these generalizations still stand. It would be tedious indeed to list all the fungi that conform to Klebs' predictions; some of the more significant modern results with pure cultures deserve, however, some attention. Suppression, relative or absolute, of reproductive stages at higher concentrations of carbohydrate — concentrations that permit heavy veg- etative growth — has been found in studies of ascomycetes (59, 137, 141), phycomycetes (267), and imperfect fungi (44, 45, 82). More ex- tensive reviews of the literature on this topic are available (140, 185). The same phenomenon can be demonstrated more neatly by transfer experiments, in which a mycelium growing in a concentrated medium is transferred to or allowed to grow into a more dilute medium. Such experiments almost always confirm the first principle of Klebs (6, 81, 82, 142, 185, 219, 296). In general, transfer to water is less successful than transfer to a dilute medium. Finally, it follows from the first principle that spore formation should occur at the point in the culture cycle at which the carbon source is substantially exhausted; studies on myxomycetes (62, 238) demonstrate this principle clearly. The same response has been shown in Melanconium fuJigineum in a different manner: heavy inoculation of an agar surface accelerates sporulation because of the more rapid utilization of nutrients (285). 368 REPRODUCTION It has often, of course, been found in studies of particular fungi that Klebs' first principle does not hold or is overridden by other factors (78, 184, 185); this is best documented in studies on fleshy basidiomycetes (185, 226), in which the relatively greater mass of the fruit body might be expected a priori to change the usual relation. The mechanism by which high nutrient concentrations inhibit spor- ulation is not definitely established. One cause which must be opera- tive in some cultures is simply the accumulation of toxic metabolites; effects of culture age and of concentration have been attributed to this phenomenon (104, 251). It seems, however, unlikely that such accumulation can be a general explanation of the concentration ef- fect. For the present, we may posit, no doubt oversimplifying the picture, that a limitation of growth per se is the stimulus to reproduc- tion, and that the effect of high nutrient levels is merely to postpone the onset of starvation. The second principle of Klebs is a corollary of the first. Granted that some minimum amount of growth must precede fruiting, and that too high concentrations delay or inhibit fruiting, then the range of concentration suitable for reproduction must necessarily be nar- rower than that suitable for vegetative growth. The third principle, of specificity of requirements, is obvious insofar as different species are concerned. Even within a species, however, different spore forms have different requirements; examples include the sexual and asexual stages of Sporodinia grandis (13, 96) and the alternative asexual stages of Blakeslea trispora (112). The concentration of the carbon source affects the type of asexual spore formed in Cytosporina ludibunda and related fungi (218) and the septation of macrospores of Fusarinm sp. (157). The type of carbon source used strongly influences the formation of reproductive structures, both sexual and asexual. In general, it is found that oligosaccharides and polysaccharides support more fruiting than do the simple hexoses; cellulose, a very slowly available com- pound for most fungi, is the best carbon source for sporulation of some forms (85, 319). The mechanism of specific carbon source effects has been thoroughly investigated by Hawker and her collaborators (133, 137, 141) and by Bretzloff (42). There seems little doubt that the primary mechanism is actually a concentration effect, i.e., it exemplifies Klebs' first prin- ciple. Essentially, provision of a slowly utilizable carbon compound is the same as use of a low concentration of a more available sugar. That is, the fungus hydrolyzes (or phosphorylates) the complex car- bohydrate slowly, the result being a steady maintenance of a low con- NITROGEN NUTRITION 369 Carbohydrate concentration *- Figure 2. The response of growth and fruiting to carbohydrate concentration. Mi and Flt mycelial growth and fruiting, respectively, with a readily available carbohydrate; Mz and F2, the same with a more slowly available carbohydrate; M3 and F3, the same with a very slowly utilizable carbohydrate. From Hawker (140), by permission of the University of London Press, Ltd. centration of hexose. This point is shown admirably in Figure 2, from Hawker (140). This principle explains many of the effects of particular carbon sources on reproductive activity. Thus, the optimum sucrose concen- tration for fruiting in Melanospora destruens is ten times that in M. zamiae, indicating — and this is borne out by sucrase assays — that the latter species utilizes sucrose much more rapidly than the former (141). Hawker (137, 138) has suggested that the favorable effect of sucrose is in part to be attributed to the formation of phosphorylated hexoses from it by the fungus. This, it must be admitted, seems unlikely and is certainly not established by data available at present; phosphory- lated compounds have, however, been tentatively implicated in studies on stimulation by contaminating organisms (p. 372). 7. NITROGEN NUTRITION The concentration of the nitrogen source in the medium is impor- tant for fruiting, i.e., an optimum concentration can be determined 370 REPRODUCTION under specified conditions; account must be taken, however, of the carbon source and its concentration (257, 305). In general, high con- centrations of nitrogen suppress sporulation (109, 153, 250). Specific effects of particular compounds are often reported but can- not be combined in a general theory; pH effects and contamination of organic nitrogen sources with growth factors unduly complicate interpretation of experimental data. Asparagine, so often a favorable source of nitrogen for growth, generally represses sporulation (5, 28, 208, 226, 257). The asparagine effect may arise from ammonia accumu- lation or may be another example of the competition between vegeta- tive growth and reproductive activity. It should also be noted that asparagine, of the sources of nitrogen tested, is the best for perithecium formation in Diaporthe phaseolorum (286). The effect of hypoxanthine (or guanine) on zygospore formation in Phy corny ces blakesleeanus is probably not specifically a reproductive phenomenon and is considered elsewhere (Chapter 12). The very- striking positive effect of low concentrations of histamine on zygospore formation in P. blakesleeanus (250) merits reinvestigation. 8. MINERAL NUTRITION Numerous observations, some of which have been mentioned in Chapter 9, attest to the influence of mineral nutrition on sporulation; these are reviewed in more detail by Foster (101). As expected, either deficiencies or excesses of particular elements reduce or inhibit sporu- lation; these reactions require no further comment. The important question is whether the mineral requirements for sporulation differ qualitatively or quantitatively from those for growth. There is no evidence for a qualitative difference, i.e., sporulation never requires an element which is non-essential to growth. Quanti- tatively, however, there are differences, most of which are subsumed in the generalization that sporulation is reduced by partial deficiencies in certain elements much more than growth is reduced by the same deficiency. Experiments with copper almost invariably show that sporulation of Aspergillus niger is reduced drastically by a deficiency which has a small or even no effect on dry weight (205, 211, 246, 269, 270, 313). This copper effect is found also in work with Penicillium spp. and Phoma betae (205). The effect of copper deficiency on spore pigment has been noted earlier (Chapter 9). The same phenomenon — sharp reduction in sporulation with only a small decrease in growth — occurs with partial deficiencies of iron (246) or manganese (26, 155, 193, 269, 270). It is a general rule, even VITAMINS AND UNIDENTIFIED FACTORS 371 with a major element like potassium (209) or magnesium (230), that sporulation is prevented by deficiencies which do not completely in- hibit growth. That is, sporulation requires more of a mineral than the minimum necessary for measurable growth. Much has been made of the repressive effect of added zinc on sporu- lation (38, 113, 152, 165, 246). Toxicity, of course, is possible, but it seems more likely that in the concentrated media used, provision of zinc simply postpones sporulation by allowing more luxuriant vege- tative growth. The response of Chaetomium globosum to calcium (21, 22) is an apparent contradiction to the principle that sporulation and growth have qualitatively the same mineral requirements: calcium does not affect dry weight but does increase perithecium formation. The fact that many fungi, including other Chaetomium spp., do respond in growth to calcium and the possibility that calcium is an antagonist rather than a nutrient (Chapter 9) are arguments against immediate revision of the principle. It may be noted that sporulation in Asper- gillus niger is inhibited by high levels of NaCl without any apparent effect on growth (201). One rather serious reservation attaches to most of the results men- tioned in this section — sporulation is estimated visually rather than measured quantitatively. A second limitation is that most of the work has been with Aspergillus niger; study of other organisms might cause us to modify some of the conclusions now accepted. 9. VITAMINS AND UNIDENTIFIED FACTORS Under this head, we consider only substances which exert a non- specific effect; hormones and hormone-like substances are taken up in the next section. The data available show that most fungi which require biotin or thiamine need more of the vitamin for adequate sporulation than they do for measurable vegetative growth (17, 134). Thus, Sordaria fimi- cola in a medium without biotin makes about one-third maximal growth but does not form perithecia unless the vitamin is supplied; in this species biotin deficiency results in abortion or failure to mature of ascospores (16). However, one cannot specify an "optimum" level of a vitamin with- out reference to other constituents of the medium. Ceratostomella fimbriata, for example, requires thiamine in proportion to the total concentration of the medium (17). Conversely, it is well established, for numerous fungi, that provision of thiamine raises the level of 372 REPRODUCTION carbohydrate optimal for fruiting (59, 132, 133, 134, 135, 140). The simplest explanation of this phenomenon is based on the fact that thiamine accelerates glucose utilization (136); this is in accord with our previous conclusion that exhaustion of the carbon source is re- quired for the initiation of reproduction. There is no conclusive evidence that any fungus requires a known vitamin specifically for the reproductive process and independently of growth effects. The available data can more or less satisfactorily be explained on the basis of the two generalizations just stated: that fruiting may require more of a vitamin than measurable growth, and that vitamins may accelerate sugar utilization and so hasten the onset of reproduction. However, at least the possibility of a more direct relation is raised by the work of Barnett and Lilly (17) on Ceratosto- mella fimbriata; a thiamine-starved mycelium begins to fruit if trans- ferred to a thiamine solution containing no other nutrients. Unidentified materials in crude plant and microbial extracts often increase fruiting. Examples include both sexual reproduction (164, 186, 226, 259, 322) and asexual reproduction (121, 144, 169). It is often found, usually as a result of accidental contamination of cultures, that other fungi and bacteria encourage fungal reproduction. Many such cases are reviewed by Asthana and Hawker (6) and by Raper (236); other instances may also be cited here (24, 143, 243, 258). However, there is no assurance that these are vitamin effects — pH, for example, or nutrient concentration could be the decisive factor. Stimulation of conjugation of Zygosaccharomyces spp. by Aspergillus niger has been described; the effect of the fungus is replaced by a mixture of riboflavin and an organic acid (216, 217). The association in nature of Gonatorrhodiella highlei with Nectria coccinea may be explained by some chemical influence of the latter on the reproduction of G. highlei (7). Another series of investigations, reviewed by Buston and Khan (60), suggests that the effect of contaminating organisms on perithecium formation in Melanospora destruens and Chaetomium globosum is to be attributed to organic phosphorus compounds, particularly 3-phos- phoglyceric acid, liberated by the inducing fungus. Whether this is a direct effect is, however, not yet clear. In C. globosum, perithecial production is associated with a high level of organic phosphate esters (61). Several apparently unrelated synthetic organic compounds are re- ported to increase sporulation without a corresponding increase in growth; possibly the effect is an antagonism of inhibitors present in the growth medium (158, 295). SPECIFIC REPRODUCTIVE HORMONES 373 10. SPECIFIC REPRODUCTIVE HORMONES Hormones — endogenous chemical substances which influence or de- termine reproductive processes — have been unequivocally demon- strated in certain of the Oomycetes, notably Achlya spp., by J. R. Raper, who reviewed the entire field in 1952 (236). Certain strains of Achlya bisexualis and A. ambisexualis are "heter- othallic," i.e., the male and female sex organs — antheridia and oogonia, respectively — are borne on different thalli. This type of heterothallism is perhaps more clearly described as haplodioecism. When thalli of opposite sex are in the same medium, the development of the sex or- gans is under hormonal control. The hormones whose existence and action must be inferred from convincing physiological and morpho- genetic evidence are summarized in Table 1. It is apparent that the Table l.The Sexual Hormones of Achlya Spp. Hormone Produced by Action A, A2 Female vegetative plant Induces antheridial hyphae on male plant A1 Male vegetative plant Augments A and A2 A3 Male vegetative plant Depresses action of A and A2 B Antheridial hyphae Induces oogonial initials on female plant C Oogonial initials (a) Induces antheridial hyphae to grow along a concentration gradient toward the oogonium; (b) induces the forma- tion and delimitation of the antherid- ium in the male plant D Antheridia Induces (a) delimitation of the oogonium and (b) differentiation of oospheres * From Raper (236). entire sequence of events leading up to fertilization is under specific and sequential hormonal control. The hormones are produced in ex- tremely small amounts, so small that only a start has been made toward chemical fractionation (237). Biological activities indicate that the battery of hormones produced by A. bisexualis is not fully identical with that of A. ambisexualis (234). Others of the Saprolegniacae — Thraustotheca clavata and the "homo- thallic" Achlya americana — are governed in reproduction by similar but not identical hormone systems (235, 236). Preliminary evidence of a hormonal system in Sapromyces reinschii has also been obtained (30). 374 REPRODUCTION Burgeft (56) studied Mucor mucedo, a heterothallic Zygomycete which copulates by the fusion of identical gametangia when two strains of opposite mating type are brought together. The two mor- phologically identical types are called for convenience plus (+) and minus (— ); historically, at least, the term heterothallism is appropri- ately applied to this situation. Burgeff showed that at least two of the steps in reproduction are initiated by a chemical, diffusible stimulus. Zygophore development in each strain is begun only when the opposite- strain is close enough for the stimulus to reach receptive hyphae. The somewhat clumsy term "telemorphosis" has been used for this reaction. A second reaction, also initiated by a diffusible metabolite, has been called "zygotropism": zygophores of each strain bend toward those of the opposite strain, preparatory to contact and fusion. These reactions in Mucor mucedo have been confirmed (167, 176, 225) and found also to prevail in Pilobolus crystallinus (177, 178). Limited evidence suggests that the zygotropic reaction is effected by a volatile product (15, 167). Such a material would have to be of very high activity to induce a differential growth response in an organ as small as the zygophore. Banbury (14) reports evidence that zygophores of the same strain in Mucor mucedo exert a mutual repulsion, also by a volatile substance. This recalls the numerous examples of "aversion" or "barrage": a zone of inhibition develops between certain colonies of the same or of different species and not between others. This phenomenon is re- viewed by Cayley (73) and by Takemaru (282); little physiological work has been done, although it has been suggested that a volatile ma- terial is responsible (293). A final type of interstrain chemical influence is shown by a slight in- crease in oxygen uptake when (+) and (— ) mycelia occupy the same respirometric vessel (57). None of the hormonal activities of the Zygomycetes has been sepa- rated physiologically or biochemically from the others; it is not yet possible, therefore, to describe any such elegant sequential mechanism as that of Achlya spp. At this point we may briefly mention the numerous attempts to find physiological or biochemical differences between (+) and (— ) strains of species of the Mucorales. Various workers have claimed that the strains differ in growth rate on certain sugars, sucrase or catalase forma- tion, lipid or carotenoid content, sugar content, reductive capacity, susceptibility to poisons, and so on. Most of these are reviewed by Ronsdorf (250) and by Raper (236). Study of the original papers and of the divergent results obtained leaves one skeptical of all this work. METABOLISM AND REPRODUCTION 375 The most recent claimed difference, a color reaction with strong alkali (292), was found invalid almost immediately (46). Enzyme differences between opposite mating types in Neurospora crassa (103) and growth differences between (+) and (— ) strains of Ustilago spp. (281) are exposed, until confirmed, to a similar skepticism. The long debate over the existence of perithecium-inducing hor- mones in Neurospora sitophila is reviewed by Raper (236); the work of Aronescu (3, 4) in particular seems conclusively negative. This con- clusion must stand unless challenged by new evidence. However, in Glomerella cingulata, like Neurospora a member of the Sphaeriales, there is preliminary evidence for a diffusible metabolite which induces the formation of perithecia (92, 200). Since the various steps of sexual reproduction in G. cingulata are gene-controlled (308), it is perhaps not too fancifuf to envision a one gene-one hormone relation. In Ascobolus stercorarius the induction of ascogonia and other early steps in sexual reproduction are controlled by diffusible materials, presumably hormones (31). The branching of the trichogyne in Neu- rospora sitophila (9) and the suppression of conidiation in Glomerella cingulata by filtrates from perithecial cultures (309) are also very probably hormone effects. There is no evidence as yet for hormonal mechanisms in basidiomycetes. The literature on the chemical stimulus for the aggregation of myx- amoebae in the Acrasiae has been thoroughly reviewed by K. B. Raper (238) and by Sussman (279). The substance responsible has been termed acrasin (34) and has now been separated from the cells; it was found to consist of two components and to be destroyed by some sys- tem, probably an enzyme, of the cell (260, 280). It is especially interest- ing that acrasin not only attracts scattered myxamoebae but also stimulates them in turn to produce acrasin; the effect is that of a chain reaction which extends the range of the chemotactic system much be- yond any possible range based only on diffusion from a single point. 1 1 . METABOLISM AND REPRODUCTION Throughout this chapter we have stressed the concept that the major stimulus to reproduction is exhaustion of the medium. However, many different metabolic changes occur at the same time in a culture, and the possibility must be kept in mind that one or more of these changes is the primary stimulus to spore formation. In particular, the mycelium begins to autolyze (Chapter 1). In yeast colonies on agar, ascospores appear in that part of the colony which is undergoing autolysis (191). Now, it is possible that some factor in normal cells is 376 REPRODUCTION released by their death and reaches a threshold concentration high enough to induce speculation. This possibility also relates to the stimulation of reproduction by ultraviolet radiation; it has been sug- gested (286) that cells killed by the radiation release into the medium substances which favor perithecial development. This line of investi- gation deserves more attention than it has so far received. Other metabolic changes are often coincident with the onset of reproduction, e.g., changes in respiratory rate (118), appearance of tyrosinase (153) or other enzymes (100), and formation of carotenoids (57a, 150a, 288, 289). The correlation between tyrosinase formation and protoperithecium development in Nenrospora crassa is the most striking, in that a wide variety of environmental factors suppress both enzyme activity and reproductive capacity, but it cannot yet be said that the relationship is causal (304). One obvious physiological necessity in sporulation is the transport of materials — presumably in a soluble form— from the mycelium to the developing spores. In Aspergillus flavus the appearance of conidia is coincident with a marked fall in mycelial nitrogen, easily interpreted as resulting from transport (224). Phosphate transfer is even more dramatic in A. niger; during spore formation mycelial phosphorus de- clines as much as 80 per cent, and at least some of this phosphorus appears in the spores (12). The transport of materials into fruiting structures may be more complete than it is usually thought to be. Thus, microscopic observa- tion of Ascochyta pisi in an infected leaf shows the mycelium ap- parently devoid of protoplasm at the time of formation of pyenidia (43). Blastocladia pringsheimii in its natural habitat forms two types of sporangia: thin-walled zoosporangia and thick-walled resting sporangia. In pure culture, resting sporangia form only at very high carbon di- oxide pressure, specifically in 99.5 per cent C02 at pH 5.5 (63). This discovery initiated an important series of investigations by Cantino and collaborators of members of the related genus Blastocladiella. These investigations have been reviewed by Cantino (64), and a brief summary is in order at this point. Blastocladiella emersonii zoospores develop into a two-celled thallus consisting of a basal rhizoidal cell and an apical sporangium. The sporangium is either a thin-walled papillate zoosporangium or a thick- walled resistant sporangium. In pure culture the type of sporangium formed is controlled by bicarbonate; virtually all thalli develop resist- ant sporangia if 0.01 M bicarbonate is supplied to the medium. The problem, thus, is not that of initiation of reproduction but of the type SPORULATION OF PLANT PATHOGENS 377 of reproduction which occurs. The explanation proposed by Cantino for the effect of bicarbonate is that the ion in some way interferes with the oxidative decarboxylation of a-ketoglutaric acid. Concurrently, several other enzyme activities of the plant disappear and a new poly- phenol oxidase becomes evident. The upshot of these changes is a reorientation of metabolism resulting in the formation of the resistant sporangium; this involves necessarily the synthesis of the chitin, me- lanin, and y-carotene characteristic of the resistant sporangium. These conclusions have been buttressed by study of a mutant strain of B. emersonii which does not, even with bicarbonate, form resistant sporangia. Crude cell-free extracts from the mutant strain are devoid of aconitase and of the enzyme system oxidizing a-ketoglutarate via the cytochromes (65). Precisely these enzymes are postulated to be re- quired in the normal strain for the expression of the bicarbonate effect. The rather elaborate metabolic hypotheses which have been erected in the course of this study of reproduction in Blastocladiella are some- what provisional and speculative; further investigation is clearly neces- sary. But, however the hypothesis may have to be modified later, it should be recognized as a bold and imaginative attempt to correlate biochemical events and patterns of morphogenesis. 12. THE SPORULATION OF PLANT PATHOGENS Reproductive processes in fungi pathogenic to or deriving their food from higher plants are difficult to study but pose at least two interesting questions: the influence of the host on sporulation in general, and spe- cific relations between the host and particular stages in the life cycle of the pathogen. It is a commonplace that plant pathogens often lose their capacity to sporulate during continuous artificial culture. Of course, this may only be an example of the very common phenomenon, not at all re- stricted to plant pathogens, of the selection in culture of non-sporulat- ing variants (77, 127, 128, 138, 207, 213, 223, 244, 310, 311). However, there is still the question of how contact with the host — often only in a single passage — acts to select for restoration of reproductive capacity. It seems probable that specific features of the food plant promote sporulation over and above any selective process. We have already noted the important observation that the host tissue replaces a light requirement for the sporulation of HelmintJiosporium gramin- eum. Experiments with autoclaved plant extracts are of little real help in working out the problem, since labile materials are probably involved. Cold sterilization of natural substrates by, for example, 378 REPRODUCTION propylene oxide appears much more promising as a technique (129, 265). Analogously, the insect pathogen Cordyceps militaris forms peri- thecia on dead pupae but not on autoclaved pupae (261). The relation of the host to sporulation has one other dimension, the influence of the host on the intensity and on the type of sporulation. This has been most thoroughly studied in relation to the appearance of the telial stage of rust fungi. From the work of Morgen thaler (210) and Waters (301) we may list factors that encourage or hasten the de- velopment of the telial stage: 1. Exposure of the host to low temperature or to slow drying. 2. Starvation of the host by dark incubation. 3. Growth of the fungus on a partially resistant host. 4. Wounding of the inoculated leaf. 5. Transfer of detached leaves from sucrose to water. Obviously, effects of this type must be indirect for the obligate para- sites, but the pattern seems clear that telia form under conditions of privation; the analogy to Klebs' conclusions is close. The factor that is dominant in nature is not known; it is most probable that drying out in autumn is dominant (91, 108), but simple starvation cannot be dis- missed. Perithecial formation in Erysiphe graminis — also an obligate parasite — is more rapid if the relatively resistant older leaves of the host are infected (114). Here again, we may speculate that resistance of the host accelerates the reproduction of the pathogen by depriving it of some essential factor. Host influences on spore size merit brief mention. Uredospores of rust fungi are smaller on a resistant host than on a susceptible one, and they are smaller if the host is held under conditions unfavorable to its growth (187, 188, 268). Similar findings on other fungi have been summarized by Fischer and Gaumann (98). 13. CONCLUSIONS The major barrier to generalizing about reproduction in the fungi is the repeated failure of experimental work to discriminate adequately between growth and sporulation; too often, only one is measured quan- titatively. Consequently, many of our hypotheses on reproduction must leave open the possibility that the factor concerned may act on growth rather than specifically on the formation of spores. The most inclusive generalization is that reproduction is initiated by factors which check the growth of an established mycelium without CONCLUSIONS 379 too drastically poisoning its metabolism. Studies on the elTect of light, especially ultraviolet light, are the most important evidence, in view ol the well-established lethal action of radiation. Gross mutilation by scraping or cutting induces sporulation in a few fungi (195, 206, 233); in scraped cultures of Alternaria solani Kunkel (179) observed that conidiophores arise from broken hyphac. Initiation of sporulation as the result of temperature changes, exhaustion of nutrients, and various treatments of the host plant of a pathogen are all consistent with the injury hypothesis. Granted that a check in growth initiates sporulation, we can only speculate on the intimate mechanism. Perhaps the most promising suggestion is that injured or moribund cells release substances which act on surviving cells and divert them into a new developmental path. It must be noted that a contrary hypothesis, that sporulation requires specific positive stimuli rather than the negative stimulation of injury, could easily be built up. In particular, the ready sporulation of many fungi on the living host plant or on cold-sterilized plant materials ar- gues for nutritional factors which must be present in some threshold amount to permit sporulation or which must be synthesized by the fungus before reproductive activity can begin. Less inclusive, but important and more firmly based, generalizations may be listed briefly in conclusion: 1. Reproduction occurs, in general, over a narrower range of envi- ronmental conditions than does growth; this is especially clear in find- ings on temperature, acidity, mineral, and oxygen requirements. The optimum for reproduction may be the same as or different from that of mycelial growth. 2. Klebs' principle, that reproduction is favored by depriving an established mycelium of one or more nutrients, is valid in general, especially for carbon and nitrogen sources. 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Spore Germination s, 'pores — specialized, self-contained, microscopic struc- tures capable of initiating new growth — are the principal agent of dispersal of fungi. Consequently, any consideration of ecology or of the spread of economically important fungi must take spore germina- tion into account. The control of plant diseases by protectant fungi- cides is in essence the problem of inhibiting the germination of spores. And, the transformation of a relatively inactive structure into an ac- tively growing thallus poses some fundamental problems of interest to biology as a whole. In this chapter, the several cytological and morphological types of spore have been distinguished in the discussion only if some important physiological aspect is specific to the type. In the long run, we may suppose, such a practice — treating the rust fungus teliospore and the myxomycete swarmspore alike — will prove indefensible. For the pres- ent, however, the available information does not permit separate treat- ment of the different spore forms, except in such special cases as the dormant "resting" spores. It will be seen that, as usual in the fungi, the corpus of knowledge is seriously out of balance. We find literally hundreds of studies of temperature and water relations of the economically important fungi, but such groups as the aquatic fungi, the fleshy basidiomycetes, and the actinomycetes are virtually unknown in this dimension. More serious still, concentration on well-known and easily determined environmen- tal factors has meant relative neglect of the fundamental metabolic 387 388 SPORE GERMINATION processes which control germination and of the physiological and bio- chemical changes consequent upon germination. It is reasonable to expect that investigations into these and related topics would in turn throw light on some of the more obscure environmental and ecological problems in spore physiology. 1. MEASUREMENT The most commonly used criterion of spore germination is the frac- tion of spores that in a given time forms a germ tube; the time period is usually chosen so as to allow all viable spores to germinate. Spores are sown on water, on agar, or on nutrient media, incubated under controlled conditions, and counted microscopically; or, the spores may be germinated in an aerated or shaken liquid medium (65). Self-in- hibition and other associative effects (p. 413) make it imperative that the concentration of spores be held constant and be known. It is usu- ally convenient to bring the spore suspension to a known turbidity, measured optically and previously standardized against a direct micro- scopic count (240). Spore concentration can also be estimated by capillary volume (192, 289). Clumping of spores introduces error and must therefore be minimized (130). The rate of germination may be determined by periodic counts dur- ing the experiment and often provides valuable supplementary infor- mation. A second major criterion is the latent period of germination, the time required for germination to begin or to reach some specified low value (25, 118, 287). Limited evidence suggests that this is fundamen- tally different from total germination, in that temperature affects it differently (p. 410) and toxicants may exert greater or less effect on the latent period than on total germination (287). The rate of increase in the length of the germ tube is again a differ- ent criterion of spore activity. In general, it is more sensitive to tem- perature than is total germination, responding more like mycelial growth; similarly, the pH range for optimum germ tube growth in Alternaria solani is rather narrower than that for best total germina- tion (159). The rate of elongation can be expressed as a velocity con- stant (126). No one of these criteria can be said to be "best." The method used must take account of the problem which is under investigation. Fun- damental studies on the germination process, for example, might make use of the rate of germination and the total germination. On the other hand, an ecological or epidemiological problem might be served better THE GERMINATION PROCESS 389 by measurements of germ tube growth. Infection of a plant by a path- ogen is dependent usually on at least some extension of the germ tube; if this process is more sensitive to unfavorable conditions than is total germination, a false picture of the potentialities of the organism will be deduced from observations on total germination alone. Unfortu- nately, germ tube measurements are rather more laborious and are perhaps avoided for that reason; a rapid, semi-automatic method of determining germ tube lengths should not be beyond the capacity of modern instrumentation and would be a real contribution to the study of fungi. 2. THE GERMINATION PROCESS Just as we have had to lump together the various morphological types of fungus spore, it is also necessary, with some exceptions, to consider the several germination types as if they were all alike. Of these, the most common and the most frequently studied is formation of one or more germ tubes which grow indeterminately and develop into a mycelium in culture or in a parasitized plant or animal. Among the phycomycetes several types of spore germinate by the formation of smaller endogenous spores, i.e., the parent structure is functionally a sporangium. In Phytophthora injestans the same structure — perhaps best called a conidiosporangium — germinates either by a tube or by formation of swarmspores, depending upon the environment, particu- larly temperature, and perhaps on the age and previous history of the structure (19, 63, 279, 291). The chlamydospores of the smut fungi germinate somewhat differently under different external conditions of temperature (140), concentration (132), and nutrients (264). Basidio- spores of Rhizoctonia solarii form secondary spores on water agar, germ tubes on a nutrient agar (131). The macroconidium of Tliielaviopsis paradoxa similarly germinates very differently on water and on nutri- ent agar (149). Multicellular spores often germinate by the simultaneous formation of two or more germ tubes (179); in Pestalotia spp., however, the basal cell normally gives rise to the germ tube (300), and germination in Helminthosporium sativum may be either polar or bipolar (137). Conidia of the higher fungi may have one, two, or several nuclei at the time of their formation; a few examples may be cited: 1. Predominantly uninucleate: Aspergillus spp. (8, 12), Colic tot ri- chum destructivum (49), Trichoderma viride (12), Verticillium albo- atrum (128). 390 SPORE GERMINATION 2. Predominantly multinucleate: Aspergillus echinulatu§ (8), Botry- tis cinerea (128), Helminthosporium sativum (54, 137). Sporangio- spores of Rhizopus nigricans are uninucleate (12), those of Mucor dis- persus multinucleate (68). Nuclear condition is of particular importance in genetic studies, since a heterokaryotic mycelium which produces uninucleate conidia — more accurately, conidia from uninucleate conidiophores — essentially regen- erates the parent types in the absence of gene exchange (112). By extension of this principle, it is possible to infer from genetic evidence in Streptomyces spp. that the conidia are uninucleate or arise from uninucleate cells (28). The number of nuclei in macroconidia of Neurospora crassa is re- duced by cultivation in a minimal medium (138) or in a sorbose me- dium (5). The nuclei of the higher fungi are, of course, normally haploid; Roper (236) has, however, succeeded in inducing the forma- tion of diploid conidia in Aspergillus nidulans, and camphor treatment of Neurospora crassa induces the formation of possibly diploid cells (243). 100 2.5 3.3 Time, hours 10 100 Figure 1. The time course of spore germination (probit scale) in Monilinia fructicola (curves 1) and Alternaria solani (curves 2). Arithmic function of time plotted in A, reciprocal function in B. Redrawn from Wellman and McCallan (299), by permission of the Boyce Thompson Institute for Plant Research, Inc. THE GERMINATION PROCESS 391 A given population of spores germinates over a period of time; if the population is standardized as to age, there is good evidence that ran- dom variability is the cause of the spread in time of germination. As shown in Figure 1, a plot of per cent germination on a probit scale against the reciprocal of time is linear (provided a correction is made for non-viable spores); i.e., time of germination is normally distributed. Similar data have been reported in studies of Neurospora crassa (240). The over-all rapidity of germination is a species characteristic: Melamp- sora lini begins germination in less than one hour (129), but Phoina apiicola spores require 24—48 hours (13). Other data on rapidity of germination are reviewed by Gottlieb (113) and by Doran (73). The three distinguishing morphological events of typical spore ger- mination are: nuclear division (8, 12), swelling of the spore, and emer- gence of the germ tube. Swelling, with one significant exception, appears to be universal in fungi and actinomycetes, having been de- scribed in sporangiospores (41), ascospores (121), basidiospores (32, 135), and conidia (25, 114, 125, 192, 255, 260a, 306). The exception— apart from the fact that observations on Sclerotinia fructicola conflict (192, 306) — is found among the powdery mildew fungi; conidia of these do not swell prior to germination (306). The significance of swelling in relation to water requirements will be considered later. There is scattered evidence that physiological changes also occur during the germination process, although the data cannot be general- ized to all fungi. Anion toxicity is more marked in the early stage of germination of the resistant sporangium of Blastocladiella sp. (45) than in a later stage; oxygen uptake by ascospores of Neurospora tetrasperma becomes more sensitive to iodoacetamide after appearance of the germ tube (109); and in the same material cation permeability appears to change at the beginning of germination (274). Other evidence of separable physiological states during the germination process appears in studies of oxygen uptake and of temperature responses. Germ tube growth presumably follows the same rules as any other mycelial growth. Extension occurs at the tip only (259, 263). For at least some time, growth is exponential, i.e., the logarithm of germ tube length increases linearly with time (220, 259); germ tube growth in Ustulina vulgaris, however, is reported to follow a different course (301). Germ tube tropisms toward water and toward nutrients have been reported, but the evidence is conflicting and inconclusive (9, 55, 100, 1 17, 203). It is not, in particular, necessary to postulate a chemotropic reaction in the penetration of plant pathogens into host tissue (39, 40, 392 SPORE GERMINATION 62). Here an unspecified contact stimulus, "thigmotropism," is sup- posed to operate. The possibility of chemotropism has been reopened more recently in studies on Saprolegnia ferax (87). One type of germ tube tropism is well documented — the negative tropism of germ tubes toward each other (55, 100, 117). As described by Stadler (263), germ tubes of Rhizopits nigricans form on the side of the spore distant from other spores and, in addition, actually bend away from other germ tubes during their growth. The stimulus is almost certainly chemical, and it is tempting to consider an auxinlike growth factor in explanation of the bending phenomenon. This possibility is also suggested by the finding that germ tubes of rust fungi are longer when germinating spores are crowded than when they are more dis- perse (83,314). However, Stadler (263) presents some evidence against an explanation based on growth factors; instead, he postulates an un- stable inhibitor which has the effect of thickening or strengthening the wall of the spore or germ tube. The last word on this problem has clearly not been spoken. Germ tubes of sporidia and of uredospores of Puccinia spp. react negatively to strong light; studies with filters indicate that the active wavelength is in the blue region of the visible spectrum (90, 98, 235, 266). It will be recalled from Chapter 1 1 that it is these shorter wave- lengths which are most active in phototropic and inductive effects on reproductive structures. Negative phototropism is also shown by co- nidia of powdery mildew fungi (204, 305). 3. SPORE METABOLISM Water, minerals, carbohydrates, and other constituents of fungus spores have been mentioned incidentally in Chapter 2. In relation to spore germination the important materials are water — considered else- where in this chapter — and, probably, lipids. It should also be recalled that spores of Aspergillus niger are relatively high in total phosphorus, a part of which is in organic combination (7, 154). Although the fat content of most spores is not unusually high (248, 271), three bits of evidence suggest that fats provide energy for the ger- mination of spores which do not require an exogenous source of car- bon. First, microscopic observation shows that stainable lipids disap- pear during germination (82, 150). Second, the respiratory quotient of Neurospora sitophila macroconidia is about 0.7 (208), a value char- acteristic of, but of course not sufficient evidence for, fat oxidation. Finally, the data of Table 1 show that, at least in high-fat spores, the principal organic material disappearing during germination is the SPORE METABOLISM 393 Table 1. Chemical Changes during Germination of Uredospores of Puccinia graminis tritici* Amount, Per Cent of Dry Weight Fraction Before Germi nation After Germination Carbohydrate 21.9 22.4 Chitin 1.4 3.4 Protein 25.9 22.3 Lipid 19.7 7.8 * From Shu, Tanner, and Ledingham (254), by per mission of the Canadian Journal of Botany. lipid fraction. The conidia of Erysiphe graminis, like the rust spores of Table 1, appear to be relatively high in fat (310). During the germination of spores of Aspergillus nidulans, the most pronounced changes which occur in the first few hours are: decline in polymetaphosphate and in organically bound phosphate, disappearance of part of the free amino acids of the spore, and a progressive fall in total lipid (253). Several hydrolytic enzymes — acting on starch, oligosaccharides, glu- cosides, pectic substances, proteins, and urea — occur in crude spore preparations; the early literature is reviewed by Mandels and Norton (195). Spores of Myrothecium verrucaria have been found to possess an enzymatic system acting on ascorbic acid (185, 186), another system oxidizing sulfhydryl compounds to disulfides (191), and one or possibly two enzymes acting on sucrose (184, 187, 188). The invertase of M. verrucaria is synthesized rapidly during germination and appears in the medium almost immediately (190). These enzymes are inactivated by an acid treatment which does not kill the spore or damage its res- piration; from this fact it has been proposed that they are located at or near the spore surface (184, 185). The diphosphopyridine nucleotidase of Neurospora crassa is produced primarily or entirely by the conidia and is so easily washed out of the cell that it, too, may have a surface location (309). Although there is as yet no reason to think that spore respiration dif- fers fundamentally from that of mycelium, studies of the general prob- lem should contribute to our understanding of both spore germination and fungicidal action. Data available at present are too fragmentary to support any firm theoretical structure or even generalized descrip- tion; the work summarized here should not therefore be taken as nec- essarily applying to all fungi. The endogenous respiration of ascospores of Neurospora tetrasperma is shown in Figure 2. The curve has an upward break coincident with 394 SPORE GERMINATION 240 o Q. (/> TJ a> > -*— > o a. 3 2 3 Time, hours Figure 2. Oxygen consumption of activated (curve 1) and dormant (curve 2) ascospores of Neurospora tetrasperma. The arrow marks the time of appearance of germ tubes. Redrawn from Goddard and Smith (110), by permission of Plant Physiology. the appearance o[ the germ tube; this particular type of curve is not universal in the fungi and would not necessarily occur under different conditions of nutrient or oxygen supply. Endogenous respiration in Memnoniella echinata and Aspergillus luchuensis is affected by ammo- nium ion and by the previous cultural history of the spore population (64). Oxygen pressure also affects the endogenous rate (110, 194). Spores generally, but not always, respond to added substrates by an increase in respiratory rate; increases may also be induced by provision of nitrogen or of complex nutrients in addition to a carbon source (173, 193, 254, 255). The data of Table 2 are fairly typical in that the range of response is from zero to high; since, however, the substrate was su- crose, the results reflect hydrolytic as well as respiratory capacities. Just as mature mycelium, spores of a given species may or may not ferment glucose anaerobically. Uredospores of Puccinia graminis tri- tici do not form carbon dioxide anaerobically (254); at the other ex- treme, both activated ascospores and conidia of Neurospora spp. fer- ment vigorously and have demonstrable glycolytic enzymes (59, 110, 209). Not much can be said at this time of detailed respiratory pathways in spores. Acids of the tricarboxylic cycle are oxidized by conidia of Neurospora sitophila, are found in conidial homogenates, and appear in the medium during germination (174, 208, 293). Poisons which affect glycolysis and the cytochrome system usually inhibit spore res- piration, but several anomalies in the results make interpretation dif- MATURATION AND DORMANCY 395 Table 2. Endogenous and Exogenous Oxygen Uptake by Conidia of Fungi* Oxygen Uptake f Species Neurospora sitophila Monilinia fruclicola Cephalosporium acremonium Myrothecium verrucaria Stemphylium sarcinaejorme Alttrnaria oleracea Aspergillus niger Rhizopus nigricans * Drawn from more extensive data of McCallan, Miller, and Weed (173), by permission of the Boyce Thompson Institute for Plant Research, Inc. f In fil/5 hr/mg fresh spores. J Exogenous = uptake with sucrose less endogenous uptake. ficult (64, 110, 254). In general, but with some exceptions, germination is more sensitive than spore respiration to both inorganic and organic fungicides (Chapter 14). No Substrate Sucrose, Exogenous^ (endogenous) 1 per cent Endogenous 8.6 68.6 7.0 5.1 54.2 9.6 2.7 42.8 14.8 8.8 22.1 1.5 2.0 21.7 9.8 7.1 17.4 1.5 12.8 17.7 0.4 8.5 9.2 0.1 4. MATURATION AND DORMANCY It is a common observation that morphologically normal and appar- ently fully developed spores often do not germinate until after a period of time. If this period is relatively short, we speak of maturation; if it is on the contrary a matter of weeks or months, the spore is said to have a dormant stage or resting period. There is as yet no evidence which enables us to say whether these two phenomena are different or the same. The failure of newly formed spores to germinate normally is best documented in studies on the uredospores of the rust fungi (73, 200, 245, 322), the behavior of which is exemplified by the data of Figure 3. As shown, immature spores have both a slower rate of germination and lower total germination than have mature spores. In addition, imma- ture spores are more sensitive to unfavorable temperatures (57, 73). It appears from most work that immature uredospores can ripen after removal from the sorus (11, 57, 178). The very long period of poor germinability of aeciospores of Gymnosporangium spp. has been note d repeatedly but has not as yet been analyzed (20, 99, 178, 222, 282). Both asexual and sexual spores of ascomycetes germinate poorly 396 SPORE GERMINATION 100 8 12 Time, hours Figure 3. The rate of germination of mature (curve 1) and immature (curve 2) uredospores of Phragmidium mucronatum. Redrawn from Cochrane (57), by per- mission of the Cornell University Agricultural Experiment Station. when newly formed (42, 145, 240). Conidia of powdery mildew fungi do not germinate until detached from the chain in which they are formed, possibly because they are impermeable to gases until exposed (33), or because the parent mycelium forms a diffusible germination inhibitor (70); this is not, therefore, comparable to immaturity. Many phycomycetes form thick-walled resting spores; these are prob- ably always the site of meiosis, and at least part of their apparent resting period represents the time required for cytological processes (81). Whether there is in addition a further period of purely physiological dormancy is still uncertain. There can be no doubt that the environ- ment, especially temperature, influences the length of the resting pe- riod in some members of the Saprolegniales (246) and Peronosporales (3, 17, 18). However, it is not yet possible to define the locus of this effect. Many oospores, it should be noted, germinate immediately after their formation (157, 321), as do also the resistant sporangia of BJastocladiella sp. (45). The rest period of resistant sporangia of Allomyces spp. is shortened dramatically by certain cultural conditions and by indoleacetic acid (176, 177). In this and related genera the strength of the sporangium wall may be one of the decisive factors in germination (176, 270) — sporangia grown under conditions in which the wall is thin germinate without a rest period. MATURATION AND DORMANCY 397 The ascospores of many ascomycetes, particularly but not exclusively coprophilic forms, exhibit a pronounced dormancy which is broken by brief exposure to high temperature (69, 120, 121, 122, 123, 139, 251). Rather intensive study of ascospore dormancy in Neurospora spp. has revealed several features of the phenomenon: 1. Activation — breaking of dormancy — is reversible, i.e., activated ascospores become dormant again if prevented from germinating (107). 2. Activated ascospores decarboxylate pyruvic acid and respire vig- orously; dormant spores respire slowly (Figure 2) and do not decarbox- ylate pyruvate (108, 110). 3. Furfural and related unsaturated heterocyclic compounds activate fresh ascospores (79, 272, 273). Aged dormant spores are activated by furfural only if they are first heated (275). Emerson (80) proposes that furfural duplicates the action of a nat- ural catalyst which is normally induced to form or to become active by the heat treatment. It is of course equally possible that a natural in- hibitor is present in fresh spores and is inactivated by heat or by those chemicals which induce germination. Neither permeability to water nor to gases can be invoked as a factor in ascospore dormancy in Neurospora tetrasperma (107, 170). How- ever, the activating effect of alkali on ascospores of Ascobolus spp. (318) may implicate permeability to water, and dormant ascospores of N. tetrasperma are relatively impermeable to cations (169a). Sporangiospores of Phycomyces blakesleeanus are activated by a heat treatment, e.g., 15 minutes at 50°C (232). The effect is duplicated by hypoxanthine. Teliospores of the rust fungi normally do not germinate until several months after their formation. Although chemical and moisture treat- ments have been reported to hasten germination (142, 181, 196, 221, 280), the same treatments fail in other studies (129, 156). Until sys- tematic analysis has been accomplished, we must assume a long obligate rest period as characteristic in these spores. In the life cycle of the smut fungi the chlamydospore is analogous to the teliospore of the rust fungi. Periods of 200 days or more are re- quired for germination to begin in certain races of Ustilago striaejormis (66, 152). The germination of these spores is often much accelerated by chemical treatments (47, 66, 165, 205, 206), by low-temperature soak- ing (136), by high-temperature cultivation of the infected host (152), or by variations in moisture (281). Mechanical abrasion also shortens the dormant period in Ustilago striaejormis (153). It is not yet possible to envision a single explanation for all types of 398 SPORE GERMINATION long obligatory rest periods. If we define dormancy as a rest period after the spore has completed both its morphological and its cytological development, and if we assume that at least part of the rest period in the phycomycete and basidiomycete spores just considered is such true dor- mancy, then the most promising hypothesis is that some factor associ- ated with the heavy spore wall — possibly impermeability, possibly sim- ple mechanical strength — is determinative. It is not likely that the same explanation will fit ascospore dormancy in Neurospora, in which an internal, biochemical mechanism seems more probable. However, ascospore dormancy in Onygena equina develops only after the heavy spore wall is laid down — the immature spore germinates without a dormant period (30). 5. SPORE LONGEVITY Several fungi form asexual spores whose survival under the most favorable conditions is measured in days only; examples include the conidia of the powdery mildew fungi (317), the sporangium of Perono- spora tabacina (302), and the sporidia (basidiospores) of some rust fungi (178, 222, 262). The short life of the powdery mildew conidia is probably related to the high water content of the spores (317). Aeciospores and uredospores of the rust fungi are intermediate in longevity. Although in a few species these spores may under excep- tionally favorable conditions survive for as long as a year (57, 77, 89, 214), their lives under most conditions of storage are weeks or months only (6, 20, 72, 73, 74, 221) and are even less under uncontrolled nat- ural conditions (57, 237). Early work on spore survival in the rust fungi is reviewed by Maneval (193) and by Zimmerman (322). Types of spore characterized by a long or very long life include the chlamydospores of the Ustilaginales (88, 171, 278), basidiospores of Hymenomycetes (95, 127), and at least some ascospores (46, 127). Spores with long periods of inability to germinate — resting spores of phycomycetes, teliospores of the rust fungi, and certain ascospores — are ipso facto long-lived. Although the rule in the fungi is that asexual spores are relatively short-lived, the conidia of certain imperfect fungi and of actinomycetes may survive very long periods (15, 175, 212, 234, 297). Ecologically, it appears that the survival function typical of sexual spores is taken over in these forms, which lack a sexual stage, by the conidia. Dried swarm- spores of the Myxomycetes similarly are reported to survive many years (78, 258). SPORE LONGEVITY 399 Techniques of preservation of spores are briefly reviewed in Chap- ter 1. Temperature is one of the major factors in the longevity of spores. Almost without exception, survival is greatest at low temperature, i.e., near 0°C. This is particularly well documented for uredospores of rust fungi (57, 77, 214, 215, 237) and conidia of powdery mildew fungi (317). Presumably, spore metabolism is accelerated at high tempera- ture, reserves are exhausted, and death (inability to germinate) ensues. The effect of subzero temperatures on survival is considered later (Chapter 13). Relative humidity is the second major factor in spore longevity and, in nature, is probably more often limiting than is temperature. Here, however, the relation is not so simple, and we must for the present at least distinguish three types of response to atmospheric moisture. Co- nidia of Sphaerotheca pannosa (167), sporidia of rust fungi (222, 262), and conidiosporangia of Phytophthora spp. (63, 141) survive longest at saturation and are less viable at any relative humidity less than 100 per cent. Typical data appear in Figure 4. It should be noted that pre- cisely these spores have just been described as short-lived in general: their susceptibility to desiccation is almost certainly the reason for their failure to survive. Uredospores of the rust fungi exhibit a different and characteristic response to humidity. Virtually all studies agree that survival is great- 3 4 Time, hours Figure 4. The survival of sporangia of Phytophthora iiif< .sinus at different relative humidities. Redrawn from Crosier (03), by permission of the Cornell University Agricultural Experiment Station. 400 SPORE GERMINATION est at intermediate relative humidity and is less at either extreme (6, 57, 129, 214, 215, 221, 237). Chlamydospores of Urocystis tritici respond in the same way (206). Damage at low moisture levels is probably sim- ple water loss; consideration of the possible effects of high humidity leads us to the third and final category of humidity response. This last type of response is exemplified by both ascospores and en- doconidia of Endoconidiophora fagacearum (201) and by conidia of Helminthosporium oryzae (211); these remain viable longest at low humidity, especially if the temperature is unfavorably high for survival. It has been suggested that at high humidity the metabolism of this type of spore is accelerated, with a consequent reduction in longevity (201); the mechanism of such an effect is obscure, but this is at present the most attractive hypothesis and may be extended to the problem of uredospore survival at high humidity. Spores in nature are often protected from rapid loss of water by a gelatinous matrix, by the structure in which they are formed, or by host plant tissues; several such phenomena are reviewed by Gottlieb (113). Spores of Myrothecium verrucaria are strongly affected by the history of the culture, in particular by the composition of the medium (64). Spores from a mycelium grown on filter paper survive more than twice as long as those from any of three agar media; this greater viability is not, however, correlated with the rate of loss of dry weight. The growth medium also exerts a significant effect on the dry weight, nitro- gen content, and respiratory behavior of spores of M. verrucaria. Co- nidia of nutritionally deficient mutants of Ophiostoma multiannulatum die less rapidly in a starvation medium than do conidia of the parent non-deficient stock; it has been suggested that the deficient spores metabolize less and hence survive longer (96). Physiologically, senescent spores are characterized by poor germina- bility, by a slower rate of germination (36, 57, 194), and by lower re- spiratory activity (64). In Streptomyces sp., ageing of the spore population at low tempera- ture increases the recovery of mutant types; the mechanism is obscure (294). 6. NUTRITION AND GERMINATION Germination may be considered as a growth process in which some or all the necessary nutrients are present in the spore ab initio. We may therefore expect two extreme types of relation to external nutri- ents— complete independence in some fungi and complete dependence NUTRITION 401 on one or more nutrients in others — and various degrees of partial dependence on the environment. Experimentally, nutritional independence of the spore is difficult to prove; it is essential that washed spores, free of materials carried over from the growth medium, be used, or that spores be collected in such a way as to prevent contamination with nutrient materials (163). Washing, of course, must be as brief as possible in order to minimize any loss of materials from the interior of the spore. Washed ascospores of Neurospora tetrasperma germinate in distilled water (274) and are therefore to be classed as nutritionally independent. The uniformly good germination of rust uredospores and aeciospores which have been collected by shaking from the host plant suggests that these, too, are independent of the environment; it has been noted earlier (Table 1) that uredospores are unusually high in reserve lipids. Although it is perhaps justifiable to assume that the larger resting spores of fungi are nutritionally independent, critical evidence is lacking. Conidia of the powdery mildew fungi, collected, like the rust spores, by shaking, germi- nate well on water, although sugars and several natural materials im- prove germination (167, 307, 309, 317). Nutritional dependence is more easily demonstrated and is probably more common than independence. Many fungus spores germinate very poorly or not at all without added nutrients; examples include basidio- spores of Psalliota bispora and other Hymenomycetes (94, 95, 105), ascospores of Sordaria fimicola (29, 43), and conidia of several imper- fect fungi (75, 194). Other fungi, too numerous to mention individ- ually, germinate fairly well in water but do so more completely or more rapidly in nutrient media (52, 53, 75, 85, 124, 202) or in contact with complex biological products (35, 36, 38, 60, 101, 113, 147, 151, 158). Since unwashed spores have been used in many of these studies, nutri- tional deficiencies are probably more acute than they appear to be. The simplest view is that the three reactions just described — com- plete independence, partial dependence, and complete dependence — reflect the adequacy or availability of the reserve substances of the spore. Critical studies on washed spores show that requirements may be simple or complex. Washed spores of Sclerotinia jructicola require a carbon source (163), those of Fusarium roseum need both carbon and nitrogen (255), and spores of Glomerella cingulata must be supplied carbon, nitrogen, phosphorus, and sulfur (161). Responses to minerals, e.g., magnesium (163), are not necessarily nutritional. Spores are so sensi- tive to ions that minerals such as magnesium may act primarily as antidoting agents. Mutants of Neurospora crassa which are deficient for amino acids SPORE GERMINATION Log concentration L-lysine Hydrochloride, mg per liter Figure 5. The response to graded doses of lysine of macroconidia of a lysine-requir- ing mutant of Neurospora crassa. Redrawn from Ryan (240), by permission of the American Journal of Botany. produce macroconidia which require the amino acid for prompt germi- nation, although the spores will germinate in time without the com- pound (239, 240). The response of one such mutant to lysine is shown in Figure 5. The assay is complicated by the fact that amino acids are somewhat inhibitory to spore germination in both mutant and normal Neurospora crassa. Parathiotrophic mutants of Aspergillus nidulans respond in the same way to sulfur compounds (252). Hypoxanthine, partially replaced by guanine, accelerates both spore germination and early growth of Phycomyces blakesleeanus (226, 227, 228, 229, 230, 231, 233, 238). The effect of organic acids and of various physical and chemical treatments in increasing spore germination in this species has tentatively been ascribed to their influence on the avail- ability of hypoxanthine (232). Although, as mentioned above, complex biological materials improve germination in many species, relatively few vitamin requirements for spore germination have been worked out: Memnoniella echinata re- quires biotin (216), Myrothecium verrucaria also needs biotin but only in the phase following germ tube emergence (189), and Colletotrichum gloeosporioides is reported, without data, to respond to riboflavin and other vitamins (61). As discussed briefly in Chapter 10, successful use of the differential germination method for the detection of vitamin- deficient mutants implies that these mutants require the factor for spore germination as well as for growth. Promotion of basidiospore germi- nation by co-culture with Torulopsis sanguinea is at least suggestive of a vitamin effect (94, 95), as is also the observation that the parent my- celium itself stimulates spore germination in Psalliota campestris (76). Germination of uredospores of Melampsora lini is increased by methyl-p-hydroxybenzoate and by aureomycin (288); there is, however, pH 403 no evidence of a nutritional mechanism in this as yet unexplained phe- nomenon. Conidia of Erysiphe graminis respond slightly to nutrients, but the response is strongly conditioned by temperature and by the time of collection of the spores, i.e., by their previous history (316, 317). Ammonium salts, often a utilizable nutrient, are toxic to some spe- cies of Mycena, but not to all; mycelial growth is much less sensitive, or possibly insensitive, to concentrations of ammonium tartrate which completely inhibit basidiospore germination (97). Chlamydospore germination in Tilletia spp. has repeatedly been found to be inhibited by complex biological products which are nutritive for other organisms (171). Ecologically, a nutritional requirement for spore germination may be an advantage, helping to insure that germination will not occur in environments devoid of the materials needed for continued growth. 7. pH AND GERMINATION It has been mentioned earlier, in Chapter 1, that spore germination usually has a narrower pH range than growth, presumably because there is less time for metabolic products to modify an unfavorable medium. In the same chapter, some of the ambiguities of acidity data were considered; the same ambiguities attach to germination experi- ments, in particular to the finding that several fungi show a double pH optimum under some conditions (90, 283, 295). Analysis of spore germination data reveals several complicating fac- tors: (1) strains of a given species differ in response (43), (2) the type of buffer — and a buffer is essential in pH studies — affects the shape and limits of the curve (274), (3) nutrient materials often modify the pH response profoundly (43, 295), and (4) the previous history of the spore population influences its behavior (2). These considerations suggest a certain caution in the interpretation of the rather extensive literature on pH-response curves, to which we now turn. Spores of most fungi germinate best at pH 4.5-6.5, with limits at about pH 3 and pH 8. These rough figures apply to, for example, uredospores of several rust fungi (2, 90, 266), ascospores (14, 43, 301), chlamydospores of Urocystis tritici (206), and spores of myxomycetes (257). Variations from this general pattern are several: a few fungi have more acid optima (283, 295), some have optimum zones extending to about pH 8 (60, 146, 160, 161, 295), and many germinate over a some- what wider range than the typical (53, 198, 257, 295, 301). •104 SPORE GERMINATION It is probable that only in rather unusual environments, e.g., very acid soils, is pH a natural limiting factor for spore germination. Inso- far as acidity and alkalinity are ecological influences, their effects on mycelial growth and spore formation are likely to be more decisive. 8. WATER AND GERMINATION Available water is one of the primary determinants of spore germina- tion. Consequently, a considerable body of evidence has accumulated on the moisture responses of economically important fungi; several reviews of these data have been published (56, 71, 113, 256). The effect of humidity on spore survival has already been considered. Certain problems of method have received less attention than they deserve. Most investigators have reported data in terms of relative humidity; although this practice is justifiable, a clearer picture of water relations emerges when the vapor pressure deficit is taken as the cri- terion (67, 262). The absolute humidity is not a useful value (224). The importance of accurate temperature control, especially in attempts to discriminate between free water and a saturated atmosphere with no free water, has not always been realized. Both static and moving-air systems for the simultaneous control of temperature and humidity have been described (56, 57, 67); the static principle — incubation in a closed system over a humidity-regulating solution — is both simpler and more accurate. When germination is measured on solid substrates which take up water — agar, grain, textiles, etc. — the minimum humidity permitting germination is often lower than that in the absence of substrate (4, 22, 118). This, we may reasonably infer, means that the water-absorbing capacity of these substrates is greater than that of the spore, but this explanation begs the question of how the spore is able to draw water from the substrate. The response to moisture is usually conditioned by temperature. Thus, conidia of Uncinula necator are adversely affected by low humid- ity at 28 °C or above, not at 25° or 20° (67). The optimum humidity for germination of Aspergillus niger conidia is substantially higher at 40° than at 30°C (25). In Altemaria citri, both germination time and germ tube length are less affected by relative humidity at the tempera- ture optimum for germination than at non-optimal temperatures (286). Excluding the powdery mildew and the rust fungi, to be taken up later, the fungi can be classified according to the minimum relative humidity at which detectable germination occurs. Several, including species of Aspergillus, Penicillium, and a few other genera, form spores WATER 405 which germinate at relative humidities near 80 per cent (1, 25, 1 18, 132, 162, 244, 250, 320). A somewhat larger group of species germinate at humidities of 90-95 per cent and do not require liquid water; exam- ples include Altemaria brassicae, Colletotrichum spp. (51), Ustilago spp. (56), Cladosporium spp. (51, 119, 224), Botrytis cinerea (224), Fomes annosus (225), Septoria ap'ii, Fusanum spp., and Verticillium albo-atrum (217). Still higher humidity is required by such fungi as Venturia inaequalis (56) and Magnusia brachytricJia (277). Most of or all the fungi mentioned in this paragraph germinate better at high humidity than at low, regardless of the minimum value. Finally, the spores of many fungi are found to germinate only in liquid water; in this group we may mention conidia and ascospores of Endoconidiophora fagacearum (60), asexual spores of the Peronospora- les (73, 141, 302, 308), and conidia of Schrotinia spp. (56, 73). Almost all studies of uredospore germination in rust fungi agree that liquid water is required and that germination is nil even at 100 per cent humidity in the absence of free water (1, 10, 57, 111, 129, 133, 134, 267, 308, 322). It is not always realized that temperature control must be extremely accurate if a humidity of 100 per cent is to be maintained without condensation of liquid water. For this reason, we must reject those reports in which accurate temperature control is not specified and germination in the absence of free water is claimed. This stricture does not, however, apply to the work of Clayton (56), who found that uredo- spores of Puccinia coronata and P. graminis germinate at 100 per cent relative humidity, albeit less well than in the presence of free water. Aeciospores, teliospores, and sporidia of Gymnosporangium globo- sum all require liquid water for germination (174). Alone among the fungi, the powdery mildew fungi form conidia which in the experience of most workers germinate at very low relative humidity, even over desiccants (33, 34, 48, 56, 67, 306). Exposure to liquid water, in fact, inhibits germination and may cause death of the spore by plasmoptysis (67, 167). As a result of these water relations, the powdery mildew diseases are favored in nature by relatively dry conditions (26, 48, 311). Yarwood has in a series of papers (306, 310, 312) provided evidence that the ability of these spores to germinate at low humidity is a func- tion of their high water content. Two different methods of determin- ing water content agree in assigning a value of about 70 per cent water to the conidia of Erysiphe polygoni. This is much higher than the water content of other fungus spores. Correspondingly, although dif- ficulties of measurement exist, the density of the spores of powdery mildew conidia is less than that of most other spores. Finally, it has 406 SPORE GERMINATION been shown that spore volume decreases during germination in dry air. Although somewhat conflicting data and a theory based on the hypo- thetical conversion of bound to free water have been reported (33, 34), it seems that the high water content of conidia of the powdery mildew fungi explains their ability to germinate at low humidity. The occur- rence of plasmoptysis in pure water argues for a rapid entrance into the spore; this is still to be reconciled with the observation that the loss of water from ungerminated spores is very slow — approximately 0.08 per cent of the loss from an equivalent free water surface (312). These studies on the powdery mildews have been generalized by Yarwood (310) in the proposition that fungus spores of the usual type, i.e., those with a low water content, must absorb water to about the level of that in the powdery mildew conidia (70 per cent) before they can germinate. This hypothesis explains the swelling of these spores so frequently reported (p. 391), and in fact demands that all spores of low water content swell on germination. This appears to be the most promising general explanation of humidity relations. As is to be expected, fungi tolerant of low humidity are also in gen- eral tolerant of high osmotic pressure in the germination medium. This relation is brought out in Figure 6. Conidia of Erysiphe graminis germinate in concentrations of deu- terium oxide up to 100 per cent, but the heavy water is markedly in- hibitory to germ tube elongation (220); Macrosporium sarcinaeforme is unable to germinate in 60 per cent deuterium oxide (213). Figure 6. The relation of the minimum relative humidity to the maximum tolerated concen- tration of sodium chloride for spore germination in several fungi (Aspergillus spp., Penicil- lium spp., Fusarium monili- forme). Each plotted point refers to one species. Drawn from data of Armolik and Dick- son (4). 15 20 25 30 Maximum sodium chloride, per cent TEMPERATURE 407 9. TEMPERATURE AND GERMINATION The so-called cardinal temperatures for spore germination — the minimum, optimum, and maximum — have been determined for very many fungi; most data on plant pathogens reported prior to 1936 are summarized by Togashi (285), other results by Wolf and Wolf (303). The student is immediately struck by the frequent and pronounced disagreements in this literature; some of these are probably inherent in the nature of the material, whereas others are traceable to inadequate methods of study. Some examples of reported cardinal temperatures are shown in Table 3. The total range of temperatures permitting at least some germina- Table 3. Cardinal Temperatures of Spore Germination Minimum, Optimum, Maximum, Species °C °c °C Albugo Candida (199) 0 ~10 25 Peronospora effusa (102) /-»^4 9 ~27 Mastigosporium album (24) — 10 20- -25 Cronartium ribicola uredospores (71) 8 14 25 Puccinia malvacearum teliospores (71) 3 14 30 Phragmidiurn mucronatum aeciospores (57) 3-6 15- -21 27- ■30 Phytophthora infestans, direct (63) 6 24 27- ■30 P. infestans, indirect (63) 1.5 12 21- ■24 Phoma apiicola (13) 10 16- ■18 23 Tranzschelia punctata uredospores (111) 8 13- -26 38 Herpobasidium deformans (115) 1 18- -26 33 Endoconidiophora jagacearum (85) 3 21- -32 33- -36 Colletotrichum indicum (166) 16 20- -32 — Erysiphe polygoni (317) 5 25 35 Allomyces arbuscula, meiosporangia (261) 14-16 20- -26 32- -34 Glomerella cingulata (299) 15 27 35 + Magnusia nitida (276) 1.5 ~32 43 Ustilago zeae (144) 8 26- -34 36- -38 Rhizopus nigricans (298) 1.5 26- -28 33 R. chinensis (298) 10 43- -45 51 tion varies in these data from 13 centigrade degrees, in Phoma apiicola, to 40, in Rhizopus chinensis and Magnusia nitida. Optima range from a low of 9°C in Peronospora effusa to a high of 43-45° in Rhizopus chinensis. In general, it appears from these and other data, and is to be expected on theoretical grounds, that fungi with a high optimum have a broader total range than those with a low optimum; that is, fungi 108 SPORE GERMINATION with a high optimum have in a sense solved the problem of developing at high temperature without losing their capacity to germinate at low temperature. Spore germination in the truly thermophilic fungi and actinomycetes has not been studied quantitatively. Primarily from the data collected by Togashi (285), we can estimate that the "average" optimum for plant pathogenic fungi is about 25°. No similar estimate can be made for saprophytic fungi; probably the figure is somewhat higher. Certain groups of fungi can be roughly characterized as a whole by their temperature preferences. Thus, species of Aspergillus tend to have high optima (21, 25), those of Peronospora to prefer rather low temperatures (84, 285). Uredospores of most rust fungi germinate best at 22° or lower (44, 57, 72, 183, 266, 268, 285, 322), although exceptions to this rule have been reported (111). Limited data on Coprinus spp. suggest a rather high optimum, 30-35° (146). Conidia of the powdery mildew fungi as a rule germinate best at a moderately low temperature, about 21°. However, reported optima for different species range from 6° to 28°, and within one species, Ery- siphe cichoracearum, strains with optima of 15° and 28° occur (317). Similarly, physiologic races of other parasitic fungi, e.g., Puccinia gluma- rum (268) and Tilletia spp. (171), may differ considerably in their re- sponses to temperature. Intrageneric variations in response are exem- plified in Rhizopus: individual species have optima as low as 27° and as high as 44° (298). Figure 7 depicts representative types of curves of total spore germi- nation versus temperature. The most frequent shape of curve is roughly symmetrical — or is slightly skewed to the right — with a wide optimum zone; this curve is usually found in studies of the rust fungi (44, 57, 266, 268, 269) and has been reported for such divergent types as Fusarium lini (31), Ustilago spp. (16), Coccomyces hiemalis (148), and several Hymenomycetes (260). An extreme example is found in the response of conidia of Magnusia spp. (276). No one type of curve can, however, be said to characterize all fungi. The two most common modifications are (1) curves which are skewed to the right, like most mycelial growth curves (53, 115, 167, 317, 318), and (2) curves with a sharp optimum rather than a broad optimal zone (71, 93, 129, 143, 296). More rarely, a curve skewed to the left has been reported (48, 84) from studies of fungi with low optimum tem- peratures. The diversity of types of curve becomes less surprising when one considers the number of factors that have been shown to affect the shape of the curve. Of these, time of observation is the most impor- TEMPERATURE 409 Temperature >■ Figure 7. Idealized types of temperature-response curves of spore germination. The distinctive features are exaggerated for emphasis. Curve 1, symmetrical with a broad optimum; curve 2, skewed to the right; curve 3, symmetrical with a sharp optimum. tant: early observation yields a curve with a sharply defined optimum, but in time the germination at less favorable temperatures catches up and the optimum becomes much broader. This phenomenon appears to be general (13, 23, 84, 166, 219, 241, 260, 299). Second in importance to the time effect as a factor in determining the shape of the temperature curve is the general principle that the limits of germination are narrower if some other factor is non-optimal. Thus, conidia of Sclerotinia fructicola at an unfavorably acid pH ger- minate only at 13-29°C and the curve is sharply peaked at 21°; at a more favorable reaction, pH 2.4, both the total range and the optimum are much broader (283). The depressant effect of low temperature on Erysiphe gram in is germination is virtually abolished in the presence of nutrients (317), and the minimum temperature of germination in Col- letotrichum lagenarium is lower in a nutrient medium than in water (101). The principle applies also to relative humidity, i.e., the tempera- ture curve becomes narrower and sharper as the humidity becomes more unfavorable to germination (118, 286). Although different spore forms of the same species usually have at least similar temperature responses (148, 178, 285), the endoconidia and macroconidia of Chalaropsis thielavioides show optima at 9-30° 110 SPORE GERMINATION and 12°, respectively; in this organism the age of endoconidia has a pronounced effect on the shape of the temperature-germination curve (168). So far, we have considered the response of germination, i.e., initiation of the germ tube, to temperature. The further growth of the germ tube may be plotted against temperature. When such curves are compared to germination curves, there is often, perhaps usually, a close corre- spondence (42, 86, 93, 103, 146, 241). With many fungi, the optimum is rather narrower than that for germination; examples include Ery- siphe graminis (317), Physalospora obtusa (92), Clasterosporiarn car- pophilum (103), and several rust fungi (57, 20, 266). In still other fungi, the shape of the curve or the position of the optimum, or both, are different (84, 102, 116, 148, 260). In general, it appears that germ tube growth is closer in its temperature response to mycelial growth than it is to spore germination; the data of Snell (260) on Lenzites sepiaria are especially instructive in this connection. For this reason, there are many problems, especially in plant pathology, in which the temperature characteristics of germ tube growth are more illuminating than those of germination. The preceding discussion emphasizes the limitations of the conven- tional temperature study of spore germination. Results often depend upon the criterion used, and upon such sometimes uncontrolled vari- ables as age of spore, humidity, and pH. The studies are not, however, to be dismissed as useless; thus, it is often possible to correlate the eco- logical niche of a parasitic fungus with the response of its spores to temperature (44, 57, 317). In this discussion we have tacitly assumed that temperature affects all processes up to the appearance of the germ tube in the same way. There is indirect evidence, from two types of temperature reaction, that this assumption is not valid, that the processes which initiate spore ger- mination have a different sensitivity from later stages. Although it might be expected a priori that at lower temperatures the rate of all processes is slower, the data of Figure 8 show a more complex situation: to be sure, the rate of germination at suboptimal tempera- tures is somewhat less than the rate at the optimum (18°), but the prin- cipal effect of low temperature is to delay the onset of germination, i.e., to increase the latent period. Once germination has begun, it is almost as rapid at low temperature as at high. Similar data may be found in studies of Fusarium moniliforme (241), Sclerotinia jructicola (299), and Magnusia spp. (276). Both rate and latent period are strongly affected by low temperature in Phytophthora infestans (63) and Unci- nula necator (67). The latent period, and by inference the metabolic TEMPERATURE 100 411 Time, hours Figure 8. Temperature and the rate of germination of uredospores of Phragmidium mucronatum. Redrawn from Cochrane (57), by permission of the Cornell University Agricultural Experiment Station. reactions occurring in the very earliest phases of germination, is uni- formly extremely sensitive to low temperature. At supraoptimal tem- peratures there is again evident a differential sensitivity of these early reactions (284). Sensitivity to low temperature of the processes concerned in the ini- tiation of germination is paralleled by sensitivity of the same processes to a favorable temperature. The germination of uredospores of Phrag- midium mucronatum at non-optimal temperatures is markedly accel- erated by exposure for a short time to a moderate temperature; for example, germination at 9°C is increased about 160 per cent by a pre- exposure for one hour at 18°, during which exposure there is no visible sign of germination (58). The same phenomenon in Puccinia chrysan- themi is suggested by the data of Campbell and Dimock (44). Simi- larly, direct germination, by germ tube formation, of the conidiosporan- gia of Phytophthora infestans at 20° is greatly increased by prior exposure for 5-10 minutes to 40° (279). It can be suggested, therefore, that early metabolic reactions in ger- mination have a different and more sensitive response to temperature than do the later stages. Obviously, the evidence is far from complete. It is perhaps analogous that the cracking of the sporangium wall in Blastocladiella sp. responds somewhat differently to temperature than do the later stages of germination (45). Parenthetically, it may be noted that these results have a methodo- 412 SPORE GERMINATION logical significance: in studies of temperature, the medium and its container must be adjusted to the experimental temperature before spores are sown. We may assume that this precaution has often been overlooked and deduce that many temperature-germination curves are in error, in that they exaggerate the extent of germination at sub- and supraoptimal temperatures. As noted in Chapter 1, a given fungus usually has about the same temperature response whether mycelial growth or spore germination is the criterion (24, 86); a few exceptions have been reported (84, 298). 10. OXYGEN AND GERMINATION It will be recalled that mycelial growth in fungi is often possible at very low oxygen pressures but that truly anaerobic growth is unlikely (Chapter 1). Virtually the same applies to spore germination in gen- eral, with a few exceptions to be noted here. Failure of submerged spores to germinate or to germinate normally has often been observed and credited, without proof, to oxygen deficit. As pointed out by Gottlieb (113), too many other factors are affected by submergence for this simple evidence to be aceptable. Ideally, experimental studies on the oxygen requirements for spore germination should include, first, demonstration of the response to graded oxygen pressures which are accurately measured and, second, proof that the spores which fail to germinate at a given p02 are still viable. Relatively few experiments meet this ideal standard. Uredo- spores of Puccinia graminis tritici (2) and chlamydospores of Ustilago zeae (217) fail to germinate at zero oxygen pressure and reach maxi- mum or near-maximum germination at 30-38 mm of oxygen. Conidia of Botrytis cinerea germinate as well in 5 per cent oxygen as in air (37), but spores of Synchytrium endobioticum are severely inhibited at 3 per cent oxygen (292). Removal of oxygen by pyrogallol prevents germination in, for ex- ample, uredospores of rust fungi (6, 129) and chlamydospores of Usti- lago avenae (143). Although conidia of Erysiphe graminis are reported to germinate at a low level in an atmosphere of nitrogen (70), other studies on powdery mildew fungi indicate that germination is pre- vented by anaerobiosis (34, 167). Ascospores of Neurospora tetra- sperma also fail to germinate anaerobically (107). Conidiosporangia of three species of Phytophthora can germinate by swarmspore formation — but not by germ tube — under anaerobiosis, although other members of the Peronosporales fail completely to ger- minate without oxygen (291). In the Mucorales, it appears that spores BIOLOGICAL EFFECTS 413 of strongly fermentative species have a very low oxygen requirement and those of less strongly fermentative species a higher one; all, how- ever, probably require some oxygen. In this study of the Mucorales (304), it was also found that the swelling of the spore of Mucor spp. can occur without oxygen, i.e., oxygen is required only at a later stage in development. Pure oxygen is at least slowly toxic to spores of Streptomyces scabies (242); in short-term experiments inhibition of germination in Botrytis cinerea by high oxygen pressure is measurable but small (37). It appears, to conclude, that spore germination usually requires at least some oxygen, but that the atmospheric level is more than ade- quate. In nature, it seems unlikely that spore germination is prevented by oxygen deficits except under such extreme conditions as flooded soil or heavily polluted water. 11. CARBON DIOXIDE AND GERMINATION From the observations cited in Chapter 7, that carbon dioxide often accelerates the early growth of fungi, we might guess that spore germi- nation is accelerated by carbon dioxide. This proves to be true in Aspergillus niger (223). Effects of high levels of carbon dioxide in increasing chlamydospore germination have been interpreted, however, as arising primarily from the effect of the dissolved gas on the acidity of the medium (218). At high concentrations, carbon dioxide is partially or even com- pletely inhibitory to spore germination. Brown (37) determined that the spores of seven different fungi, if sown in water, are completely inhibited by an atmosphere of 20-30 per cent carbon dioxide; sensitiv- ity is markedly less if the spores are sown in a plant decoction. Other reports agree that carbon dioxide is inhibitory and that the level at which germination is affected may be low (180, 266) or high (139, 169). Inhibition at concentrations of 1 per cent or so is probably specific; effects of the higher concentrations could be indirect, e.g., on acidity or on oxygen supply. 12. BIOLOGICAL EFFECTS ON SPORE GERMINATION It is commonly observed that excessive crowding of spores on a sur- face results in reduced germination; the phenomenon is known in many fungi (27, 37, 70, 73, 148, 160, 172) and may be described by the general term self-inhibition. In Neurospora crassa, crowding of conidia re- duces the germination rate, but not total germination (240). These 414 SPORE GERMINATION interactions of spore populations are formally analogous to the self- inhibition of growth reported in species of actinomycetes (207, 290) and inhibition of spore germination in Phycomyces blakesleeanus by products of mycelial growth (249). However, we may anticipate that these reactions of growing mycelium are more complex and more de- pendent on the environment than is self-inhibition in spores. Bacillus globigii endospores produce a substance, antagonized by alanine, which inhibits germination of fresh endospores of the same species (265). Self-inhibition has been most clearly demonstrated in studies of rusts, Uromyces phaseoli (313, 314), Puccinia graminis tritici (2), and P. he- lianthi (315). The inhibitory effect of a dense concentration has been demonstrated in vivo, on the host plant, as well as in vitro (Figure 9); most of our information indicates that the active material is volatile. Allen (2) has determined several characteristics of the substance — possibly substances — responsible for self-inhibition of Puccinia grami- nis tritici uredospores. The active material is stable to heat, is inacti- vated or removed from solution by glass surfaces, and is more effective at high pH than at low. The inhibitor is a spore metabolite produced under aerobic conditions. This differs somewhat from the situation 0.4 0.8 1.2 1.6 Weight of spores, mg per ml 2.0 Figure 9. The effect of uredospore concentration on germination in Puccinia graminis tritici. Curve 1, spores in open vessels; curve 2, spores in sealed vessels. Redrawn from Allen (2), by permission of Phytopathology . BIBLIOGRAPHY 415 in Aspergillus niger (155) and Coccomyces hiemalis (148), in both of which limited evidence suggests than an inhibitor is present in the spore at the time of its formation and can be removed by washing. Preliminary evidence has been reported by Forsyth (91) that Puccinia graminis tritici uredospores produce 2-methyl-butene-2 (trimethylethyl- ene), and that this compound inhibits spore germination. Whether this is the effective substance remains to be seen. Self-inhibition has at least one obvious ecological function, that ger- mination of spores within the fructification is minimized. The unger- minated spore is much more resistant to the environment than the germinated and is, of course, the effective agent of dispersal. Although germination in situ has occasionally been reported, it appears to be the exception; this suggests that self-inhibition is a general principle in spore physiology, and that it is not limited to the few fungi so far studied. At the other extreme from self-inhibition is the situation in Ophiobo- lus graminis (210); ascospores germinate slowly if isolated singly, more rapidly if many spores are present. 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Hydrostatic pres- sure has hardly been studied in relation to the fungi; very young in- fections by plant pathogens are prevented from developing by the application of pressure (156), and reduced pressure somewhat fa- cilitates germination in Erysiphe graminis, perhaps by accelerating carbon dioxide loss (12). It may be expected that studies of cellular reactions and of the action of fungicides under high pressures will contribute to our understanding of the fungi, as similar studies have contributed to the knowledge of bacteria (76). 1. HIGH TEMPERATURE The practical problem of heat sterilization is primarily that of killing bacterial endospores; fungal structures, with some exceptions, are less resistant. Consequently, there has been little interest in fundamental studies on the death of fungi at high temperature. However, destruc- tion of fungi within host tissues has proved practicable for a few patho- gens which are present as mycelium in seeds or cuttings (148). The thermal death point, defined as the least temperature at which 423 424 PHYSICAL AGENTS all cells are killed in 10 minutes, has been determined for spores of several fungi as 40-60°C, a value close to that usually reported for vegetative cells of mesophilic bacteria (1, 19, 21, 24, 88, 128). How- ever, spores of some species are much more resistant; known examples are the conidia of Nocardia sebivorans (38) and ascospores of Bys- sochlamys fulva (70), Neurospora crassa (40), and Penicillium sp. (153), Conidia of thermophilic actinomycetes appear to be heat-resistant (37). Ascospores are more resistant than conidia in Neurospora crassa (40, 125) but are equally susceptible in Endoconidiophora fagacearum (24). Data on mycelium are too scanty for generalization; Fusarium lini is killed in 10 minutes at 55°, but Colletotricluim lini survives 3 or 4 hours at this temperature (139). A culture of Nocardia sebivorans withstands 10 minutes at 90° (38), and suspended sclerotia of Penicil- lium sp. are completely killed at 82° only in about 16 hours (153). Spores of Erysiphe spp. are somewhat more resistant to heat than my- celium (157), but comparative studies of spores and mycelium in other fungi have not been made. Neither the thermal death point nor the related thermal death time has proved adequate in studies of bacteria (124), and there is no reason to hope that they will prove to be any more satisfactory for work with fungi. Development of a better index will, however, have to wait until we have more knowledge of the order of death. Characteristically, the killing of vegetative cells of bacteria is exponential, i.e., a plot of the logarithm of survivors against time is linear (118, 124), although non-exponential orders of death by heat are known (77, 119). Data on the killing of fungi by heat are few in number; Figure 1 shows a non-exponential order of heat killing of ascospores of Byssochlamys fulva; ascospores of Penicillium sp. respond similarly (153). At lower temperatures the curve becomes distinctly sigmoid, i.e., short exposures have proportionately less effect (128). These few statements refer to studies of spores; sclerotia of Penicillium sp. respond to heat as one would expect in a multicellular structure (153). Tentatively, we may propose that the non-exponential order of death of fungus spores reflects genetic heterogeneity of the material, but the data are too limited at present to justify a firm conclusion, and in particular the occurrence of bi- or multinucleate cells may influence the reaction. Death by heat is most drastically affected by moisture. Cells die much more rapidly at high temperatures if they are wet (22, 37, 74) or if the relative humidity is high (24, 53). Heat resistance in Bys- sochlamys fulva is affected by the pH of the menstruum (70). I.O W T I M V I R AT I' RE 425 Figure 1. The order of killing of ascospores of Byssoclilarnys fulva by moist heat (85°C). Replotted from Hull (70), by permission of the Cambridge University Press. 40 Time, minutes 80 The mechanism of death at high temperature is usually thought to be denaturation of proteins; the high Q10 of thermal death (128) is consistent with this explanation. Whatever the validity of the de- naturation theory at high temperatures, some other explanation must be invoked to explain death at temperatures which are just above the maximum for development. Thus, mitosporangia and sporophytic mycelium of Allomyces sp. die within 24 hours at 30° (98), and uredo- spores of Phraginidiam mucronatum survive only 12 hours at 27° (22). Presumably some fraction of metabolism continues under these condi- tions, and either endogenous reserves of essential materials are de- pleted or essential cofactors decay and cannot be resynthesized. Less likely, but possible, is the accumulation of one or more metabolites in toxic concentrations. 2. LOW TEMPERATURE No systematic study of damage to fungi by low temperature has been reported. Even more important, the scattered data have been obtained under such different experimental conditions that comparison and generalization must be very tentative indeed. Too often we are not in- formed of essential factors, particularly (1) the type of cells present, (2) the quantitative degree of injury, (3) the rate of freezing, and (4) the rate of thawing. It must be realized that survival can only be 426 PHYSICAL AGENTS assayed after thawing the material, and, as shown later (Figure 2), the rate of subzero warming is critical. Exposure to very low temperatures, e.g., that of liquid air, does not completely kill mycelium, fruiting bodies, or dry spores of most fungi (8, 16, 79, 89). The mycelium of some fungi does not survive this treatment (89), and wet conidia of Neurospora crassa are killed almost immediately at -170° to -190°C (40). Exposure to moderately low temperatures — from 0 to — 40°C — is of immediate ecological interest and has frequently been investigated. The results, however, defy any neat generalization; either fungi differ very widely in their tolerance or, as suggested above, methods of investi- gation are so different that comparison is not legitimate. Mycelium of Phycomyces nitens and Botrytis cinerea is killed by exposure to —15 to —22° (6), and neither mycelium nor sclerotia of Phymatotrichum om- nivorum survives 24 hours at —13° (39). But mycelium of Phacidium infestans and Collybia velutipes is still viable after 138 days at —21° (114), and fruit bodies of Schizophyllum commune withstand exposure to -15 to -40° (17). Similar differences are reported in the survival of spores at these moderately low temperatures. Uredospores of the rust fungi are usu- ally found to survive no more than 2 months or so at —15° to —40° (22, 42, 104, 149), but at least some uredospores of Melampsora lini are still viable after 1295 days at —10° (41), and uredospores of Uromyces phaseoli live as long as 2 years at —20° (59). Both spore forms of Endoconidiophora fagacearum are germinable after 83 days at —10° (24). From these reports and from the partial list compiled by Luyet and Gehenio (91), it appears that spores are in general less damaged by low temperature than mycelium, dry spores less than wet. Most of the reported experiments on the true fungi are probably examples of relatively slow freezing, in which the primary injurious factor is dehydration; crystals of ice form extracellularly and withdraw water from the cell (105). Damage to dry cells is less than to wet (14, 40), as would be expected. Furthermore, it was early found that solutes protect the mycelium against freezing injury (6); hydrogen bonding substances — sugars, glycols, etc. — essentially lower the amount of water lost to the crystallization process (105). Visual observation suggests that hyphal tips are more susceptible than basal mycelial cells and that aerial hyphae are more resistant than submerged (87); the water content may be a factor in these differences. Rapid freezing, in which crystal formation occurs throughout both cells and medium, must obviously be so rapid as to limit the size of VISIBLE LIGHT 427 Figure 2. The influence of the rate of warming on viabil- ity (germinability) of conidia of Aspergillus flavus frozen at — 70 °C. Each curve represents a separate experiment. Re- drawn from Mazur (103), by permission of the Rockefeller Institute for Medical Research. 1 10 100 1000 Rate of subzero warming, °C per minute the individual crystals (105). It follows that thawing must be equally rapid, in order to prevent crystal growth. This is shown especially well by the data of Mazur (103), part of which is reproduced in Figure 2. The lethal effects of slow, as compared to rapid, warming were par- ticularly evident in the range —20 to 0°C; i.e., the rate of subzero warm- ing, not the rate of thawing of ice, is the critical factor. The possibility of freezing material so rapidly as to prevent crystal formation entirely has been considered at length by Luyet and Ge- henio (91). The experimental difficulties are formidable, and there is some problem in determining whether or not success has been achieved (105). In any event, it is clear that prevention of crystal formation is not essential to survival at low temperature. We may conclude that fungi, especially vegetative mycelium and certain types of spores, are relatively susceptible to freezing injury, particularly to the slow freezing which occurs in nature; repeated freezing and thawing are even more damaging (115). The principal factor is almost certainly the moisture content of the material; it fol- lows that preservation in the frozen state will be most successful if ap- plied to dry spores or other cells which can withstand a preliminary desiccation. Although it is unlikely that freezing can eliminate all cells of a given species, cold injury must be an important ecological factor at least for soil fungi and plant pathogens (22, 39). 3. VISIBLE LIGHT Light of the visible range, 400-800 m/x, exerts undoubted, but so far ill-defined, influences on mycelial growth, spore germination, and other phenomena; effects on reproduction have been discussed in 428 PHYSICAL AGENTS Chapter 11. It must be recalled that exposure to "white light" may, depending on the source, involve exposure to some ultraviolet; heating effects, too, cannot de disregarded unless specifically excluded. Several early observations, reviewed by Smith (127), and later data (20, 58, 75, 113, 129) agree that light retards mycelial growth; dry weight measurements confirm this for Sclerotinia fructigena (55) and Karlingia rosea (60). The growth of Botrytis squamosus on agar is completely inhibited by light (112). Light stimulation of growth, infrequently reported (60, 127), has been most carefully analyzed in Blastocladiella emersonii (18). In this organism dry weight increases of up to 141 per cent, at constant temperature, are induced by illumination. It is observed further that fixation of carbon dioxide is increased by light, and it is proposed that light acts in some way to accelerate the reductive carboxylation of succinic acid and arketoglutaric acid. Vegetative mycelium, like reproductive structures, may be photo- tropic (122) and may show zonation in response to light and dark periods (25). Pigmentation is frequently dependent upon or enhanced by light (127); Centrospora acerina forms its red pigment after a very short exposure (107). Melanogenesis in Neurospora crassa is some- what reduced by illumination (123). The effect of light on carotene formation has been mentioned elsewhere (Chapter 6). It has been recognized at least since 1860 (61) that strong light in- hibits spore germination. Whether spores are more or less sensitive than mycelium is in doubt (127). Obviously, any ultraviolet con- tamination is especially serious, and there is no doubt that many of the lethal effects of light can be credited to a short wavelength com- ponent. However, in both Erysiphe graminis (117) and Puccinia gra- minis (30, 31) experiments with filters implicate the longer wave- lengths, in the yellow-red region, and exclude ultraviolet. Moist spores are more susceptible than dry (23). A number of reports — too many to disregard — suggest that spore germination is increased by light or even requires light (47, 48, 71, 106, 130, 155, 160). The mechanism of such an effect is difficult to imagine, and it can only be said at this time that further quantitative work under accurately controlled conditions is necessary. Photodynamic action has been demonstrated by Kaplan (78) in Penicillium notatum: conidia vitally stained with erythrosin are sensi- tized so that visible light, normally innocuous, becomes mutagenic. Eosin-stained cells of Neurospora crassa appear from limited data to respond similarly (34). Methylene blue intensifies the zonation re- sponse of Penicillium notatum to light (120a). ULTRAVIOLET RADIATION 429 4. ULTRAVIOLET RADIATION The lethality and mutagenicity of ultraviolet radiation are prob- lems of general biological interest; studies on fungi have made sig- nificant contributions toward their solution. The early, and now largely obsolete, literature is reviewed by Smith (127), more recent work by Pomper and Atwood (116). Techniques for the irradiation of spores and isolation of radiation-induced mutants are well developed (7, 120, 138, 152). Virtually all studies of the action spectrum of killing and of muta- genesis in fungi, as in other organisms, agree that the most effective wavelength is in the region 260-265 m^. The action spectrum of lethality to Trichophyton mentagrophytes is shown in Figure 3; similar spectra prevail in Neurospora crassa (67, 109), Penicillium notatum (69), Ustilago zeae (85), and Aspergillus oryzae (102). The primary peak is generally accepted to be consequent upon the specific absorption of this wavelength by nucleoproteins. McAulay and associates (92, 93, 94) have reported that biological activity for Chaetomium globosum is greatest at 280 m^, in the region of absorption by proteins. These observations cannot be reconciled with any other data and merit re-examination. Aspergillus terreus conidia mutate after exposure to higher wave- lengths (297 and 313 m/j(,) or to sunlight (65). Efficiency, however, is Figure 3. The action spectrum of killing of spores of Tricho- phyton ynentagropliytes. Re- drawn from original of Figure 4, A. Hollaender and C. W. Emmons, Journal of Cellular and Comparative Physiology, Vol. 13, p. 391-402 (1938), by permission of the Wistar In- stitute of Anatomy and Biol- ogy- 240 260 280 Wavelength, mju 430 PHYSICAL AGENTS very low, and at still longer wavelengths most organisms survive very large doses (50), although bactericidal action can be detected at high intensity (62). The requirement for high intensity poses serious methodological problems in attempts to demonstrate the mutagenicity of the long ultraviolet (43). Very short ultraviolet — the Schumann region, below 200 m^ — has not been studied, primarily for technical reasons; indications are that lethal action at least is very great (85, 111, 159). The lethality of ultraviolet radiation is most easily expressed by the fraction of irradiated spores which fail to germinate. Doses that are not lethal delay germination or induce abnormalities (33, 64, 67, 85). Different fungi vary widely in susceptibility; thus, spores of Mucor dispersus require almost 20 times the energy per spore that is sufficient for an equivalent degree of inactivation of Aspergillus meUeus spores (32, 33). In surveys of fungal susceptibility it is often noted that 100 Figure 4. The survival of microconidia of three mutant strains (inositolless) of Neurospora crassa as a function of ultra- violet dosage. Redrawn from Giles (51), by permission of the Long Island Bio- logical Association. 100 200 400 800 Irradiation time, seconds ULTRAVIOLET RADIATION 431 Figure 5. The survival of macroconidia (average number of nuclei 2.67 per conidium) of Neurospora crassa as a function of ultraviolet dosage. Redrawn from Atwood and Norman (5), by permission of the National Academy of Sci- ences. 60 120 180 Irradiation time, seconds 240 species with dark spores tend to be more resistant (36, 46), and a pro- tective action of wall pigments in Puccinia graminis uredospores has been claimed (29). Pigmentation alone, however, cannot explain spe- cific differences in sensitivity. The curve generated by a plot of survival against dose is usually one of two types: 1. The exponential curve, in which the logarithm of the fraction surviving is a linear or approximately linear function of dosage. This curve is characteristic of microconidia of Neurospora crassa (Figure 4) and spores of Penicillium spp. (2, 46), Aspergillus melleus (33), and Streptomyces flaveolus (80). 2. The sigmoidal curve (Figure 5), which is the more common type, reported in studies of Aspergillus spp. (73, 158), Mucor dispersus and Rhizopus suinus (33), Ustilago zeae (85), Glomerella cingulata (95), Fusarium eumartii (126), Trichophyton mentagrophytes (64), and Streptomyces griseus (121). The significance of these curve shapes has been debated vigorously, and it may be too early for a categorical explanation. By itself, the sigmoidal curve could be interpreted as expressive of variability in the spore population, and Markert (95) has shown such variability in sensitivity in Glomerella cingulata. However, this interpretation ap- plied to the exponential curve requires a highly improbable distribu- tion of sensitivity within the population. Studies on Neurospora crassa 1:;l' P H Y S I C A L AGENTS (5, 109, 110) suggest instead that the shape of the dose-response curve is a function of the number of nuclei per cell: exponential for uninu- cleate spores, sigmoidal for bi- and multinucleate spores. Death in a multinucleate spore requires that several targets must absorb radiation; the sigmoidal curve follows from this requirement. Two mechanisms of lethality have been proposed (109): induction of recessive lethal mutations and non-genetic damage to nuclei. Ob- viously, the first of these cannot be of much significance in the death of conidia having more than one nucleus. The induction of morphological and biochemical mutations by ultraviolet radiation responds rather differently as dosage is increased. As shown in Figure 6, mutation frequency increases with dosage only to a point, then levels off or declines at higher dosage. Dose-response curves of this general type have been reported from several studies in which all morphologically visible mutations are scored (63, 66, 67, 73, 100, 135). However, in one instance in which a known mutation at a single locus was scored, the response curve was strikingly different (51), and the mutation frequency in Streptomyces flaveolus does not drop at high dosage (80). Ultraviolet-induced mutagenesis is affected by several modifying environmental factors. Pretreatment before irradiation with infrared radiation, 2,4-dinitrophenol, or nitrogen mustard increases mutagenesis (133, 134, 135, 136). Survival and mutation rate in Streptomyces sp. are modified by treatments applied after irradiation (143, 145, 146). Respiratory poisons and anaerobiosis decrease both the lethal and the mutagenic effects of ultraviolet radiation on Penicillium chrysogenum Figure 6. Morphological muta- tions in Penicillium notatum as a function of ultraviolet dosage. Plotted from data of Hollaender (63). 30 60 Dosage, ergs per spore IONIZING RADIATIONS 433 (152). Most important, perhaps, is the observation of McElroy (102) that postirradiation treatment with high hydrostatic pressure reduces the recovery of morphological mutants in Nenrospora crassa and Asper- gillus terreus. This observation suggests that the primary action of ultraviolet (and chemical mutagens) is to cause the formation of a semi-stable intermediate which may decay to a new, mutant, state or may revert to its original, normal, state (103). The hypothetical inter- mediate may be the gene itself or a metabolite or metabolic system (143, 146). Peroxides and unknown products in irradiated media are mutagenic to Neurospora crassa (27, 142). However, results published so far fail to implicate products formed by ultraviolet irradiation of the medium, at the wavelengths employed in mutation studies, as important factors in the induction of mutations. Photoreactivation was discovered in Streptomyces griseus (82) and has been observed in several of the true fungi (13, 17a, 52, 81, 109). As usually studied, photoreactivation means that lethal effects of ultra- violet radiation are counteracted in part by subsequent exposure to light of longer wavelength, 330 to 480 m^; however, other changes induced by ultraviolet are also counteracted. The action spectrum of photoreactivation in S. griseus has a pronounced peak at 436 m^, dif- fering significantly from the action spectrum of the same process in Escherichia coli (83). The theories and problems of photoreactivation are reviewed by Dulbecco (35). Wainwright and Nevill (144) present evidence that photoreactivation exerts independent effects on survival and mutagenesis in Streptomyces sp. 5. IONIZING RADIATIONS Ionizing radiations, as the name implies, are radiations whose pas- sage through a material causes the production of ion-pairs. Most studies on fungi have employed X-rays or the equivalent y-rays emitted by radium and other radioactive materials; a few data are available on a-rays (from polonium, but also produced by the cyclotron), /3-rays (from radioactive materials or from an ion source), and cyclotron-pro- duced neutrons. All these types of radiation, we assume, have the same mode of action but differ, of course, in energy, ionization per unit path length, and ability to penetrate (86). Consequently, physically equivalent doses of different types of ionizing radiation are not bio- logically equivalent. However, it is not yet clear how such differences in biological effect are to be explained. Thus, in the killing of spores of Aspergillus terreus, the densely ionizing radiations (protons and 434 PHYSICAL AGENTS a-rays) are more effective than the less densely ionizing X-rays and y-rays, but the order of effectiveness is reversed if mutagenesis in A. terreus is the criterion (132). The killing of A. niger spores by «-irra- diation is similarly most effective at high ionization density (161). On the other hand, lethality to vegetative cells of Escherichia coli is great- est with radiation of low ionization density (159). Lethal and mutagenic effects of types of radiation other than X-rays have been reported, viz., neutrons (4, 56, 99), a-particles from polonium and radium (9, 10, 11), y-rays from radioactive elements (26, 137), and ^-particles from carbon- 14 or an ion source (84, 150, 151). Earlier studies are reviewed by Smith (127). The lethal effect of X-irradiation on spores of fungi and actinomy- cetes can usually be expressed as an exponential or approximately ex- ponential dose-response curve (3, 72, 80, 121). Sigmoid survival curves have only occasionally been reported (45, 90). It seems probable, es- pecially from preliminary work on yeasts, that the form of the curve may be primarily determined by the number of nuclei per cell (116). This consideration cannot apply to the striking response of dry spores of Aspergillus terreus: X-irradiation follows the sigmoidal pattern but survival after irradiation with a-particles is exponential (132, 162). Mutation frequency is linear with dose over a range; as shown in Figure 7, some material shows a plateau in the curve at high dosage (54, 80, 108). Presumably this plateau will be reached with any or- ganism provided the dose is high enough. Theoretical models for this saturation effect are considered by Harm and Stein (57). 100 200 Dose, kiloroentgens 300 Figure 7. Mutation frequency in Streptomyces flaveolns as a function of X-ray dosage. Each curve represents a separate experiment. Redrawn from Reiner (80), by permission of the Williams and Wilkins Company. LONG WAVELENGTH RADIATION 435 Lethality and mutagenic action are not the only effect of irradiation. In particular, irradiated spores may germinate but fail to make con- tinued growth; this phenomenon, reported in several fungi (11, 28, 90, 141) is analogous to the delayed effect of X-irradiation on yeast (116) and to the "microscopic mutant" induced in Chaetomium glo- bosum by ultraviolet or ionizing radiation (44, 93). The irradiation of Aspergillus niger spores with low (non-lethal) intensities of a-parti- cles results in the development of asporogenous colonies in an unspeci- fied number (11). These delayed effects are perhaps best described as lethal mutations, but something more than a descriptive analysis would surely be of interest. Stimulation of spore germination by ionizing radiation has been reported (15, 154). Other less well-defined stimulatory effects claimed by earlier workers are reviewed by Smith (127). The environmental factor most significant in modifying responses of fungi and bacteria to X-irradiation is the ambient oxygen pressure (68, 131): both lethality and mutagenesis are less at low oxygen tension. Sensitivity of Aspergillus terreus to X-irradiation is also increased by previous exposure to infrared radiation (133, 135) and by high spore moisture content (131). The lethality of X-irradiation to Strepto- myces sp. is decreased by various postirradiation treatments (147). Radiation biology is undergoing rapid advances at this time, and it is to be expected that generalizations will have a short life. In the fungi it appears that the primary effect of radiation is on the nucleus, and this effect — which may be itself complex — is best visualized on the basis of the target theory so strongly urged by Lea (86). 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The Action of Chemical Agents i .nhibition by chemical agents has probably been the single most active field of investigation of the fungi. Of necessity, however, much of this work has been directed to the practical prob- lem of controlling the fungi pathogenic to plants and to man and other animals and the fungi that destroy economically valuable com- modities. Theoretical problems have, of course, not been neglected; the important work has been summarized by Martin (278) and more recently by Horsfall (172, 174). In a study of the physiology of fungi, such as this, it seems appropri- ate to concentrate attention upon those materials, regardless of their practical importance, about which we have some information as to mode of action. This approach neglects many fascinating problems in the chemical inhibition of fungi: the role of antibiotic substances in soil (152, 425) and the use of purified antibiotics (218, 337, 396, 430), the expanding field of chemotherapy of plants (90, 175, 176, 402), and many of the developments in the use of organic compounds as fungistatic or fungicidal agents. It is to be hoped that sufficient attention will be devoted to mode of action so that these topics can be treated theoret- ically in the near future. The chapter has been organized so as first to consider some general problems and then to give detailed attention to a few types of com- pounds— heavy metals, sulfur, quinones, and the chelating agents — which have been most actively investigated. 440 RESPONSE AND ITS MEASUREMENT 441 1. THE RESPONSE TO TOXICANTS AND ITS MEASUREMENT For practical reasons most of our information on the response of fungi to toxic agents relates to the inhibition of spore germination or of mycelial growth. However, other effects are occasionally observed and may be briefly described. The most important aspect of response is, of course, its measurement, since any investigation of mode of action presupposes an accurate, reproducible assay of the biological effect of a fungicidal or fungistatic agent. Morphological Responses. Morphological abnormalities of growing hyphae are caused by many toxicants, including such diverse agents as diphenyl (340), dyes (363), benzothiazole (142), solanin (2), and sodium tungstate (256). Other instances, as well as data on abnormal development of germ tubes, are tabulated by Horsfall (174). Thymol and carvacrol cause swelling of conidia of Aspergillus niger (450), as does phenol (420). Griseofulvin, an antibiotic produced by Penicillium spp. (Chapter 6), causes stunting, branching, distortion, and other abnormalities of growth in virtually all chitin-walled fungi tested, not in the yeasts or Oomycetes, which have non-chitinous walls (Chapter 2). Brian (42) suggests that griseofulvin either acts as a growth regulator or interferes with a growth regulator controlling wall extension; spore germination of Botrytis allii is not prevented by a concentration 500 times that which affects germ tube growth (43). Griseofulvin is taken up by green plants (74, 403) and protects to a considerable degree against infection by Alternaria solani (44). A few fungi which probably have chitin walls are nonetheless insensitive (304). Camphor causes the formation of large, possibly diploid, conidia (361, 362). Cholic acid and sodium taurocholate induce the formation of uninucleate cells in the dikaryon of several basidiomycetes, prob- ably converting a clamp initial to a separate branch (286). Mitotic Poisons. Many fungitoxic substances are known to inhibit mitosis in other organisms (137, 174). Exposure of basidia of Gymno- sporangium clavipes to penicillin or streptomycin results in abnormal development and interference with meiosis (27). In yeast, penicillin inhibits cell division without inhibiting growth, so that filaments are formed; reversal experiments suggest that sulfhydryl groups essential to cell division are blocked by the antibiotic (310). The Response of Developmental Stages. Specific inhibition of spor- ulation has both theoretical and practical importance. Simultaneous 442 CHEMICAL AGENTS Figure 1. The response of germination (curve 1) and oxy- gen uptake (curve 2) of Neuro- spora sitophila conidia as a function of the concentration of cycloheximide. Respiration was measured in the presence of sucrose, germination after re- moval of the toxicant. Redrawn from McCallan, Miller, and 10 ^00 1000 Weed (247), by permission of Cycloheximide, Mg per mg spores the B°yce Thompson Institute for Plant Research, Inc. and fully quantitative determinations of both growth and sporulation are difficult; preliminary data suggest that the high metal requirements of sporulation (Chapter 11) make the process especially sensitive to chelating agents, e.g., dimethylglyoxime, but many other types of toxi- cant also reduce reproductive activity (179, 332, 333, 340, 342, 399, 428, 448, 457). It is generally believed that mycelial cells are less sensitive to toxicants than are spores; this is borne out by direct comparisons for sulfanila- mide (125, 147) and cycloheximide (430a, 431). The difference may, however, be related to uptake (246a). Inhibition of Respiration. Inhibition of respiratory activity as measured by oxygen uptake is often parallel to growth inhibition (106, 269, 309, 334), and it has even been tentatively suggested that the prin- cipal activity of Captan1 is on pyruvic carboxylase (160). However, most studies, on very different compounds, show that in general endo- genous and substrate respiration is much less sensitive to a wide range of fungicides than is spore germination or growth (83, 125, 127, 132, 203, 224). Figure 1 is drawn from an extensive study of fungicidal activity, judged by killing of spores, and spore respiration; the figure shows that a compound, in this case cycloheximide, kills at concentrations which do not measurably reduce oxygen uptake. The data of Klopping (203) show a similar relation between fungistatic action and inhibition of respiration; 8-hydroxquinoline has no effect on respiration at a con- centration 100 times the fungistatic concentration. Low concentrations of toxicants often stimulate respiration (247). It seems likely that membrane permeability to substrate is increased 1N-(trichloromethylthio)-4-cyclohexene-l,2-dicarboximide. RESPONSE AND ITS MEASUREMENT 443 under these conditions, although other explanations are possible (20). Increase in oxygen consumption is also caused by poisons, e.g., 2,4- dinitrophenol, which interfere with the utilization of respiratory en- ergy (380). We may conclude that in general the activity of fungicides against respiration does not parallel their effects on growth, and infer that effective fungicides usually act on systems much more sensitive than is the gross oxygen uptake. We may speculate that a compound which acts on respiration and growth at approximately the same concentra- tion— mercuric chloride is an example (247) — is a general enzyme poison. It should also be clear that, although a respiratory system may be inhibited by a fungistatic agent, this fact alone is not sufficient evidence that the inhibition observed is the primary locus of toxicity. Sclerotinia laxa grown in contact with partially inhibitory concen- trations of several fungicides is affected physiologically in its formation of respiratory enzymes, some being increased and others decreased in amount by specific compounds (55). Mutagenic Effects of Chemical Agents. Cultural variants of unde- termined genetic status are induced by high levels of zinc (89) and nitrite (Chapter 8). Mutation in fungi, verified by genetic studies, is caused by a wide variety of chemicals, e.g., nitrogen and sulfur mus- tards and related compounds (16, 286a, 400, 409), peroxides (87, 188), epoxides (208), diazomethane (189), and caffeine (115). Some of these are sulfhydryl reagents, others possibly generate free radicals, but no single mode of action can be proposed at present. Fungistatic Action of Chemical Agents. Both practically and theo- retically, the most important effect of toxicants on fungi is the inhibi- tion of growth. This fungistatic action is the most frequently measured and should not be confused with killing, fungicidal action sensu stricto. We may distinguish two classes of fungistatic assay: (1) determination of my- celial growth on a natural or artificial medium, and (2) determination of spore germination. In both methods, the test fungus and the toxi- cant are in contact throughout the period of incubation. The early development of agar plate methods is described by Bate- man (22), who established a satisfactory quantitative treatment of the data obtained with Fomes annosus. This treatment has since been extended to other fungi (330, 426). After a more or less protracted lag period, which is itself an index of toxicity, the rate of growth across the agar surface is linear with time. Comparison with a control inocu- lation on toxicant-free agar yields the basic datum, degree of inhibition. 444 CHEMICAL AGENTS 0.8 i i i i i i i 1 | 1 _ • .x" .^' c .^ ■B 0.6 15 ^ IE mS* c mS* IP 0.4 ~ ^ _j • 0.2 - 0 1 1 1 I 1 1 ! I I ! 1 1 Figure 2. The inhibition of Aspergillus niger by p-hydroxy- benzoic acid in an agar medium. Drawn from data of Vincent (426). -0.5 0 +0.5 Log millimolar concentration p-hydroxybenzoic acid This is related to toxicant concentration by the equation: e6 y = ax" (1) where y stands for degree of inhibition, x for concentration of the fun- gicide, and a and b are constants. A plot of the logarithm of the degree of inhibition against the logarithm of the concentration yields curves of the type shown in Figure 2. Not all curves are as clearly linear as this one (426). A variety of special methods are essentially similar to the assay of Bateman just described (26, 102, 108, 109, 114, 151, 203, 271, 283, 296, 297, 317, 318, 359). Criteria used include the least concentration preventing all growth, the colony diameter at a fixed time, or the dry weight in liquid culture. A rather different approach is found in the use of filter paper discs impregnated with the toxicant (218, 415); here, of course, diffusion becomes an important factor. These methods all have particular and limited value; thus, the medi- cal mycologist has a practical interest in the inhibition of established infections rather than in prevention of spore germination. Fungi which do not sporulate in culture may have to be studied as mycelium. For theoretical work on mode of action, however, the presence of nu- trients and, in agar media, of undetermined contaminating substances, severely restricts their value. It is almost always found that nutrient materials reduce the apparent toxicity of fungi toxic agents (214, 352, 404). In a qualitative way, it is usually true that the agar plate method and the spore germination method agree, but they may give divergent results for particular compounds (174). As a rule, fungitoxic ma- terials are more active against spore germination and spore respiration RESPONSE AND ITS MEASUREMENT 445 than they are against the growth or metabolism of mycelium (125, 147, 271, 341, 430a, 431). Fungistatic methods adapted for the assay of wood preservatives are reviewed by Cartwright and Findlay (61). The type of assay chosen has marked effects on the apparent efficacy of test compounds (110, 223, 348). Usually, loss in weight of treated wood blocks is the best cri- terion of protection against decay; a somewhat more rapid assay based on the static bending strength of a treated wood block has been intro- duced (126). Special methods for fabric treatments are described by Siu (386). Soil fumigants are usually of interest insofar as they kill organisms, but a fungistatic method of bioassay has been devised (101). For the practical testing of plant protectants, assays employing living inoculated plants are well worked out (243, 245, 251, 280). The most progress has been made by the use of bioassays which are based on inhibition of spore germination. This fundamental tech- nique has been developed both practically and theoretically by McCal- lan and by Horsfall, whose papers must be read for a thorough under- standing of the method (172, 173, 174, 241, 242, 243, 252, 254, 258, 259, 436, 445). More or less standard procedures have been agreed upon (71, 72). A standardized spore suspension of a test fungus — Monilinia fructi- cola (=ScIerotinia fracticola) and Stemphylium sarcinaeforme (=Mac- rosporium sarcinaeforme) are the most often used, but other sporulat- ing fungi are suitable — is allowed to germinate in contact with the toxicant, which may be either mixed with the spore suspension or sprayed on the slide (33). If washed or vacuum-collected spores are used, it is usually necessary to add nutrients, and these materials, especially complex plant products such as orange juice, must be taken into account in any consideration of the mode of action; orange juice, possibly by reason of its content of citric acid, which can complex metals, reduces both the fungicidal and the fungistatic effect of copper on at least many fungi (91, 250, 287). In the spore germination assay, a simple plot of per cent inhibition (or germination) against toxicant concentration or the logarithm of toxicant concentration yields a sigmoid curve. This can be converted to a straight line in the so-called logarithmic-probability system of coordinates. The abscissa of this curve used in most studies is the logarithm of the dose, the ordinate a calculated "probit" or "normal equivalent deviation." Use of this coordinate system is based on the general phenomenon that resistance in a population to a toxic agent is distributed normally with respect to the logarithm of the dose (30, 446 CHEMICAL AGENTS 1.0 10 100 Concentration of metallic ion, ppm 1000 Figure 3. Dosage-response curves for the action of heavy metals on Stempliylium sarcinaeforme. Ordinate is probit of per cent inhibition of germination, abscissa is logarithm of concentration. Redrawn from more extensive data of McCallan, Wellman, and Wilcoxon (252) and selected to show different types of curve. Re- produced by permission of the Boyce Thompson Institute for Plant Research, Inc. 174, 244, 446). Representative curves of this type appear in Figure 3. Three characteristics of the log-probit curve are of greatest interest: position, shape, and slope. Lateral position is, of course, indicative of absolute toxicity; either the ED50 or the ED95 — the concentration re- quired to inhibit germination by, respectively, 50 and 95 per cent — may be determined from the curve and used in the rating of fungi- cides. Position is affected by several factors in the assay — the presence of nutrients, the age of spores, the concentration of spores, extremes of temperature, the germination time, and the physical or chemical form in which a toxicant is supplied (49, 91, 144, 174, 252, 260, 265, 436). Non-linear curves are not, however, uncommon; thus, a detailed study of the action of copper sulfate on Monilinia fructicola and Alter- naria oleracea shows for both fungi a J-shaped curve, concave upward (242). Parker-Rhodes (323) proposed that this curvature represents the cumulative effect of more than one chemical or physical process in the inhibition reaction and also detailed an exponential transformation of the independent variable which converts the J curve to a linear form. Use of the transformation does not require acceptance of the multi- process theory of fungistatic action, some of the difficulties of which are discussed by McCallan (244). Non-linear curves of other types are frequently encountered (242, 244, 252). The slope of the log-probit curve is its most interesting feature. Since the ordinate is based on the distribution of sensitivity in the population of spores, a steep slope means that there is relatively little RESPONSE AND ITS MEASUREMENT 447 difference between the concentration which inhibits the most sensitive spore and that required to prevent germination of the most resistant spore. Conversely, a low slope, or flat dosage-response curve, indicates that the difference in dose required for inhibition of the two extremes of resistance is large (446). Slope is most conveniently measured as the reciprocal of the difference between ED84 and ED50. Although several factors of the assay influence slope, the compound itself is the most significant (252). From this it has been argued that the slope is an indication of the fundamental mode of action (174, 241, 279, 346, 416). That is, compounds with the same mode of action should generate dosage-response curves of the same slope. Rich and Horsfall (346) show that homologues of 2-imidazoline yield curves of similar slope and different position; the ED50 varied from 0.0037 to 3.57 ;u,M per square centimeter, but the slopes were essentially alike — 9.37, 11.01, and 9.97. From this and other experience, it is argued that position measures ability of the compound to reach its site of action, and that slope indicates the mode of action once the site has been reached. Attractive as this hypothesis is, there are other factors that must give us pause. First, as has been mentioned, non-linear curves are not un- common. Second, there is as yet no measure of mode of action which is truly independent and which could serve as a check on the hy- pothesis. Finally, virtually all the available data are dosage-response curves in which external concentration is the independent variable. The discovery that fungi accumulate very large amounts of toxicant (p. 450) does not by itself defeat the hypothesis, but this fact does necessitate a re-evaluation of the whole problem (246). Spores of many fungi increase markedly in size during the first stages of germination (Chapter 12); inhibition of this growth process in Myrothecium verrucaria has been adapted to the bioassay of fungistatic agents (268). Fungicidal Action. Fungicidal action, the killing of fungi by a toxicant, requires an assay method in which cells are exposed to a compound for a known period of time and are then removed from contact with the toxic agent and tested for viability. Such assays have been worked out for a variety of theoretical and practical problems (26, 69, 120, 128, 151, 246a, 250, 360, 463). The plot of probit of per cent germination against logarithm of time is linear for the action of copper, silver, arsenate, and crystal violet (250). The same plot of the data of Muller and Biedermann (302) shows, however, that the strict exponential relation is evident only at 448 CHEMICAL AGENTS 100 Uranium, ppm 10,000 Figure 4. Dosage response curves for the action of uranium acetate on spores of Alternaria solani. Curve 1, fungistatic action; curve 2, fungicidal action at 20 hours; curve 3, fungicidal action at 10 minutes. Redrawn from McCallan and Wellman (250), by permission of the Boyce Thompson Institute for Plant Research, Inc. relatively high concentrations of copper — at lower concentrations there is no perceptible damage for a considerable period of time, after which the exponential form of curve appears. Similarly, the shape of the time-inhibition curve for the action of phenol is affected by the con- centration of the toxicant (391). Killing is also a function of temperature; the fungicidal action of a number of relatively mild materials is sharply and non-linearly in- creased as the temperature is raised, with an especially sharp break as the temperature approaches its lethal value for spores in the absence of toxicant (183). Although a strongly fungicidal agent is fungistatic, the reverse is not necessarily true. In particular, several compounds of high fungi- static activity — potassium dichromate, uranyl acetate, imidazolines, and 8-hydroxyquinoline are examples — have only very weak fungicidal activity (250, 437). In general, dosage-response curves for fungicidal action are much flatter than those for fungistatic, i.e., the range in resistance of the population is greater (Figure 4). 2. TOLERANCE AND RESISTANCE Species of fungi differ widely in their sensitivity to toxicants. This has been especially noticed since the introduction of antibiotics and organic fungicides; differences in sensitivity of 50- to 100-fold are com- mon (203, 270, 349, 440). However, strains tolerant to more general poisons are also encountered in studies on mercury (124, 232, 423), fluoride (227, 423), arsenate (303), and creosote (65). Copper tolerance is often associated with tolerance to hydrogen ion or other cations (195, 387, 397). This suggests that the mechanism of copper tolerance is a TOLERANCE AND RESISTANCE 449 general exclusion of cations from the cell. As Horsfall (174) points out, an organism producing metabolic acids may be less sensitive to a toxi- cant which must compete with hydrogen ion for its site of action. Fungi may also be "trained," by serial culture in media containing low doses of a toxicant, to tolerate a normally inhibitory concentration. Such adaptation has been demonstrated to arsenite (157), borax (205), fluoride (222), and copper (186, 195); other reports are cited by Leben etal. (217) and Horsfall (174). Such adapted strains and strains which appear spontaneously in culture are usually unstable and lose their resistance on subculture in toxicant-free media (158, 195). Spon- taneously arising sectors of Fusarium caeruleum appear in plates ex- posed to the vapor of 2,3,5,6-tetrachloronitrobenzene, and these variants are somewhat less sensitive to the poison than is the parent strain (263); they are, however, almost equally sensitive — by rather inexact criteria — to 2,3,4,5-tetrachloronitrobenzene (47). Genetic analysis has been performed only infrequently on resistant strains of fungi. Resistance to indole toxicity in Neurospora crassa (77) and to acriflavine in Aspergillus nidulans (355) is genetically con- trolled. This is in line with the usual experience in studies on bacteria and yeast (85, 216, 371). Copper-adapted yeast is reported to contain a ribonucleic acid which can be transmitted to, and which confers copper resistance on, normal unadapted yeast cells (293). Physiological mechanisms of acquired tolerance are possibly of di- verse types. Work on the resistance of trypanosomes to arsenicals sug- gests that resistant strains are less permeable to the drug (38), possibly by virtue of an altered distribution of surface charges (366). Fluoride tolerance in clones of Propionibacterium pentosaceum appears also to be a permeability phenomenon (427). Other mechanisms are, however, possible: an antimycin-resistant variant of Venturia inaequalis probably lacks the normal respiratory system sensitive to the antibiotic (219). Detoxification of fungicidal agents has been observed and is, of course, a potential mechanism of resistance. Methylation of selenium, tellurium, and arsenic (Chapter 6) conceivably fits in this category. The effects of cycloheximide on both spore germination and respira- tion decline with time, indicating an inactivation (431, 440). Oxida- tive processes have been suggested as the basis for resistance to phenols and quinones (200, 347); ^-oxidation of a fatty acid side chain or hy- droxylation of a ring in some instances causes detoxification (54, 57). Other possible detoxification mechanisms are reviewed by Gottlieb (134); it seems likely that the most common is the complexing of toxic metals by chelate-forming metabolites such as amino acids and hydroxy acids. 450 CHEMICAL AGENTS 3. SELECTIVE ACCUMULATION We have customarily thought of the inhibiting of an unwanted or- ganism as a problem in selective toxicity, to find materials which are inherently more toxic to one form of life than to others. It has been assumed that the ultimate basis for such a selective action must be the existence of some sensitive protoplasmic reaction in the undesirable organism which does not exist, or is dispensable, in other species. This approach has, of course, had its successes, notably in explaining the chemotherapeutic value of the sulfonamide drugs, but the major effort of modern biology has been to show how similar are all forms of life in fundamental biochemical aspects and thus to render less and less likely the prospect of finding truly selective agents at the protoplasmic level of action. It now appears that another selective principle may be equally im- portant: the differential accumulation of a toxicant by the unwanted organism. The term selective accumulation has been used by Yarwood (460) to describe the finding that spores of Uromyces phaseoli accumu- late more sulfur from a fungicidal treatment with the vapor phase than do the leaves of the host plant. That is, we may hope that some materials which are perhaps equally toxic to all organisms may be accumulated selectively by a fungus; the result, of course, is equivalent to selective toxicity and is a form of it, but does not depend on an inherent protoplasmic or enzymatic sensitivity. Selective accumulation does, of course, require that the sensitive organism differ physiologically from the insensitive, but it does not require us to postulate, for ex- ample, that protein synthesis or energy metabolism of the susceptible species differs from that of the resistant. The problem becomes nar- rower and more manageable: by what mechanism does the sensitive organism accumulate, or the insensitive exclude, the toxicant? In the previous section, some evidence has been cited that resistant strains of microorganisms owe their resistance to the failure of the toxicant to reach sensitive sites. This is in a sense the reverse of selective accumulation, to be considered provisionally as selective ex- clusion. The idea of selective accumulation is not, of course, new; it has been known for some time that the lethal action of heavy metals on bacteria follows upon accumulation of the toxicant by cells in amounts far in excess of their concentration in the external medium (455). It is apparent that fungus spores can similarly remove from the medium very large amounts of many different toxicants. Marsh (273) found SELECTIVE ACCUMULATION 451 that spores of Monilinia fructicola exposed to copper accumulate the metal to a concentration 2000-4000 times its external concentration. Even more striking results are reported by Miller, McCallan, and Weed (290, 291), using different fungi and such diverse materials as silver, cerium, 2-heptadecyl-2-imidazoline, and ferric dimethyldithio- carbamate. Several features of these data warrant emphasis: 1. Uptake is extremely rapid; spores of Neurospora sitophila take up their limit of the imidazoline within 15 seconds from a dilute solu- tion, within 5 minutes from a more concentrated solution. Uptake of dithiocarbamates by yeast is similarly rapid (127). 2. Fungicidal action is a linear function of the amount of toxicant taken up. 3. Some toxicants are concentrated as much as 10,000-fold. 4. A material which has low inherent toxicity on a spore weight basis may be so extensively concentrated by spores as to be an effective inhibitor. The application to bioassay is obvious — external concen- tration is not necessarily a measure of effective dose. Valko (419) dis- tinguishes "apparent toxicity," a function of external concentration, from "specific toxicity," a function of the amount of toxicant absorbed by the cell. 5. Species differ in the amount taken up of a given toxicant. 6. Unrelated toxicants are taken up independently, i.e., previous ex- Figure 5. The relation between uptake and antifungal activity (test organism Neurospora sito- phila) in chlorine derivatives of 2,4dichloro-6-anilino-5-tria- zine. Curve 1, fungicidal ac- tion; curve 2, fungistatic action. Compounds: u = unsubstituted, o — ortho-chloro, rn = meta- chloro, p = para-chloro deriva- tive. Drawn from data of Burchfield and Storrs 51). 1.0 1.5 2.0 2.5 Relative uptake by spores 452 CHEMICAL AGENTS posure to one material does not materially change the results with another. Closely related chemicals appear, however, to compete for a common receptor (246, 288). 7. Absorbed toxicants of some types are held firmly in or on the spore and can be released in quantity only by rather drastic treatment. Silver, by contrast, is easily removed by exchange (292). The relation of uptake to effectiveness is shown in Figure 5 for a series of related s-triazines. Fungitoxic action is seen to be correlated with the capacity of the spore to remove the toxicant from solution. In general, two mechanisms suggest themselves as alternative ex- planations of the phenomenon of rapid uptake against a concentra- tion gradient. First, it is, of course, known that "active transport" of nutrient materials into cells occurs commonly and results in accumu- lation against a gradient, and Pramer (336) has shown that strepto- mycin is so accumulated by Nitella clavata. However, to suggest active transport as the mechanism of entrance of a variety of toxicants raises the difficulty that active transport as usually understood necessarily involves the expenditure of metabolic energy and is prevented by many inhibitors. As discussed later in connection with the heavy metals, it seems at present more promising to look toward a second mechanism, the adsorption of the toxicant by a solid phase in or on the cell. For both these alternative mechanisms, it should be noted, the same general relation between uptake and external concentration holds; in both there is some saturation value of the external concentration. Conse- quently, whether or not the concentration-uptake relation is described by the adsorption isotherm does not distinguish between active trans- port and adsorption. More important results will come from studies of the effect of metabolic state upon uptake. Drawing primarily from studies on the action of heavy metals (see below), we may suggest that a toxic material which does not move freely across the semi-permeable membrane undergoes a two-stage process. In the first phase there is a rapid sorption of the toxicant outside the permeability barrier, or perhaps both outside it and on it. In the second phase, the material is released at a slower rate to the interior of the cell from the saturated peripheral sites; only in this phase, for most materials, does interference with growth occur. This hypothesis has many difficulties which are, however, better con- sidered in connection with heavy metal toxicity. It ignores, for con- venience, the possibility that the sorbed toxicant may exert specific effects before it enters the cell at all; this possibility is treated as a sepa- rate topic. SURFACE ACTION 453 4. SURFACE ACTION It has been suggested immediately above that the first phase of toxi- cant action is an accumulation of the active material in a peripheral layer or zone outside the semi-permeable membrane. We have further argued that, for most toxicants, the primary inhibitory effect is not at this stage but later, after some of the toxicant has desorbed from the outer zone and penetrated the protoplasm proper. However, such a sharp separation of phases is only a convenience, since accumulation of a foreign material is very likely to have some effects on the cell mem- brane. Hypothetically we may set up three possible classes: (1) effects of the sorbed toxicant are themselves lethal; (2) sorption contributes to lethality by damaging the osmotic barrier and thereby facilitating or making possible entrance of the toxicant into the protoplast; and (3) sorption has only minor effects. A number of surface agents are known to be lethal to bacteria, at least in part, as the result of destruction of the semi-permeable mem- brane (118, 181). The antibiotic polymyxin has the same effect — disruption of the osmotic barrier — in bacteria (307). The contribution of lysis to lethality has, however, been questioned (398). Surface toxicity need not, in principle at least, be confined to effects on permeability. If surface anabolic reactions are essential to growth, for example, inhibition of them by a non-penetrating toxicant would be sufficient. Finholt et al. (110) interpret the different toxicity of ali- phatic amines in malt agar culture vis-a-vis wood block tests as indi- cating that a toxicant could act externally by preventing the action of an extracellular hydrolytic enzyme essential to survival. This sug- gestion deserves further exploration; perhaps the most promising pos- sibility is that the extracellular enzymes are actually synthesized ex- teriorly to the osmotic barrier and that a toxicant might interfere with the synthesis. Such an effect would be missed completely in the usual assay which provides diffusible nutrients and thereby renders the fungus independent of extracellular enzymes. The second possibility, that sorption of a toxicant might contribute to lethality without being the primary event, comes to mind particu- larly in relation to a few findings that toxicants at sublethal concen- trations alter cell permeability. Cellular phosphorus is lost from spores treated with subfungistatic doses of silver (288) or of substituted s-triazines (51). Lysozyme treatment causes an increase in permeability to water and, probably, to furfural (235). In bacteria, phenol increases permeability to glucose; it is probable that "one-way" effects on per- IM CHEMICAL AG K NTS meability occur, a compound increasing the loss of cellular nitrogen and phosphorus but not increasing the entrance of substrates (182). Finally, we may expect relatively minor effects of sorbed materials, especially on permeability and accumulation. We have referred earlier (Chapter 8) to the possibility that amino acid toxicity is exerted by the exclusion of one acid by another. Among other metabolic effects of yS-propiolactone on Blastomyces dermatitidis is the inhibition of amino acid uptake (119), although this could be secondary. Reduction of the rate of solute movement is shown especially well by the studies of Rothstein (356) on the poisoning of yeast respiration by uranium. This appears to be a specific interference with the up- take of hexoses; endogenous respiration and the utilization of pyruvic acid or ethanol are very little affected. Rothstein suggests that uranyl ion complexes with groups at or near the cell surface which are es- sential to sugar uptake, and suggests further that these groups are cellular polyphosphates which are also capable of binding other ions (357). Similar responses to uranium have been found in animal ma- terial (315). 5. HYDROGEN-ION CONCENTRATION In any survey of the literature on fungicides, one is forcibly struck by the common failure to determine or to report the pH of the test solution. For practical purposes this may not be important, at least if the pH is that of the projected use situation, but conclusions on mode of action of ionizable materials are difficult indeed to draw if the acidity is unknown. Some effects of pH are obvious: a fungicide may break down more or less rapidly and in so doing yield products of greater or less toxicity; thus, thiosulfate forms a more toxic product (162), and dithiocarba- mates form less toxic materials (432). A second possibility is that the toxicant itself changes the pH to a value unfavorable for growth or spore germination; this can of course be compensated for by specific controls. The chelating agents, as mentioned later, become less effec- tive in binding metals as the pH is lowered; uranyl ion forms complexes with hydroxyl ion at pH values above 4.5 and becomes therefore una- vailable (356). Competition between hydrogen ion and toxic cations is considered in connection with the heavy metals. When weak acids or bases are dissolved in water, the degree of ioniza- tion is pH-dependent. This is evident by inspection of the equation for the dissociation of an acid: HA ^± H+ + A- (2) HYDROGEN-ION CONCENTRATION 455 The negative logarithm of the ionization constant of Equation 2, signi- fied by pKa, is numerically equal to the pH at which the acid is 50 per cent dissociated. The pKa is characteristic of the molecule, and one of the commonly overlooked effects of addition of substituents to an organic molecule is that the substituent may change pKa. Exactly the same considerations, of course, apply to weak bases. Acidic toxicants almost invariably are more effective at low pH than at high; as discussed later, the effect of pH is most noticeable near pKa, so that data on very strong and very weak acids will not normally show an effect of hydrogen-ion concentration under the usual experi- mental conditions. Data on inhibition of fungi which fit the rule of greater activity at low pH are extremely numerous, although they do not include spore germination results. As examples, we may cite the action of fatty acids (161, 350, 354, 456), aromatic acids (75, 76, 78, 162, 339, 390), acid dyes (205), substituted phenols (41, 187, 383), and nitrous acid (Chapter 8). The usual explanation for the effect of dissociation on toxicity is that only the undissociated molecule enters the cell. This reflects the general experience that ions penetrate the cell at least much more slowly than do neutral molecules (84). Several experiments with fungi have shown that the ester of a toxic acid is more effective than the free acid (24, 62, 140, 178, 301). The ester almost certainly pene- trates the cell more rapidly than the partially ionized acid and is then split by esterases. More direct evidence would, however, be desirable, since esterification of an acid may have other effects, for example on reactivity of groups near to the carbonyl carbon. It is often stated that one can conclude from these pH-activity studies that it is only the undissociated molecule which is toxic, i.e., action within the cell is visualized as caused solely by some combination of the neutral molecule of the acid or base with a sensitive element. For this conclusion to hold, the biologically effective dose in terms of neutral molecules must be invariant with pH; Simon and Blackman (383) review several studies on echinoderm eggs in which this relation prevails for both weak acids and weak bases. However, studies on a wide variety of compounds — phenols, cresols, fluoride, azide, cyanide, etc. — show quite conclusively that the ion must also play a role in toxicity within the sensitive volume of the cell (382, 383). Figure 6 exemplifies the data: it is seen that as the ionization increases with increasing pH the equi-effective concentration of undissociated molecules actually declines. The total drug — ions plus neutral molecules — rises of course, but it is the fall in undissoci- ated toxicant which is decisive. 456 CHEMICAL AGENTS Two conclusions flow from this analysis. First, inhibition of the sensitive cell element must be effected by both neutral molecules and ions, although they need not be equally effective. A chemical model for this behavior is analyzed by Briggs (45). Second, the pH at the site of action must be affected by changes in the external pH, in order for the equieffective concentration of the penetrating species to decline as found (these are long-time experiments in which we can assume equi- librium conditions). As mentioned in Chapter 1, the effect of external pH upon cytoplasmic pH is not known with any certainty or precision. The significance of pKa can now be assessed (Figure 7). The rise in required concentration for an equal effect begins at about pKa; at lower pH (higher for a base) there is much less effect of acidity (383). It follows that in theoretical or comparative studies on weak acids or weak bases the effect of ionization can be minimized by assaying activity at a pH at least two units below pKa (for an acid) or above pKa (for a base). There are exceptions to this rule, the sulfonamide antibacterials being the outstanding and best-known example (6, 382). The acridine antibacterials exemplify a rather different relation of toxicity to dissociation and pH (4, 6), one which has so far not come to light among organic fungitoxic materials. In this series only the cation is biologically effective. This fact, and observations on the Figure 6. The effect of pH on the concentration of 2,4-dinitro- phenol (pKa — 4.0) required to effect a 50 per cent reduction in the growth rate on agar of Trichoderma viride. Curve 1, total concentration; curve 2, concentration of undissociated molecules. Redrawn from Simon and Blackman (383), by permission of the Cambridge University Press. STRUCTURE AND ACTIVITY 457 Figure 7. The general form of the relation between pH and the toxicity of a weak acid. Redrawn from Simon and Beevers (381), by permission of the American Asso- ciation for the Advancement of Science. 1 r i i i i i 1 1 1 c o PK r c a> u iZ o " o o> > o Hg > Cu > Cd > Cr > Ni > Pb > Co > Zn > Fe > Ca. For any one organ- ism the order may differ slightly (23, 256, 300). Roughly the same order of effectiveness is found in experiments on animals (373). Because of the reactivity of the heavy metals, growth experiments in complete media are of dubious value; the spore germination technique is to be preferred. Proteins and peptones are especially active in de- creasing fungitoxicity (153, 214, 458), but other materials present in culture media have the same effect (247). Present information allows us to consider the three most toxic heavy metals — silver, mercury, and copper — together, i.e., to assume provi- sionally that all have the same general mode of action; silver may, how- ever, differ. We can also, but perhaps with less justification, consider that the final toxic effect of heavy metal compounds is to be ascribed to the metal itself. However, some problems are raised by this second assumption. Different inorganic salts of copper, for example, are not always of equal toxicity (28, 279); when copper is supplied as cupric ammonium sulfate, it is more firmly bound by spores than copper sup- plied as the sulfate (34). These differences cannot be explained as yet in detail, but we may guess that the form in which copper is supplied affects its uptake by the spore. Provision of silver as the iodide sharply reduces both uptake and toxicity as compared to those of other silver halides (288). The toxicity of coordination complexes of copper deserves special attention. For years the effectiveness of Bordeaux mixture as a plant protectant was a mystery, inasmuch as the copper is present on the leaf in an insoluble complex. A number of investigations, reviewed by Martin (278) and by McCallan (244), established firmly the proposi- tion that materials from the spore solubilize the copper. The most important of these substances are the amino and hydroxy acids, com- pounds which form soluble chelate complexes with copper. Mono- methylamine liberation by spores of Tilletia caries also results in solu- bilization of copper (327), and seeds of higher plants liberate copper from Bordeaux mixture (429). This hypothesis requires that soluble copper complexes be toxic. Some data show that, under specified conditions, the glycine and malate action may not be so great (130). Concentration factors are critical: complexes are as fungistatic as copper sulfate itself (257); fungicidal HEAVY METALS 467 if the ratio of complexing agent to metal is high — five to ten — toxicity disappears (28). This is consistent with the concept that cell and com- plex compete for copper; it is significant that ethylenediaminetetraace- tate (EDTA), which forms very stable metal complexes, abolishes tox- icity at a 1:1 molar ratio to copper (28, 302). Unfortunately, these same observations are consistent also with a second explanation, that the malate and glycine complexes enter the cell whereas the EDTA complex does not. Organic mercurials are generally more toxic than inorganic, both to bacteria (39) and to fungi (174, 372). Part of the answer to their practical effectiveness lies in the appreciable volatility of some of them (17, 88, 220). There is some evidence, however, that the effective or- ganic mercurials penetrate the cell as such, i.e., that they are more readily taken up than mercuric ions (35, 324). Against this may be cited limited evidence from pH studies in a complete growth medium (187) that phenylmercuric acetate is more toxic as the ion. In general, both inorganic and organic mercurials affect the same enzymes and are equally antagonized by thiols, although occasional secondary effects complicate the picture, as in the case of pancreatic amylase (319). The extreme case of increase in toxicity by provision of a metal in an organic molecule is found in a study of the fungitoxicity of tin com- pounds (199). Stannous and stannic chlorides are essentially non-toxic, but tri-n-butyltin acetate prevents the growth of fungi at 0.1-0.5 ppm. McCallan and Wellman (267) find that copper, silver, and mercury are both fungistatic and fungicidal. Fungicidal action is, of course, a function of time of exposure and of the method by which the spores are washed free of toxicant. The action of heavy metals on fungi may be conveniently divided into two topics: the uptake of metals by spores and the metabolic or other effects of the metal once it has reached a sensitive site. We have already mentioned that metals, in common with other fungi- static materials, are accumulated by fungus spores in very high concen- tration and very rapidly (288, 290, 291). Some further light is cast on this by earlier and technically less satisfactory studies on the effect of copper and mercury on Tilletia tritici (34, 35, 36). Copper uptake was found to follow the Freundlich adsorption isotherm, i.e., to be non- linearly related to external copper concentration. Copper supplied as an ionizable salt is removable by acid, but the copper of cupric ammo- nium sulfate, a coordination complex, is not so removed. Finally, ionic copper disappearing from solution is almost quantitatively ac- counted for by other cations — H+, Fe++, Mg++, etc. — liberated from the spore. K.S CHEMICAL AGENTS Figure 9. The effect of pH on the toxicity of copper chloride to spores of Alternaria tenuis. Curve 1 (solid line), control; curve 2 (broken line), 0.0001 M CucL. Drawn from data of Biedermann and Midler (28). Divalent cations generally reduce both uptake and toxicity of the heavy metals (1, 28, 150, 226, 232, 273, 308). The uptake of silver by spores is reduced by copper and, more completely, by mercury (288). Competition by hydrogen ion is equally important; this is shown in Figure 9 and also by the fact that absorbed copper is removed from spores by acid (3, 34, 174, 291). There are at present two ways in which to interpret the data on up- take of metals by spores. First, we may assume that the entire volume of the spore protoplasm accumulates the metal. This assumption re- quires, in view of the rapidity of the uptake, that the metal move rela- tively freely across the semi-permeable membrane; such a rapid move- ment is suggested by the speed with which cellular zinc of Aspergillus niger exchanges with extracellular zinc (288), and by some data on the loss of potassium from bacteria (209). Staining methods show that copper does enter spores (129, 418), but this approach has not been developed sufficiently to be of critical importance. The second mode of interpretation is to consider metal uptake as an adsorption, defined as a process that concentrates one component of a system at an interface, and to limit the locus of this adsorption to peripheral regions of the cell, i.e., outside the permeability barrier. HEAVY METALS 469 This approach has the advantage that we need not postulate that ions are able to penetrate the cell rapidly; although, as just mentioned, there is some evidence for rapid passage of metals through the membrane, it is generally true, considering all the organisms studied, that cations, especially di- and trivalent cations, do not move easily into cells by dif- fusion (84). The concept that the initial uptake is an adsorption, or ion exchange, process has the additional advantage of explaining both ion competition effects and the fact that uptake of copper is accom- panied by roughly equal extrusion of other ions. Nor does a surface adsorption require that the cell be living or even metabolizing nor- mally; it has been noted (291) that uptake of several materials con- tinues long after enough toxicant has been absorbed completely to prevent spore germination. Against an adsorption hypothesis, it may be urged that the uptake of some materials is so large that the classical monomolecular film could not accommodate the observed toxicant (290). To test these two hypotheses, the most direct approach is provided by model experiments on the uptake of methylene blue by bacteria (238, 239) and by ascospores of Neurospora tetrasperma (407). The uptake reaction is independent of temperature; a metabolically powered ac- cumulation would have a high temperature coefficient. Dead cells adsorb dye as rapidly and completely as living cells. Different cations compete with each other for a limited number of sites; as in yeast (356, 357), it appears that the cell surface has a countable number of more or less strongly negative binding sites. Exploration of the uptake-con- centration relation does not appear so profitable; any type of accumula- tion would show saturation effects as the external concentration is raised. If it proves possible to regard the initial rapid uptake of metals as a surface adsorption, then the toxic action of heavy metals can be visu- alized, as already mentioned, as composed of a rapid stage of uptake at the surface followed by a slower penetration, perhaps through a dam- aged membrane, into the interior of the cell. Alternatively, the metal may enter as a coordination complex. Effects of sorbed metals upon the cell surface must be considered as a possibility, although it seems doubtful that the major site of toxic action is outside the permeability barrier. Copper reduces the perme- ability of the erythrocyte to glycerol (84), and heavy metals, especially mercury, depress phosphate uptake by bacteria (294). Silver increases the loss of phosphorus from fungus spores, although mercury does not (288). Interference with catabolic, anabolic, or carrier mechanisms out- side of or on the plasma membrane is also a possibility, as in the action 470 CHEMICAL AGENTS of uranyl ion on yeast (p. 454). Increases in respiratory rate are in- duced by sublethal concentrations of metals (247); the explanation may lie in an increased permeability to exogenous substrate or, less likely, in inactivation of regulatory enzyme systems (20). Present theories of the ultimate action of heavy metals visualize them as enzyme poisons and admit only the question of whether they are primarily effective against sulfhydryl enzymes or generally inhibitory to enzymes of all types. The reaction of a divalent metal with a sulfhydryl enzyme is depicted conventionally as mercaptide formation: SH S / /\ En 4- HgCl2 -+ En Hg 4- 2HC1 (3) \ \/ SH S The resemblance to a five-membered chelate complex — there are two points of attachment on the enzyme — is obvious. A monovalent metal or an organic mercurial like p-chloromercuribenzoic acid reacting as such presumably forms a mercaptide with only a single sulfhydryl group. The argument for the importance of this reaction is based primarily on the observation that heavy-metal poisoning of both bac- teria and fungi can be prevented or, within certain time limits, re- versed by such thiol compounds as cysteine, glutathione, and 2,3-di- mercaptopropanol (107, 185). In addition, it can be shown that mercaptide-forming reagents react stoichiometrically with free sulfhy- dryl groups of proteins (13, 316). Shaw (373) proposes a model for the inhibition of vital processes that is based on these considerations and the added assumption that the sulfhydryl groups with which cations combine are part of the catalyt- ically active site of the inhibited enzyme. The driving force of Equa- tion 3 is regarded as proportional to the free energy of the simple reac- tion: Me++ 4- S— ±=; MeS (4) That is, toxicity should be correlated with the insolubility of the metal sulfides (MeS). The order of insolubility is in fact very much like the order of toxicity: Hg = Ag > Cu > Pb > Cd > Zn. This is also the order of affinity of metals for the sulfhydryl group of purified serum albumin (204). Emphasis on reaction with sulfhydryl groups may, however, divert attention from other possibilities. In particular, the simple demon- stration that thiol compounds antagonize the toxicity of metals to in- HEAVY METALS 471 tact cells cannot by itself prove that the primary effect of metals in vivo is on sulfhydryl enzymes. Thiols form stable covalent compounds with metals and would be expected, therefore, to compete successfully with almost any bond formed between enzyme and metal. That the sulfhydryl hypothesis may be too limited is indicated above all by the observation that many enzymes which are not apparently dependent upon sulfhydryl groups for their activity are inhibited in vitro by one or more of the heavy metals. These include sucrase, /3-glu- cosidase, arginase, asparaginase, carbonic anhydrase, catalase, and cyto- chrome oxidase (319, 388, 405). As already mentioned, inhibition of respiration and spore germination at approximately the same concen- tration by heavy metals is also suggestive of a general poisoning of many different enzymes. If we grant that heavy metals are general enzyme poisons, we may inquire about the sites at which they might act on an enzyme. Obvi- ously, as Horsfall (174) emphasizes, the order of toxicity closely resem- bles the order of stability of metal chelate complexes. From this, it is apparent that the heavy metals might act by displacing from an enzyme a metal like magnesium which is essential to its activity and which is bound normally in a coordination complex. However, the order of stability of chelate complexes is, for obvious reasons, much the same as the order of insolubility of metal sulfides just described, and either series may with equal probability be considered the more important. Several potential binding sites are available on proteins to react with transition metals like mercury, silver, and copper. These include car- boxyl, phosphoric, imidazolium, ammonium, and phenolic hydroxyl groups (204). The primary carboxyls and the a-amino groups of the amino acids are, of course, tied up in peptide bonds, and such side chains as the guanidine and amide groups cannot be expected to bind metals at biological pH values. In principle at least, inactivation of enzymes by metals is not limited as to location; the enzyme or enzymes may just as well be at the surface of the cell as in the interior. However, it seems more probable that the metals reach the interior of the cell and are not confined in their action at fungistatic concentrations to the surface. This discussion of toxicity of the heavy metals makes it clear that there are numerous unsolved problems, especially with regard to the penetration of heavy-metal ions into the cell. Some of the more im- portant points may be listed in conclusion: 1. Differences in toxicity between various compounds of a given metal are ascribed to differences in the rate at which the toxic metal 472 CHEMICAL AGENTS reaches the site of action. The contrast between ionized salts and unionized lipid-soluble organic compounds is especially marked. 2. Toxicity of the metals is affected by hydrogen ion and divalent cations and by the presence of natural substances — proteins, amino acids, hydroxy acids, etc. — which bind metal ions. 3. Metals are accumulated by spores in large amounts from dilute solutions. 4. If metal ions cross a permeability barrier slowly, it is possible that accumulation occurs by an adsorption process and is followed by a slower entrance of the ion or a complex of it into the protoplast. Ion antagonism, according to this hypothesis, is exerted in the adsorption phase. For the present, however, it must be admitted that this mecha- nism is speculative. 5. Although the primary effect of metals is probably on enzymes in the interior of the cell, effects on the permeability barrier itself cannot be excluded entirely. 6. Heavy metals inactivate enzymes. Combination with essential sulfhydryl groups is the favored explanation, but it is likely that other inactivation reactions, involving the formation of electrostatic or co- ordinate bonds, also contribute to enzyme inhibition. 8. THE ACTION OF SULFUR The most important organic sulfur fungicides are the dithiocarba- mates, considered later as chelating agents. Many other organic sulfur compounds — dithiazolidines (12), thiourea (80, 112), mercaptobenzo- thiazole (100), xanthates (82), arylthioalkanecarboxylic acids (102), phenothiazone (131), 3-pyridinethiol (394), benzothiazoles (142), com- pounds with the N-trichloromethylthio grouping (201), substituted thio- cyanates (215, 444), thiobisphenols (328), and such antibiotics as acti- thiazic acid and gliotoxin — are more or less fungitoxic. However, there is no reason to suppose that sulfur is necessarily directly involved in the reaction of all these diverse compounds with cellular constituents. In some organic molecules, the presence or absence of sulfur has no influ- ence on toxicity (37, 82); in others it has (102). Some of the sulfur compounds probably act primarily as chelating agents (11, 174). In others sulfur may exert an effect by modifying the reactivity of nearby functional groups or by affecting solubility. Inorganic sulfur, like copper, has been in use as a fungicide since the beginning of serious efforts to control plant disease. The various forms used, including liquid lime sulfur, all act ultimately as elemental sulfur. Sulfur is, of course, virtually water-insoluble, and a great deal of effort SULFUR 473 was earlier devoted to proving that some more soluble derivative is the active agent; the history of these investigations has been reviewed else- where (174, 244). It is now agreed that the form in which sulfur reaches the fungus spore is the vapor phase of elemental sulfur (246). Fungitoxicity in any particular situation of use is dependent upon tem- perature (459), the particle size of the sulfur deposit (370, 442), and, to some degree, the allotropic form of the sulfur (103). For some time following a series of critical studies, the details of which are reviewed by McCallan (244, 246) and Horsfall (174), ele- mental sulfur was considered to be reduced by the fungus spore and the effective toxic agent was taken to be the hydrogen sulfide formed by the biological reduction. Hydrogen sulfide is fungistatic (442, 443) and somewhat fungicidal (248, 249). It appears now, however, that the toxicity of elemental sulfur on a weight basis is greater than that of an equivalent amount of hydrogen sulfide, as shown in Table 2. Conse- Table 2. The Relative Toxicities of Sulfur and Hydrogen Sulfide* f Species Monilinia fructicola Cephalosporium acremonium Aspergillus niger Glomerella cingulata Neurospora sitophila Rhizopus nigricans Alternaria oleracea Stemphylium sarcinaeforme * From Miller, McCallan, and Weed (289) , by permission of the Boyce Thomp- son Institute for Plant Research, Inc. f Toxicity is expressed as the dose, in parts per million, required to kill 50 per cent of spores in an exposure of 24 hours; concentrations are of the external solution or suspension. % Highly significant difference between colloidal sulfur and hydrogen sulfide. quently the second part of the older theory — that hydrogen sulfide is the ultimate toxicant — is untenable. Attention has therefore been drawn to the process of sulfur reduc- tion itself as a possible primary event in toxicity. The reduction of sulfur to hydrogen sulfide by fungus spores is a general phenomenon and has been shown to be brought about by spores of many different fungi, under both anaerobic and partially aerobic conditions (253, 289). The activity of species varies widely: Cephalosporium acremo- nium under anaerobic conditions forms 6.39 mg hydrogen sulfide per lettable Colloidal Hydrogen Sulfur Sulfur Sulfide 54 0.5 2.8J >1000 0.3 12t >1000 0.3 15| >1000 0.4 20 1 >1000 1.0 38J >1000 2.7 5.9 >1000 18 15 >1000 31 8.8 474 CHEMICAL AGENTS gram of spores per hour, almost 300 times the rate of the same process in Rhizopus nigricans. In these experiments there was no external hydrogen donor, so that cell constituents must have been oxidized; it is found, as would be expected, that under such circumstances high rates of hydrogen sulfide evolution are not maintained for long. An- aerobically, the formation of hydrogen sulfide from sulfur is accompa- nied by an equivalent release of carbon dioxide; aerobically, sulfur increases oxygen uptake (246b). Mycelium of Fusarium lini is similarly capable of reducing elemen- tary sulfur in the presence of isopropyl alcohol as hydrogen donor (369). Fungi of different species differ very markedly in their sensitivity to sulfur under field or laboratory conditions; of the common test fungi for example, Stemphylium sarcinaeforme is about 60 times as resistant as Monilinia fructicola (Table 2). However, it has not yet been possi- ble to correlate the enzymatic reduction of sulfur with its fungitoxicity — fungi which are relatively tolerant of sulfur are numbered among both those most active and those least active in reducing sulfur, whereas the more sulfur-sensitive species tend to be intermediate in reducing capacity (289). However, this type of study has only begun, and it is not yet possible to exclude any hypothesis with finality. The production of large amounts of hydrogen sulfide from elemental sulfur in the absence of external hydrogen donors suggests that some relation may in the future be established between the derangement of metabolism associated with this process and the toxicity of sulfur (289). It is clear that sulfur is competing with normal hydrogen acceptors, and two speculative hypotheses are suggested by this conclusion. The first may be expressed, rather crudely, by the proposition that in the pres- ence of very large amounts of a suitable hydrogen acceptor, sulfur, de- hydrogenation reactions escape from the normal control imposed upon them under anaerobic conditions. This normal control may be either the limited supply of acceptors for "high-energy" phosphate or a poi- soning of metabolism by the accumulation of such fermentation prod- ucts as ethanol; the first of these is the more likely. The physiological effect of such an escape from regulation would be the disappearance of the cell's reserves. According to this hypothesis, sensitivity would be a function of the amount of utilizable reserve materials available to the spore and should be reduced by provision of an external hydrogen donor. A related hypothesis to explain a deleterious effect of the reduction process itself is that utilization of reserves per se is less important than the fact that provision of sulfur results in a series of oxidations which are not coupled to the energy storage and energy utilization mechanisms QUINONES, ETC. 475 of the cell. That is, sulfur diverts metabolism into non-productive or inefficient pathways. Anaerobically, methylene blue prevents the reduction of sulfur by spores of Neurospora sitophila (289). It is known that methylene blue does not accept hydrogen directly from substrates or the pyridine nu- cleotide coenzymes but from hydrogen (electron) carriers, such as the flavoproteins and, possibly, cytochrome b (116). If the sulfur reduc- tion process is in itself damaging to the cell, it is at this level that we should look first. It may be noted that sulfur reduction is also inhib- ited by 2,3-dichloro-l,4-naphthoquinone, the oxidation-reduction po- tential of which is presumably close to that of methylene blue. Selenium and tellurium are much less toxic than sulfur, and their reduction to hydrides by yeast cells is correspondingly much less rapid (443). Selenium is accumulated in organic form by the mycelium of Aspergillus niger in competition with sulfur, and selenate inhibition is partially or wholly counteracted by reduced sulfur compounds (435). These and similar data on Chlorella vulgaris (378) can be interpreted in terms of competitive inhibition by a metabolite analogue. 9. QUINONES AND OTHER UNSATURATED COMPOUNDS A number of actively fungistatic materials are characterized chemi- cally by a system of conjugated double bonds. Of these, the most im- portant are the quinonoid compounds, but the a,/?-unsaturated ketones are also of interest. The most generally active quinonoid compounds are the halogenated p-benzoquinones (266, 411) and 1,4-naphthoquinone and various de- rivatives of it (228, 229, 280, 364, 413), including synthetic vitamin K (2-methyl- 1,4-naphthoquinone) (15, 70, 133). The triphenylmethane dyes, e.g., malachite green and crystal violet, are fungistatic and, sig- nificantly, the leuco form (benzenoid) is not active against either fungi (93) or bacteria (111). The chromophoric group of the actinomycins is quinonoid (Chapter 8), and the antibacterial javanicin is a naphthoquinone (Chapter 6). The tropolones, some of which are produced by fungi (Chapter 6), have a modified quinonoid structure (325); some of the thujaplicins present in the decay-resistant heartwood of Thuja plicata show mod- erate fungistatic activity (19, 344, 345). A few examples of quinonoid fungicides are shown in Figure 10. It will be recalled that many fungi possess part or all of an enzymatic system which oxidizes phenols to quinones and further metabolizes the quinones to black melanin pigments (Chapter 6). It follows therefore 476 CHEMICAL A O F. NTS Tetrachloro-p- benzoquinone (Chloranil) 2,3-Dichloro-l,4- naphthoquinone (Dichlone) (CH3)2— N C=< )=N— (CH3)2— cr Malachite green Figure 10. Fungistatic quinonoid compounds. that appropriate hyclroquinones, themselves non-toxic, may be con- verted to toxic quinones. Thus, 2,3-dichloro-l,4-naphthohydroquinone inhibits Monilia laxa, presumably because it is oxidized by the fungus to the corresponding naphthoquinone; esters of the hydroquinone are first hydrolyzed, then oxidized (56). Detoxification of quinones would be expected of a fungus which forms melanin pigments; StempJiylium sarcinaeforme, which is nor- mally black-pigmented, appears to convert many quinones to non-toxic products (347). The reactivity of quinones is so great that more than one mode of action may easily be envisaged. Although any quinone affects the oxi- dation-reduction potential, it does not seem possible to correlate either bacteriostatic or fungistatic effects with this property (266, 321, 434). As a,/?-unsaturated ketones, quinones can react with both sulfhydryl and amino groups (372), and these possibilities have enlisted consid- erable interest. Quinones react, by addition, with simple thiols (212, 292). Corre- spondingly, the antimicrobial activity of quinones is often reversible by sulfhydryl compounds (70, 121). However, we have already noted that thiol reversal is not by itself sufficient proof of the nature of the in vivo reaction. Many of the enzymes known to depend upon sulfhy- QUINONES, ETC. 477 dryl groups for their activity or known to be inhibited by sulfhydryl reagents are, it is true, inhibited in vitro by quinones and naphtho- quinones (113, 159, 21 1, 213, 228, 319, 320, 364) and protected from this inhibition by thiols (319). However, a series of investigations shows, first, that quinones inhibit enzymes other than sulfhydryl-dependent types and, second, that the order of effectiveness of different quinones as enzyme inhibitors is not correlated with their antimicrobial activity (163-171). It is also to be noted that quinone toxicity to Gram-positive bacteria is not antagonized by thiols (121). These considerations indicate that some other reaction with essential proteins should be considered, possibly an inactivation of amino groups. For the present, it is not possible to specify any particular ultimate site of action for the quinones, and the evidence suggests that they may be general rather than specific enzyme inhibitors comparable to the heavy metals. The relation of structure to fungistatic activity in the quinones is re- viewed by McNew and Burchfield (266). Toxicity generally decreases in the series naphthoquinone > phenanthraquinone > benzoquinone > anthraquinone. Halogenation improves effectiveness; the effect of other substituents depends upon specific type and position. Compari- son of the effect of structure on inhibition of amylases reveals several instances in which a given structural feature affects fungitoxicity in one way, enzyme inhibition in another (320). Among the naphthoquinone antimalarials, activity is generally correlated with low water solubility; that is, not only a reactive grouping but also some non-polar properties are essential (105). Although some quinones are natural products of fungi and could in principle participate in terminal respiration (Chapter 6), there is as yet no firm evidence that fungitoxic quinones act as metabolite ana- logues, although, as mentioned earlier, their action may be reversed by natural quinones. Non-quinonoid a,/3-unsaturated ketones are not so active as the qui- nones against fungi; many, however, are antibacterial. Several, e.g., patulin and penicillic acid, appeared first as antibiotics produced by fungi. These compounds inhibit sulfhydryl enzymes, and their inhibi- tion of both isolated enzymes and intact cells is reversed by thiols. Consequently, it is believed that antibacterial action depends upon some reaction, perhaps addition across the reactive double bond, with sulfhydryl groups (122, 123, 351). Patulin, however, does not inhibit urease, a sulfhydryl enzyme (168), and the action of dehydroacetic acid is not reversed by thioglycolate (451). Studies of the action of unsaturated compounds on Bolrytis allii and 478 CHEMICAL AGENTS other fungi have a direct bearing on the toxicity of quinones and other o,/?-unsaturated ketones. It is proposed that any substituent which lends to withdraw electrons from a double bond increases toxicity (262, 301). Thus, derivatives of w-nitrostyrene, CCH5 — CH = CH— N02, are much more active than styrene, C6H5— CH = CH2. The similarity to the a,/3-unsaturated ketone structure, O \ I H c=c— c— is obvious. The same relation holds among the lachrymators: keto, aldehyde, ester (but not carboxyl), and nitro groups all confer ability on the double bond to attack sulfhydryl compounds; i.e., all increase activity toward nucleophilic reagents (94, 95). 10. CHELATION AND FUNGISTATIC ACTIVITY A number of chemicals which are known to be strong chelating agents for metals are also found to be toxic to cells of fungi and other organisms. Since the most effort has gone into the problem of the mode of action of the dithiocarbamates and of 8-hydroxyquinoline (oxine), we shall limit this section largely to these compounds, after preliminary consideration of general topics. Chelate compounds are compounds in which a metal ion combines with two or more electron-donating groups of a ligand in such a way that one or more rings are formed (276). As an example, the stepwise reaction of oxine with copper may be written: Cu + + Oxine 1 : 1 complex #\ \_/" \ " <5> 1:2 complex In this example, the final product, the 1:2 chelate, has no exposed polar CHELATION 479 groups and hence extracts easily into a non-polar solvent. The 1:1 chelate is polar. A polar group not involved in the union with the metal would, of course, remain unchanged and reduce lipid solubility. The number of different complexes formed by a given metal with a ligand is a function of its coordination number. Biological systems necessarily contain many different ions able to participate in chelate complexes, and the nature of competition must be considered. In general, most chelating agents form complexes of varying stability with metal ions in the order (from most to least sta- ble): Cu++, Ni++, Co++, Zn++, Cd++, Fe+ + , Mn++, Mg++ (276). Thus copper will displace any of the other listed ions, magnesium none. However, some biologically important chelating substances do not fol- low this rough rule; a,a'-dipyridyl and o-phenanthroline, for example, have a specific affinity for iron. Stability of metal chelates is usually expressed by the constant K, the formation (or dissociation) constant, a measure of the equilibrium: M + xA ±=; MA, (6) in which M = metal, A = ligand, and x = the number of ligand mole- cules per metal atom in the complex. Ideally, K is expressed by: (MA,) m K ~ (mxaT* (7) in which brackets indicate activities (276). Values of log K may be very high, e.g., 23 for the copper-oxine complex; it is obvious that negli- gible amounts of free copper can exist in the presence of excess oxine. Factors which fundamentally determine K are discussed by Calvin (59); Chabarek et al. (63) decribe the use, under biological conditions, of a plot of the negative logarithm of metal concentration against pH for prediction of the behavior of chelate systems. The ligand may be considered as a Lewis base; at low pH more of it is bound by protons and, consequently, the metal chelates are less stable under acid condi- tions. Many normal cell metabolites are chelating agents; examples of such metabolites include amino and hydroxy acids, porphyrins, peptides, and polyphosphates. Some of these may owe their biological function to their chelating properties. Chelation is almost certainly necessary to the function of the manganese-requiring peptidases (289), and quite likely it is essential to the action of other metalloenzymes and electron or oxygen transport systems (59, 97). We may approach the question of fungistatic action by first noting that chelating agents are often powerful inhibitors of metalloenzymes 480 CHEMICAL AGENTS in cell-free preparations. The poisoning of certain iron enzymes, e.g., cytochrome oxidase, by cyanide or azide presumably involves chelate bonds with the iron atom. Enzymes known to depend on metals for their activity are inhibited by chelating agents: polyphenoloxidases by diethyldithiocarbamate, azide, and other compounds able to complex with copper (184, 319, 405), zinc metalloenzymes by oxine, a,«'-dipyri- dyl, and o-phenanthroline (420). The specificity of chelating agents is, however, so low at biological pH levels that inhibition by them does not as a rule suffice to identify the metal active for a given enzyme or enzymatic system. In the systems just mentioned, it is not known whether the chelating agent combines with the metal in situ on an enzyme or coenzyme or competes with the catalyst for the limited amount of metal present. Inhibition of dialkylfluorophosphatase by 8-hydroxyquinoline and ethylenediaminetetraacetic acid (EDTA) appears to arise from removal of calcium from the enzyme (299); other enzymes dependent upon cal- cium for activity or stability are also inhibited by EDTA (86, 139). Finally, a few examples of in vivo effects of chelating agents may be mentioned: inhibition of synthesis of phenolic compounds by Phyco- myces blakesleeanus (48), prevention of the transformation of Brucella abortus from the smooth to the non-smooth form (68), and inhibition of glutamic acid assimilation by Staphylococcus aureus (117). In the two last-mentioned instances, it is believed that the biologically active metal is manganese, but the locus of the manganese chelate — within the cell or outside it — cannot be determined. Several metal complex- ing agents, including cyanide, sodium dimethyldithiocarbamate, and ethylenediaminetetraacetic acid, cause the release of subcellular par- ticulates from cells of Candida albicans (284). Although it is usually found that in any series of chelating agents many compounds are toxic (174, 180), and many empirically discov- ered toxicants turn out to be chelating agents (7, 60, 433), the associa- tion is not uniform, presumably because both the stability of the com- plex and its molecular architecture are factors in toxicity or in the ability of the molecule to reach the site of toxicity. Chelation alone, in other words, does not necessarily confer toxicity (8, 264). The best example of a close association between toxicity and chelation is in the series of compounds related to oxine: only those that complex metals are bacteriostatic (10, 11). The association of toxicity and ability to chelate was at first thought to signify only that the chelating agent deprives the cell of essential metals (461, 462). Several subsequent developments, however, make it seem unlikely that metal starvation per se is the primary mechanism CHELATION 481 of toxicity at minimally fungistatic doses. First, copper oxine is as toxic as, or even more toxic than, oxine itself (281, 313, 424). The same re- lation holds for the metal complexes of other chelating agents (272, 372, 395, 433). Second, there is some evidence that toxicity to bacteria and fungi is less in a medium low in metals than in the usual medium (5, 9, 14, 358). Finally, successful use of chelating agents to supply metals both to fungi (Chapter 9) and to higher plants (143, 401) suggests that cells in general can compete successfully for metals, with even quite stable complexes. Metal starvation cannot, however, be dismissed completely as a factor in toxicity. Chelating agents reduce the metal-dependent spore pig- ment of Aspergillus niger (338); EDTA prevents spore germination of Neurospora tetrasperma without apparently entering the cell (406), and citrate effects on enzymatic adaptation in Azobacter spp. can at least be interpreted as a depletion phenomenon (368). Inhibition of sporulation by chelating agents (179, 428) suggests that a higher metal requirement makes sporulation especially sensitive to metal depriva- tion. A number of observations, covered in detail in the original literature (9, 31, 32), support a plausible working hypothesis for the action of oxine and its metal complexes. The hypothesis starts with the equi- libria already described (Equation 5) of oxine, its charged 1:1 chelate and the neutral 1:2 chelate. It is believed that the 1:2 chelate, perhaps because of its higher lipid solubility, enters the cell more rapidly than the 1:1 chelate and in that sense is the active species. However, within the cell it seems likely that the 1:1 chelate is the more toxic, perhaps by reacting directly with essential metal-binding sites or with sulfhydryl groups essential to the cell. This hypothesis has the advantage of ex- plaining the bimodal dose-response curve of oxine (6, 31, 281), which is similar in principle to that of the dithiocarbamates (Figure 11), and the complex interactions of metal and oxine concentrations (32). Still to be demonstrated, however, are the roles of free metal ions and free oxine, the site of the proposed toxic reaction, and the role of natural chelating substances within and on the cell. Also, as with all perme- ability hypotheses, the lack of direct evidence is distressing. 8-Hydroxyquinoline-5-sulfonic acid is a chelating agent but is not antibacterial (6). It fails to inhibit Monilinia fructicola but is active against Macrosporium sarcinaeforme (177). It seems possible that the anionic group acts by preventing access of the molecule to the site of action (6); on this hypothesis, M. sarcinaeforme must be more perme- able to the compound than are bacteria and M. fructicola. The action of the dithiocarbamates and their derivatives may be ex- 482 CHEMICAL AGENTS 0.1 1.0 10 100 300 Concentration, mM per liter Figure 11. The effect of the concentration of tetramethylthinram disulfide (TMTD) on spore germination of Stemphylium sarcinaeforme. Redrawn from Dimond, Horsfall, Heuberger, and Stoddard (91), by permission of the Connecticut Agri- cultural Experiment Station, New Haven, Connecticut. plained in a similar way. Figure 1 1 illustrates the well-known bimodal dose-response curve characteristic of this family of toxicants; the gen- eral form of the curve shown has been confirmed in studies on spore germination of Venturia inaequalis (298) and on growth of Aspergillus niger (196, 198). The structures of interest are shown in Figure 12, with code abbrevi- ations. Other complexes can, of course, be prepared and have been CH3 n n CHo N — C— S— S— C— N CH3 CH3 Tetramethylthiuram disulfide (TMTD) CrL N— C— S— Na Sodium dimethyldithiocarbamate (Na DMDT) CH. \ ch/ N— C— S— Fe. S— C— N. S— C— N / .CH3 CH3 CH3 / \ CH, S ~13 Ferric dimethyldithiocarbamate (Fe DMDT) Figure 12, The structure of some dithiocarbamate fungicides. CHELATION 483 tested as fungitoxicants (174), but it is presumed that the active group- ing is the same in all. The important reactions of the dithiocarbamates from the standpoint of mode of action may be summarized (127): TMTD Oxidation Reduction metals CS2 + (CH3)2NH <- - DMDT ;± Metal complexes (8) Hydrolysis TMTM (Symbols are taken from Figure 11; TMTM = tetramethylthiuram monosulfide.) The primary problems of mode of action in this series are set for us by (1) the bimodal response curve and (2) the effect of metals on toxic- ity. Although it has been considered possible that carbon disulfide, formed under acid conditions (234) or enzymatically (191, 192, 384) from DMDT, is the active toxicant, both this and other decomposition products are of too low inherent toxicity to account for DMDT action (322, 432). The best available working hypothesis for the action of DMDT is rather similar to that just discussed for oxine. It is proposed (127, 196) that the first peak in the dose-response curve mirrors the formation of a toxic 1:1 complex of DMDT with copper present in the medium. It is suggested on the basis of work with yeast that this water-soluble charged 1:1 complex exerts its effect at or just beneath the cell mem- brane (127); this suggestion, supported by indirect evidence, avoids the problem of explaining penetration of a charged complex. At higher DMDT concentrations the insoluble and non-toxic 1:2 copper complex (2 moles DMDT per copper atom) forms and accounts for the trough in the dose-response curve. At still higher concentrations of DMDT, either the free anion becomes toxic (196) or some other toxic complex, e.g., with zinc, is formed (127). The first of these possibilities seems more probable; free dithiocarbamate inhibits enzymes in vitro (319). It is to be noted that this hypothesis explains the first peak of the bimodal response curve as a function of copper in the system; in a me- dium low in copper and other metals there should be only a single peak in the curve. This is borne out by visual estimates of mycelial growth (196) but has not yet been shown by the more precise spore germination technique. 484 CHEMICAL AGENTS Liebermeister (225) has proposed that several drugs effective against Mycobacterium tuberculosis are also active by virtue of their formation of a toxic complex. Some of the detailed data on the action of the dithiocarbamates is inconsistent with, or at least not explained by, the theory of action just outlined. These data are thoroughly reviewed by Horsfall (174), and it should be clear that many problems remain. No other hypothesis, however, explains the bimodal dose-response curve. Imidazoles, which are themselves complexing agents, antagonize the action of DMDT in the concentration range of the first peak of toxicity (198). This suggests that the locus of toxicity of the 1:1 copper chelate is an enzyme which is normally coordinated to some metal, and that the available coordination positions of the copper in the 1:1 complex re- place the metal on the enzyme. Dithiocarbamates at minimally fungistatic concentrations have very slight activity against endogenous or glucose respiration in fungi (81, 127, 203, 314). Acetate oxidation in yeast is considerably more sensi- tive (127). The respiratory enzyme triose phosphate dehydrogenase is inhibited in yeast and in Fusarium roseum by TMTD (384, 385). This may be explained on the basis that the dithiocarbamates react with glutathione (432) and that the reduction of TMTD to DMDT (Equa- tion 8) is likely to be coupled to an oxidation of such labile cell mate- rials as glutathione. Since glutathione is the prosthetic group of triose- phosphate dehydrogenase (210), it follows that this enzyme will be- especially sensitive. It must be concluded that we are at present unable to point to any specific system, the inhibition of which is the primary effect of either free dithiocarbamates or their active complexes. The present picture of the action of fungistatic chelating molecules is thus one of great complexity. A given compound exists as an anion or as an uncharged molecule and as complexes of various metal :ligand ratios with one or more metals. One or more of these molecular species reaches the sensitive region of the cell, at which point a new set of equilibrium conditions, complicated by the presence of cellular chelat- ing agents, must be envisioned. Still other complications can be intro- duced by structural alteration of the chelating agent; such structural changes influence ionization, water and lipoid solubility, and the sta- bility of metal complexes. Although the bisdithiocarbamates are superficially similar to the dithiocarbamates and are also chelating agents, the evidence to date suggests that the mode of action is not the same. In particular, fungi show different sensitivities to the two classes of toxicant, for a given fungus the slopes of the dose-response curves are different, and the BIBLIOGRAPHY 485 bimodal dose-response curve does not prevail in the bisdithiocarbamates (174, 246). Bisdithiocarbamates are unstable (234), and several break- down products have been suggested as the active toxicant. Disodium ethylenebis[dithiocarbamate] itself is completely without fungicidal action (247, 432). Carbon disulfide, one of the breakdown products, is unlikely to be of primary importance (432). It appears that three de- rived compounds, which arise by breakdown of the parent substance, all contribute to toxicity: ethylene diwothiocyanate, ethylene thiuram monosulfkle, and polymers of the thiuram monosulfide (197, 236, 237, 414). Reactivity with thiol groups has suggested that the primary toxic action is on a sulfhydryl enzyme (197). 1 1 . CONCLUDING REMARKS In his treatise on the principles of fungicidal action, Horsfall (174) emphasizes the two requirements of a toxicant, that it be able in some way to react with an essential cell constituent, and that it be able to reach the site of this reaction. It seems fair to say that for no fungicidal or fungistatic material can both these requirements be specified in terms of structure. Nor does it seem likely to me that further empirical test- ing of ever-different organic compounds will by itself allow us to draw up accurate specifications as guides to both theory and practice. In- stead, we need to know more about the toxicants we have, especially in terms of the theory of chemical reactions. On the biological side of the problem, a frontal attack on the problems of the cell surface is surely an urgent necessity. It must be realized that, at the present time, all explanations based on an assumed permeability or impermeability are no more than guesses, in the absence of independent evidence; i.e., evidence not based upon the toxic reaction itself. Although no line of investigation can, of course, be ruled out, it does seem that the search in the fungi for specific enzymatic loci of toxicity is less promising. 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Organism Index (Binomial names only) Absidia, 342, 346 blakesleeana, 149 glauca, 40 spinosa, 183 Acetobacter, 221 Achlya, 373, 374 ambisexualis, 373 americana, 373 bisexualis, 373 caroliniana, 40 prolifera, 23 Actinomyces, 17, 132, 133 i'5rae/i, 123, 302 Agaricus (see also Psalliota) campestris, 253, 365 melleus, 27 Agrocybe dura, 164 Albugo Candida, 407 Allomyces, 15, 159, 396, 425 arbuscula, 40, 132, 264, 306, 311, 407 javanicus, 68, 69, 81, 159, 162, 248, 249, 302 kniepii, 61, 331 macrogynus, 61 Alnus glutinosa, 249 Alternaria, 364, 366 brassicae, 358, 405 Alternaria, citri, 404 oleracea, 395, 446, 473 5o/am, 24, 247, 327, 359, 379, 388, 390, 441, 448 /enuw, 180, 183, 468 Amanita, 255 mappa, 274 muscaria, 273 phalloides, 255 Anabaena cylindrica, 300 Aphanomyces euteiches, 20 Apodachlya, 132 brachynema, 248, 258 Armillaria, 78 mflk, 9, 24, 27, 122, 134 Arthrobacter terregens, 344, 345 Ascobolus, 397 stercorarius, 375 Ascochyta pisi, 376 Ascoidea rubescens, 328, 335 /J^frya gossypii, 40, 214, 216, 224, 336- 337, 343 Is^ergillus, 25, 36, 42, 46, 47, 78, 86, 91, 100, 102, 107, 109, 114, 115, 119, 122, 132, 133, 134, 135, 137, 144, 149, 153, 154, 155, 156, 172, 178, 179, 180, 181, 183, 184, 206, 214, 216, 217, 246, 250, 499 500 ORGANISM INDEX 253, 264, 265, 275, 278, 302, 306, 308, 309, 325, 328, 342, 346, 347, 363, 389, 404, 406, 408, 431 aureus, 249 batatae, 249 echinulatus, 390 elegans, 122 fischeri, 43, 45, 149, 150, 390 flavus, 20, 44, 82, 179, 219, 249, 281, 282, 283, 302, 376, 427 foetidus, 118 fumaricus, 141, 147 fumigatus, 16, 17, 49, 135, 146, 167, 221, 227, 255, 267, 280 glaucus, 26, 280 itaconicus, 134, 144 janus, 364 luchuensis, 106, 108, 394 melleus, 430, 431 nidulans, 17, 45, 46, 48, 152, 169, 273, 305, 306, 339, 390, 393, 402, 449 niger, 3, 9, 20, 23, 26, 36, 37, 38, 42, 43, 44, 46, 49, 62, 63, 64, 65, 83, 88, 100, 107, 108, 109, 110, 114, 117, 119, 121, 123, 131, 132, 134, 135, 137, 139, 140, 141, 142, 144, 145, 146, 152, 170, 174, 176, 177, 180, 182, 204, 208, 209, 212, 214, 216, 218, 219, 220, 224, 225, 228, 241, 242, 244, 245, 249, 250, 261, 263, 265, 278, 279, 301, 302, 303, 305, 306, 307, 308, 310, 312, 313, 314, 316, 328, 334, 337, 343, 370, 371, 372, 376, 392, 395, 404, 413, 415, 434, 435, 441, 444, 461, 468, 473, 475, 481, 482 ochraceus, 157, 309 oryzae, 8, 9, 23, 36, 37, 40, 65, 81, 91, 107, 108, 110, 111, 112, 113, 114, 119, 153, 178, 187, 219, 224, 231, 232, 252, 253, 268, 275, 279, 302, 312, 429 parasiticus, 121, 253 quadrilineatus, 168 sydowi, 43, 46, 47, 48, 120 terreus, 133, 134, 144, 145, 183, 306- 307, 316, 429-430, 433-434, 435 versicolor, 78, 81, 83 Azotobacter, 481 Bacillus globigii, 414 subtilis, 111, 256 Basidiobolus ranarum, 40 Blakeslea trispora, 331, 368 Blastocladia, 366 pringsheimii, 23, 24, 69, 132, 133, 142, 328, 337-338, 376 ramosa, 340 Blastocladiella, 230, 366, 391, 396, 411 emersonii, 206, 224, 229, 248, 266, 306, 331, 376-377, 428 variabilis, 40 Blastomyces (see also Paracoccidioides), 11 brasiliensis, 206 dermatitidis, 10, 11, 40, 46, 47, 163, 334, 454 Boletus, 346 bovinus, 122 edulis, 40, 342 granulatus, 346 variegatus, 20, 331 Botryosporium, 20 Botrytis, 115, 133 allii, 441, 477-478 cinerea, 17, 115, 117, 132, 133, 134, 364, 390, 405, 412, 413, 415, 426 squamosus, 428 Bremia lactucae, 364 Brevilegnia gracilis, 69, 91, 305 Byssochlamys fulva, 115, 116, 122, 424, 425 Caldariomyces fumago, 133, 183 Calonectria decora, 157 Candida, 306, 336, 346 albicans, 10, 302, 480 pulcherrima (see also Torulopsis pul- cherrima), 283 tropicalis, 10 Cantharellus cibarius, 40 Capnodiutn salicinum, 15 Cenococcum graniforme, 259 Centrospora acerina, 428 Cephalosporium acremonium, 395, 473 costantii, 14 salmosynnematum, 304 Cephalothecium roseum, 156, 359 Ceratostomella (see also Endoconidio- phora, Ophiostoma), 331, 335, 346 fimbriata, 331, 363, 371, 372 ips, 334 microspora, 328 ORGANISM INDEX 501 Ceratostomella pini, 334 ulmi, 10, 132 Cercospora beticola, 18 kikuchii, 20 sesami, 363 Cercosporella herpotrichoides, 331 Chaetomium, 253, 312, 342, 346, 371 aureum, 167 funicola, 247 globosum, 107, 114, 115, 371, 372, 429, 435 thermophile, 18 Chalara quercina, 20, 65, 70, 75, 76, 91 Chalaropsis thielavioides, 331, 409 Chlorella vulgaris, 161, 210, 475 Choanephora cucurbitarum, 247, 358, 359, 364, 365 Chondrus crispus, 119 Chytridium, 20, 66, 68, 75, 132, 247, 248 Circinella, 133, 342 "Citromyces," 137 Cladosporium, 17, 405 Clasterosporium, 133 carpophilum, 410 Clavaria, 83 Claviceps paspali, 279 purpurea, 46, 47, 108, 264, 271, 273, 274, 279, 363 Clitocybe, 78 diatreta, 164 Clostridium, 157 acetobutylicum, 336 Coccidioides immitis, 43, 121, 277 Coccomyces hiemalis, 408, 415 Colletotrichum, 405 antirrhini, 156 destructivum, 389 gloeosporioides, 402 graminicolum, 49 hibisci, 20 indicum, 407 lagenarium, 409 lindemuthianum, 21, 364 Km", 424 phomoides, 115 Collybia, 78, 83 tuberosa, 331 velutipcs, 302, 331, 365, 426 Coniophora cerebella, 24, 132, 133, 134 Coniothyrium, 156 Coprinus, 14, 20, 21, 70, 246, 277, 408 atramentarius, 40 ephemerus, 311 fimetarius, 13, 17 hiascens, 258 lagopus, 331, 357, 358 radians, 37, 44, 253 similis, 167 Cordyceps militaris, 122, 282, 378 Corticium, 78, 164 centrifugum, 134, 144 Cortinarius, 172 glaucopus, 331 Cronartium ribicola, 407 Cryptococcus (see also Torulopsis), 121, 302 neoformans, 43 Ctenomyces farinulentis, 40 Cunninghamella, 133, 253, 313 blakesleeana, 156, 157 Curvularia, 156 lunata, 157 Cylindrocarpon radicola, 157 Cytosporina ludibunda, 368 Dacrymyces stillatus, 159 Dactylium dendroides, 156 Daedalea flavida, 115 juniperina, 165, 181 Dahlia, 76 Dematium pullulans, 10, 132, 134, 135, 214, 221 Dermocybe sanguinea, 168 Diaporthe phaseolorum, 339, 358, 370 Dictyostelium, 336 discoideum, 41, 206 Didymella vodakii, 156 Didymium eunigripes, 358, 362 Diplocarpon rosae, 247, 339 Diplodia macrospora, 57 natalensis, 264, 277 tubericola, 214 Drosophila subatrata, 183 Eberthella typhosa, 459 Empusa, see Entomophthora Endoconidiophora (see also Ceratosto- mella, Ophiostoma) adiposa, 63 jagacearum, 400, 405, 407, 424, 426 502 ORGANISM INDEX Endoconidiophora fimbriata, 331 moniliformis, 134 Endomyces, 46, 149 decipiens, 40 magnusii, 331 Endomycopsis, 149 fibuliger, 40 vernalis, 265, 267 Entomophthora, 66, 75, 86, 247 coronata, 91 Epidermophyton floccosum, 40 Eremascus fertilis, 40 Eremothecium ashbyii, 40, 259, 269, 276, 336, 337, 339-340 Erysiphe, 36, 424 cichoracearum, 408 graminis, 363, 364, 378, 393, 403, 406, 409, 410, 412, 423, 428 polygoni, 405, 407 Escherichia coli, 87, 209, 227, 268, 271, 310, 433, 434 Fome5, 78, 331 annosus, 20, 102, 133, 134, 405, 443 fraxineus, 20, 115 Fumago vagans, 132, 133, 183 Fusarium, 18, 46, 61, 114, 115, 118, 133, 134, 143, 145, 149, 170, 214, 215, 216, 217, 221, 228, 255, 273, 281, 283, 308, 328, 342, 346, 359, 362, 366, 368, 405, 415 aquaeductum, 20 aurantiacum, 20 avenaceum, 346 caeruleum, 449 conglutinans, 363 eumartii, 431 graminearum, 46, 133, 273 javanicum, 168 lini, 123, 132, 136, 151, 204, 210, 214, 216, 219, 221, 246, 250, 408, 424, 474 moniliforme, 406, 410 nivale, 17 niveum, 246 oxysporum, 9, 10, 71, 91, 110, 117, 163, 254, 255, 276, 279 roseum, 149, 401 solani, 6, 157, 207, 208, 327, 366 tricothecioides, 338 vasinfectum, 283 Gelasinospora tetrasperma, 231 Geotrichum, 20, 149 candidum, 302 Gibberella baccata, 156 fujikuroi, 281, 283 saubinetii, 19 Gliocladium catenulatum, 157 roseum, 37 Gliomastix convoluta, 88 Gloeosporium fructigenum, 115 Glomerella, 254 cingulata, 114, 115, 175, 176, 177, 338, 346, 375, 401, 407, 431, 473 Gnomonia, 17 Gonatorrhodiella highlei, yi2 Grifola confluens, 164 Gymno sporangium, 395 clavipes, 441 globosum, 405 Gyrophora esculenta, 169 Hansenula anomala, 245 Helicostylum piriforme, 156 Helminthosporium, 172 gramineum, 244, 359, 377 oryzae, 400 ravenelii, 168 sativum, 61, 308, 389, 390 victoriae, 254 Hendersoniella, 17 Herpobasidium deformans, 407 Herpomyces stylopygae, 41 Herpotrichia nigra, 17 Hirsutella, 347 Histoplasma, 11 capsulatum, 10, 40, 306 farciminosum, 10 Hormodendrum, 207 Hydnum ferrugineum, 167 Hypholoma fasciculare, 40, 331 Isaria farinosa, 347 Karlingia rosea (see also Rhizophlyctis rosea), 247, 428 Labyrinthula minuta, 259 vitellina, 345 ORGANISM INDEX 503 Lactarius, 47, 164 Lactobacillus, 221 arabinosus, 260 casei, 342 leichmanii, 343 pentosus, 221 Lagenidium giganteum, 40 Larix, 114 Lentinus degener, 167 lepideus, 165, 461 omphalodes, 259 Lenzites abietina, 156 sepiaria, 115, 152, 410 trabea, 17, 346 Lepiota naucina, 280 Leptographium, 258 Leptomitus lacteiis, 60, 81, 248, 258, 260 Leuconostoc mesenteroides, 216 Lobaria retigera, 167 Lophodermium pinastri, 339 Lycogala, 108 Lycoperdon giganteum, 153 Macrochytrium botrydioides, 68, 132 Macrophomina phaseoli, 115 Macrosporium sarcinaeforme (see also Stemphylium sarcinaeforme), 175, 406, 481 Magnusia, 408, 410 brachytricha, 405 nitida, 407 Malbranchea pulchella, 184 Marasmius, 21, 78, 83, 302, 346 chordalis, 134 fulvobulbillosus, 331 graminum, 20 perforans, 331 rotula, 21 Mastigosporium album, 17, 407 Melampsora lini, 391, 402, 426 Melanconium juligineum, 367 Melanospora destruens, 369, 372 zamiae, 369 Memnoniella echinata, 65, 72, 81, 91, 133, 327, 334, 394, 402 Merulius, 133, 134 confluens, 20 Merulius domesticus, 83, 302 lacrymans, 15, 119, 132 niveus, 143 Metarrhizium, 115 Micrococcus denitrificans, 244 Micromonospora, 79, 254 vulgaris, 18, 209 Microsporum, 84, 265 audouini, 337 cam's, 302 gypseum, 84, 306 Mitrula paludosa, 333 Mcmascus, 342 Monilia (see also Sclerotinia), 132, 133 formosa, 133, 135 fructigena, 115 Monilinia (see also Sclerotinia) fructi- cola, 9, 36, 390, 395, 445, 446, 451, 473, 474 /axa, 476 Monoblepharella, 132 Monotostoma lanuginosa, see Thermo- myces lanuginosus Morchella esculenta, 38 Mortierella, 133, 214 renispora, 251, 253 Mucor, 40, 46, 115, 132, 133, 156, 214, 250, 342, 346, 413 dispersus, 390, 430, 431 guillermondii, 10, 206 hiemalis, 161, 342 mucedo, 82, 133, 152, 374 plumb eus, 132 pusillus, 17 pyriformis, 133 racemosus, 115, 221 ramannianus, 331 rouxii, 132 Afycena, 27, 78, 83, 403 Mycobacterium, 35 paratuberculosis, 344 tuberculosis, 344, 484 Mycoderma vini, 40 Mycogone perniciosa, 20 Mycosphaerella citrullina, 366 pinodes, 247, 366 Myrothecium verrucaria, 106, 107, 113, 180, 205, 208, 209, 217, 231, 247, 259, 327, 393, 395, 400, 402, 447 504 Nectria cinnabarina, 40, 281 coccinea, 372 galligena, 264 Neurospora, 261, 326, 339, 342, 394, 397 crassa, 5, 7, 9, 20, 22, 25, 38, 47, 63, 67, 87, 88, 108, 115, 118, 121, 133, 134, 145, 151, 152, 156, 159, 160, 161, 163, 171, 172, 174, 175, 176, 181, 204, 210, 214, 216, 218, 219, 224, 226, 228, 229, 231, 244, 245, 256, 258, 261-273, 275, 276, 277, 280, 302, 304, 306, 308, 310, 312, 313, 325, 326, 328, 331-342, 363, 375, 376, 390, 391, 393, 401-402, 413, 424, 426, 428, 429, 430, 431-432, 433, 449 sitophila, 133, 254, 276, 335, 375, 392, 395, 442, 451, 473, 475 tetrasperma, 230, 232, 391, 393-394, 397, 401, 412, 469, 481 Nigrospora sphaerica, 365 Nitella clavata, 452 Nocardia (see also Proactinomyces), 331, 343 acidophilus, 164 corallina, 249, 275, 276 opaca, 155 sebivorans, 424 Oidium, 149 Omphalia flavida, 27, 264 Onygena equina, 84, 398 Oospora, 46, 149 color ans, 167 sulphurea-ochracea, 166 Ophiobolus graminis, 20, 23, 415 lierpotrichus, 156 miyabeanus, 461 Ophiostoma (see also Ceratostomella, Endoconidiophora), 335, 346, 363 multiannulatum, 10, 271, 276, 277, 305, 340, 400 pini, 333 Paecilomyces varioti, 49 Panus stipticus, 26 Paracoccidioides brasiliensis (see also Blastomyces), 10, 11, 40 Parmelia, 169 Paxillus atromentosus, 167 ORGAN ISM INDEX Penicillium, 42, 43, 45, 46, 48, 49, 86, 91, 100, 102, 115, 132, 133, 134, 135, 137, 141, 144, 149, 156, 165, 166, 167, 172, 173, 174, 178, 179, 180, 182, 183, 184, 214, 216, 247, 252, 253, 261, 263, 265, 278, 280, 302, 325, 328, 342, 344, 346, 347, 370, 404, 406, 424, 431, 441 amarum, 168 brevi-compactum, 43, 132, 165, 173, 221 capreolinum, 43 charlesii, 42, 43, 180 chrysogenum, 37, 44, 47, 62, 81, 107, 117, 118, 135, 136, 143, 157, 173, 174, 184, 186, 187, 203, 208, 209, 211, 212, 216, 219, 224, 225, 226, 228, 230, 260, 261, 264, 265, 268, 275, 302, 305, 328, 334, 432 cinerascens, 180 crustosum, 47 cyclopium, 166, 283 digitatum, 36, 66, 72, 75, 81, 91, 111, 134, 163, 327, 341 duponti, 18 expansum, 20 funiculosum, 119 gladioli, 166 glaucum, 20, 42, 82, 154, 155, 221, 461 griseofulvum, 8, 37, 133, 280 islaridicum, 168 janczewskii, 81 javanicum, 46, 48, 81, 86 lilacinum, 48 luteopurpurogenum, 132 luteum, 43, 306 nalgiovensis, 183 nigricans, 24 notatum, 37, 38, 153, 154, 184, 185, 216, 220, 253, 262, 269, 306, 308, 428, 429, 432 obscurum, 280 puberulum, 169 resticulosum, 133, 142, 220, 280 roqueforti, 152 roseo-purpureum, 174 rugulosus, 43 sanguineum, 261 sclerotiorum, 43 spinulosum, 107, 109 stipitatum, 169 ORGANISM INDEX 505 Penicillium terlikoivski, 280 terrestre, 180 urticae, 63, 64, 172, 309 varians, 43 viniferum, 135 westlingii, 49 Peronospora, 408 destructor, 364 ^U5«, 407 tabacina, 398 Pestalotia, 389 trial or um, 305 Peziza, 156, 164 Phacidium infestans, 17, 426 Phallus impudicus, 273 Phlyctorhiza variabilis, 20, 338 Pholiota aurea, 250 Phoma, 249 apiicola, 17, 363, 391, 407 fcefae, 370 terrestris, 283 Photobacterium fischeri, 27 Phragmidium mucronatum, 396, 407, 411, 425 Phycomyces, 328, 331, 360, 361, 362 blakesleeanus, 13, 20, 40, 46, 48, 151, 156, 159, 160, 162, 165, 171, 258, 263, 276, 302, 324, 328, 330, 332, 361, 363, 370, 397, 402, 414, 480 nitens, 361, 364, 426 Phycopsis, 17 Pfiymatotrichum omnivorum, 16, 37, 42, 43, 63, 67, 76, 246, 247, 426, 461 Physalospora obtusa, 410 Physarum polycephalum, 203, 253, 366 Phytophthora, 40, 114, 331, 399, 412 cactorum, 70, 91 cinnamomi, 332 erythroseptica, 115 infestans, 389, 399, 407, 410, 411 primulae, 17 Pichia membranaefaciens, 40 Pilobolus, 344, 345, 360, 362 crystallinus, 374 kleinii, 361 Piricularia oryzae, 247, 253, 258, 265, 331, 363, 365 Plenodomus meliloti, 19 Pleurotus lunaillustris, 27 Polychytrium aggregatum, 66, 68, 84, 91 Polyporus, 78, 115, 158, 247, 331 anceps, 133 annosus, 83 arcularius, 357 benzoinus, 165 betulinus, 107, 302 eucalyptorum, 158 hanedai, 27 texanus, 342 tumulosus, 134, 165 vaporarius, 14, 132 versicolor, 176, 331 Polystictus, 78, 115 Polytomella caeca, 60 Poria cocos, 158 vaillantii, 134, 336 Proactinomyces (see also Nocardia), 81, 82, 83 erythropolis, 155 opacus, 82, 162, 163 roseus, 156 Propionibacterium pentosaceum, 449 Psalliota (see also Agaricus) bispora, 71, 75, 80, 81, 85, 91, 247, 401 campestris (see also P. bispora), 365, 402 Pseudomonas, 221, 222, 227 fluorescens, 174, 220 putrefaciens, 60 Pseudoperonospora humuli, 358 Pseudopeziza ribis, 363 Puccinia, 159, 392 chrysanthemi, 411 coronata, 309, 405 glumarum, 408 graminis, 46, 163, 393, 405, 412, 414, 415, 428, 431 helianthi, 414 malvacearum, 407 Pullularia pullulans, 14, 79 Pyronema confluens, 357, 365 Pythiacystis citrophthora, 278 Pythiogeton, 20, 70, 91, 132 Pythium, 4, 247, 305 butleri, 326 debaryanum, 14, 40, 115 Rhipidiurn, 132 Rhizoctonia, 115 so/am, 18, 389 506 ORGANISM INDEX Rhizophlyctis rosea, 75, 246, 247 Rhizopus, 18, 115, 132, 133, 136, 141, 156, 214, 215, 216, 251, 311, 328, 342, 346, 363, 408 bovinus, 40 chinensis, 17, 407 delemar, 111 japonicus, 37, 38, 122 nigricans, 9, 115, 134, 157, 203, 221, 227, 228, 229, 302, 342-343, 390, 392, 395, 407, 473, 474 oryzae, 9, 136, 215, 216, 228 suinus, 57, 263, 328, 339, 431 Rhodotorula aurantiaca, 340 gracilis, 150, 151 rubra, 40, 159, 160, 161 Romanoa terricola, 10 Russula aurata, 273 Sabouraudites audouini (see also Micro- sporum audouini), 40 Saccharomyces, 40 cerevisiae, 345 Saprolegnia delica, 69, 91, 305 ferax, 392 lapponica, 40 mixta, 6, 363, 367 Sapromyces elongatus, 132, 248 reinschii, 373 Scenedesmus obliquus, 313 Schizophyllum, 78 commune, 25, 182, 281, 426 Schizosaccharomyces, 40 Schizothecium longicolle, 74 Sclerospora, 358, 364 Sclerotinia (see also Botrytis, Monilia, Monilinia), 134, 405 camelliae, 326, 339, 340 cinerea, 80, 115 fructicola, 15, 40, 175, 307, 316, 359, 391, 401, 409, 410 jructigena, 428 /axa, 443 Sclerotium (see also Rhizoctonia), 331 bataticola, 17, 115 delphinii, 72, 132, 133, 134, 144 ro//sn, 328, 332 Scopulariopsis brevicaulis, 5, 182, 210, 242, 243, 244, 246, 255 Sepedonium, 17, 20 Septoria apii, 405 £e£K, 20 Sordaria fimicola, 9, 88, 326, 334, 371, 401 Sphaerobolus, 365 Sphaerotheca pannosa, 363, 399 Sphaerulina trifolii, 331 Spicaria violacea, 119 Sporobolomyces roseus, 40 Sporodinia grandis, 7, 364, 367, 368 Sporotrichum, 17 schencki, 10, 40, 333 Stachybotrys atra, 60, 63, 73, 91, 113, 115, 122, 332 Staphylococcus aureus, 480 Stemphylium (see also Macrosporium), radicinum, 214 sarcinaeforme, 395, 446, 473, 474, 476, 482 Stereum, 78 frustulosum, 25, 331 gausapatum, 71 hirsutum, 132 murraii, 331 purpureum, 115 Streptococcus faecalis, 270 /aciis, 342 Streptomyces, 2, 9, 17, 21, 23, 41, 43, 61, 79, 80, 84, 106, 111, 114, 119, 132, 133, 136, 156, 157, 249, 251, 252, 254, 255, 275, 277, 281, 282, 311, 312, 328, 343, 346, 390, 400, 432, 433, 435, 462 albo-niger, 283 albus, 120 aureofacie?is, 155, 183 coelicolor (see also 5. violaceus-ruber), 20, 63, 73, 76, 91, 133, 142, 143, 204, 208, 216, 218, 219, 222, 224, 225, 228, 243, 256, 283, 302 flaveolus, 431, 432, 434 flavus, 47 fradiae, 6, 143 griseus, 36, 110, 208, 225, 226, 228, 243, 264, 266, 278, 342, 343, 366, 431, 433 limosus, 283 microflavus, 112 nitrificans, 249, 316 olivaceus, 344 proteolytics, 252 ORGANISM 1NDKX 507 Streptomyccs scabies, 20. 175, 2fi3, 413 thiolutenSj 280 venezuelae, 166, 260, 264, 269 violaceus-ruber (see also S. coelicolor). 251 Syncephalastrum racemosum, 115, 183 Synchytrium endobioticum, 412 Tetrahymena piriformis, 228 Thamnidium, 363 elegans, 115 Thelephora, 167 Thermoascus aurantiacus, 18 Thermomyces lanuginosus, 18 Thielaviopsis basicola, 88, 115 paradoxa, 389 Thraustotheca clavata, 373 Thuja plicata, 475 Tilletia, 403, 408 car»'e5, 216, 218, 266, 166 horrida, 331 fewis, 273 tritici, 331, 467 ToruZa, 40, 46, 149 cremoris, 302 u£t7t5 (see also Torulopsis utilis), 265 Torulopsis, 43, 149 neoformans, see Cryptococcus neo- formans pulcherrima (see also Candida pul- cherrima), 308 rotundata, 120 sanguinea, 402 uijKs, 210, 246, 268, 278, 302 Trametes, 115 cinnabarina, 283 suaveolens, 38, 165 Tranzschelia punctata, 407 Trichoderma, 114, 115 koningi, 251 Ugnorum, 44 t/m'de, 40, 183, 280, 309, 312, 328, 389, 456 Tricholoma, 21, 44, 78 equestre, 302 flavobrunneum, 66 fumosum, 113 gambosum, 251, 258 imbricatum, 75, 258 nudum, 20 Trichophyton, 84, 258, 259, 846 discoides, 328, 331, 335 equinum, 338, 339 faviforme, 339 ferrugineum, 40 gypseum, 252 mtntagrophytes, 258, 429, 431 persicolor, 20, 256 rosaceum, 251 ru brum, 123, 302 tonsurans, 134, 259, 331 Trichosporon capitatum, 10 cutaneum, 20 Trichothecium, 114 roseurn, 156 Typhula, 17 Uncinula necator, 410, 414 Urocystis tritici, 400, 403 Uromyces phaseoli, 414, 426, 450 Ustilago, 250, 331, 346, 375, 405, 408 avenae, 412 major, 346 scabiosae, 258 sphaerogena, 230, 231, 309, 325, 330, 344 striaeformis, 397 violacea, 12, 40, 71, 75, 86 zeae, 3, 44, 47, 108, 134, 151, 206, 219, 276, 305, 407, 412, 415, 429, 431 Ustulina vulgaris, 132, 391 Venturia inaequalis, 44, 63, 257, 306, 338, 342, 405, 415, 449, 482 Verticillium, 247 albo-atrum, 12, 115, 389, 405 coccorum, 119 malthousii, 20 psalliotae, 20 tricorpus, 14 Xylaria malt, 247 Zygorhynchus, 346 moelleri, 143, 224, 226 vuilleminii, 24 Zygosaccharomyces, 372 t/mij 40 Subject Index Accumulation, of amino acids, 259-260 of metals, 316, 472 selective, 450-452, 467 Acetaldehyde, 213, 214 Acetamide, 258, 265 Acetanilide derivatives, 464 Acetic acid, 161, 337 as buffer, 22 formation of, 133, 143, 221 metabolism of, 138, 143, 145, 223, 224, 227, 462, 484 utilization of, 68-73, 81 Acetic dehydrogenase, 227 Acetoacetic acid, 153, 161, 263 Acetoin, 123, 214 Acetone, formation of, 154 Acetonedicarboxylic acid, 146 N-Acetyl-p-aminobenzoic acid, 341 Acetyl choline, 273 Acetyl-coenzyme A, 138, 143, 222-223, 227 N-Acetyl-D-glucosamine, 39, 121 Acetyl phosphate, 221, 225, 227, 228 Acids, toxicity of, 455 Aconitase, 223, 227 cis-Aconitic acid, 134, 138, 223 m-Aconitic decarboxylase, 145 Acrasin, 375 Acridines, 456-457 Acriflavine, 449 Actinomycins, 254, 475 Actithiazic acid, 282, 283, 472 Acylases, 265 Adenine, 275-277, 283, 337 Adenosine, 275, 276 Adenosine diphosphate, 275 Adenosine diphosphate ribose, 275 Adenosine phosphates, 38, 203, 213, 301 Adenosine-3'-phosphate, 275 Adenosine-5'-phosphate, 275, 277, 302 Adenosine triphosphate, 302 Adenyl deaminase, 275 Adenylic acid, see Adenosine-3'-phos- phate, Adenosine-5'-phosphate Adenylosuccinic acid, 277 Adonitol, 68-73 Adsorption, 452, 468-469, 472 Aerobic respiration, 202 Agar, impurities in, 325 in culture media, 1, 9-10, 12-14, 324 Agaric acid, 47 Age, and respiration, 206-207 Agmatine, 273 Agroclavine, 279-280 508 SUBJECT INDEX 509 Agrocybin, 164, 283 o-Alanine, metabolism of, 257, 260, 267 /3-Alanine, 342 Alcohols, effect of on acid formation, 141 toxicity of, 457-459, 461 utilization of, 83 see also individual compounds Aldolase, 204, 213, 216 Alkylation, and toxicity, 463 N-Alkyl-a-bromoacetamides, 461 N-Alkylpyridines, 461 Allantoic acid, 45, 278-279 Allantoicase, 279 Allantoin, 278-279 Allantoinase, 279 Allithiamine, 331 Alternariol, 180 Aluminum, 38, 307, 313 Amanitins, 255 Amicetin, 266, 283 Amidases, 265 Amides, 253, 258, 265 Amines, 273-274, 453, 461 d- Amino acid oxidases, 260-261 l- Amino acid oxidases, 261-262 Amino acid racemases, 259 Amino acids, acyl, 265 Amino acids, as autolysis products, 8 as chelating agents, 315, 479 biosynthesis of, 138, 265-272 deamination of, 260-262 effect of on respiration, 211 of mycelium, 44, 45 oxidation of, 260-265 requirements for, 248, 259 toxicity of, 258, 259 transport of, 259-260 utilization of, 83, 256-265 a-Aminoadipic acid, 270-271 p-Aminobenzoic acid, 171, 326, 327, 329, 340-341, 345, 461 biosynthesis of, 271-272 7-Aminobutyric acid, 265-267 Aminoethanol and methylated deriva- tives, 181 o-Amino-e-hydroxycaproic acid, 271 4-Amino-5-imidazole carboxamide, 276 5-Aminolevulinic acid, 344 Aminopeptiduses, 253 L-2-Amino-l-propanol, 279 Amino sugars, 121-122 Ammonia, as autolysis product, 8 in amino acid metabolism, 262, 265, 266, 370 physiological effects of, 249, 328, 365 requirement for, 326, 335 Ammonium dehydrogenase, 249 Ammonium ion, physiological effects of, 19, 247-248, 394, 403 utilization of, 210, 243-244, 246-249, 333 Ammonium nitrate, utilization of, 243- 244 Amphomycin, 254 Amygdalin, 65-66 Amylases, 110-112, 477 Anaerobic respiration, 202 Aneurin, see Thiamine Aniline, 83 Anisaldehyde, 165 Anthranilic acid, 271-272 Anthraquinones, 168, 170, 172, 477 Antibiotics, 440 Antimalarials, 477 Antimycins, 449, 462 Aquatic fungi, 23, 24, 347 Arabans, 114 D-Arabinose, 63, 68-73 L-Arabinose, 63, 68-73 D-Arabitol, 122 Arabogalactan, 114 Arabonic acid, 132, 136 Arachidic acid, 48 Arginase, 278, 471 Arginine, metabolism of, 260, 264, 268- 269, 278 requirement for, 259 utilization of, 257 L-Argininosuccinic acid, 269 Aromatic compounds, metabolism of, 164-177 Arsenate, 447, 448 Arsenic, 182, 449, 463 Arsenite, 449 Arsenous acid, 182 Arthrobacter terregens factor, 344-345 Arylthioalkanecarboxylic acids, 472 Ascorbic acid, 180, 345, 393 Ascosin, 462 510 Asparaginase, 265, 471 Asparagine, 265, 324, 370 requirement for, 258 utilization of, 256-268 Aspartase, 262, 266 Aspartic acid, metabolism of, 262, 265, 266, 267, 269, 270, 333 utilization of, 256, 257 Aspergillic acid, 282, 283 Aspergillin, 308 Asperthecin, 168 Asperxanthone, 170 Atromentin, 167 Aureomycin, 183, 402 Aureothricin, 280 Autoclaving, effects of, 57-58 Autolysis, 7, 41, 241-242, 303, 304, 375- 376 Auxin, see Indoleacetic acid Aversion, 374 Avidin, 335 8-Azaguanine, 277 Azaserine, 280, 281 Azelaic acid, 334 Azide, 231, 455, 480 Barbituric acid, 275 Barium, 306 Barrage, 374 Bavendamm reaction, 83-84, 175 Behenic acid, 48 Benzaldehyde, 173-174 Benzamide, 265 Benzoic acid, 174, 265 o-Benzoquinone, 175 p-Benzoquinones, formation of, 167, 170 toxicity of, 475-477 Benzothiazoles, 441, 472 Beryllium, 307 Betaine, 181, 273 Betulin, 157 Bicarbonate, effect of on reproduction, 376-377 Bioassay, of minerals, 313-314 of vitamins, 328-329 Biocytin, 334 Bioluminescence, 26-27 Biotin, 283, 325, 326, 327, 328, 329, 332- 335, 345, 371, 402 Biotin sulfone, 335 SUBJECT INDEX Biotin sulfoxide, 331 Bisdithiocarbamates, 484-485 Bisphenols, 464-465 Bisulfite, 305 Borax, 449 Bordeaux mixture, 166 Boric acid, as buffer, 22 Boron, 313 Bound water, 26, 36, 406 Bran, as culture medium, 3 Bromide ion, 183 Buffers, 22 Bufotenine, 274 1,2-Butanediol, 123 2-Butanol, 214 2-Butanone, 214 2,3-Butylene glycol, 123, 214 Butyric acid, 153, 154 Cacodylate, sodium, 182 Cadaverine, 273 Cadmium, 308 Caffeine, 276, 277, 443 Calcium, 19, 38, 311-312, 371 Calcium cyanamide, 278 Caldariomycin, 183 Callose, 41 Camphor, 390, 441 Canavanine, 268, 270 Capreolinose, 43 Capsular polysaccharides, 43, 121 Captan, 442 O-Carbamyl-D-serine, 280 Carbohydrate, of mycelium, 37, 42-43 of spores, 393 Carbon, total, 36 Carbon balance, 100-101 Carbon dioxide, fixation of, 138, 142, 146, 222, 225, 228-229, 333, 337 physiological effects of, 24. 209, 365- 366, 376-377, 413 Carbon disulfide, 483, 485 Carbon monoxide, 231 Carbon nutrition, and reproduction, 367-369 and spore germination, 401 Carbonic anhydrase, 471 Carboxylase, see Pyruvic carboxylase Carboxymethylcellulose, 77-78 m-m-y3-Carboxymuconic acid, 174 SUB J ECT INDEX 511 Carboxypeptidase, 2.53 Carlic acid, 180 Carlosic acid, 180 Carnosine, 250—25 1 Carolic acid, 180 Carolinic acid, 180 a-Carotene, 159, 161 jS-Carotene, 158, 159, 161 rCarotene, 159, 161, 377 ^-Carotene, 159, 161 0-Carotene, 159 Carotenoids, 374, 376 biosynthesis of, 160-162 formation of, 158-160, 337 function of, 162, 361 Carvacrol, 441 Casein, 251, 301 Catalase, 221, 231-232, 307, 308. 374, 471 Catechol, 174, 175, 263 Cell disruption, 205 Cell wall, 39-41 Cellobiase, 108, 113 Cellobiose, 107, 108, 113 utilization of, 56, 66, 68-73 Cellotriose, 113 Cellulase, 113 Cellulose, 40-41, 368 hydrolysis of, 112-113 utilization of, 68-73, 77-79. 83-84, 90 Cephalins, 47, 153 Cephalosporin N, 184 Cephalosporin Pj, 158 Cerebrins, 47-48 Cesium, 303, 316 Chelating agents, 306, 312, 315-316, 449 toxicity of, 442, 454, 466-467, 471, 472, 478-184 Chemical potential, 458-459 Chemostat, 3 Chemotherapy, 440 Chemotropism, 391-392 Chitin, 35, 37, 39-41, 377, 393 utilization of, 79-80 Chitinases, 119 Chlamydospores, 356, 366 Chloramphenicol, 160, 166, 183 Chloranil, see Tetrachloro-p-ben/oqui- none Chlorate, toxicity of, 245 Chlorination, biological, 183-184 Chlorine, 31 3 p-Chloromercuribenzoic acid, 463, 470 Chloromycetin, see Choramphenicol 2-Choro-l, 4-naphthoquinone, 344 Chlortetracycline, 183 see also Aureomycin Cholesterol, 155 Cholic acid, 441 Choline, 38, 181-182, 273 Choline sulfate, 45 Chromates, 466 Chromium, 313 Chrysophanic acid, 168, 170 Citric acid, 22, 312, 316 formation of, 133, 224, 225, 308-309 metabolism of, 138, 219, 223-227 utilization of, 68-73, 81 Citric acid cycle, 138, 222-226 Citrinin, 308 Citrovorum factor, 342 L-Citrulline, 269, 277 Clavacin, see Patulin Clavatin, see Patulin Claviformin, see Patulin Cobalamins, see Vitamins B12 Cobalt, 253, 312-313, 316, 343 Cocarboxylase, see Thiamine pyrophos- phate Cocoa butter, utilization of, 82 Coconut oil, utilization of, 82 Coenzyme A, 152, 161, 342 Colchicine, 170 Compactose, 43 Competitive inhibition, 461—162 Condensing enzyme, 223, 226, 227 Copper, 314, 315 physiological effects of, 139, 184, 309- 310, 370 requirement for, 309-310 toxicity of, 307, 446, 447, 449, 451, 465-472 uptake of, 316, 450-452, 467-472 Copper oxine, 481 Copper sulfate, 466 Coprogen, 344-345 Cordycepin, 122, 282, 283 Corn steep liquor, 186 Cotton, as culture medium, 3 Creosote, 448 Cresols, 455 512 SUBJECT INDEX < ryptoxanthin, 159 Crystal violet, 447, 475 Culture, shaken, 2 Cupric ammonium sulfate, 466, 467 Cyanide, 231, 250, 455, 480 Cvanocobalamin, 337, 343-344 see also Vitamins Bu Cycloheximide, 282, 283, 442, 449 Cyclopaldic acid, 166, 172 Cyclopenin, 283 Cyclopolic acid, 166, 172 Cycloserine, 280 Cystathionine, 270 Cysteine, 186, 205, 470 metabolism of, 270, 305-306 utilization of, 257, 305 Cysteine desulfhydrase, 264 Cysteine-sulfinic acid, 306 Cystine, 186, 305 Cytase, see Protopectinase Cytidylic acid, 277 Cytochrome oxidase, 230-231, 471, 480 Cytochrome peroxidase, 232 Cytochrome reductases, 230 Cytochromes, 204, 229-231, 309, 330, 344, 377, 462 Cytosine, 276, 283 Dechlorogriseofuhin, 183 Dehydroacetic acid, 172, 477 Dehydrofusaric acid, 283 A'-Dehydropipecolic acid, 262 5-Dehydroquinic acid, 171 5-Dehydroshikimic acid, 171 2-Deoxyglycose, 64, 220 4-Deoxypyridine, 335 Deoxyribonucleic acid, 45, 275 Depsides, 169, 170 Depsidones, 169, 170 Dermatophyte fungi, 16, 21, 84, 252, 278 Despeptido-actinomycin, 254 Desthiobiotin, 327, 333-334 Detoxification, 449, 476 Dextranase, 119 Dextrin, 68-73, 76 Dextrinase, 119 Dialkylfluorophosphatase, 480 2,6-Diaminopurine, 276-277 Diastase, see Amylases Diatetrynes, 164, 283 Diazomethane, 443 Dichlone, see 2,3-Dichloro-l,4-naphtho- quinone 2,4-Dichloro-6-(o-chloroanilino)-s-tria- zine, 457 l,2-Dichloro-4,5-diamonibenzene, 337 2,3-Dichloro-l,4-naphthoquinone, 344, 462, 475, 476 2,6-Dichlorophenolindophenol, 204, 205 Dichromate, potassium, 448 Diethanolamine, 209 Diethylarsonic acid, 182 Digallic acid, 176 Dihydroxyacetone phosphate, see Triose phosphates a,/3-Dihydroxy-£-ethylbutyric acid, 270 a,/3-Dihydroxy-|3-methylbutyric acid, 270 Dihydroorotic acid, 277 2,5-Dihydroxyphenylacetic acid, 174 2,4-Dihydroxyphenylalanine, 263 2,5-Dihydroxyphenylpyruvic acid, 263 3,5-Dihydroxyphthalic acid, 165 2,3-Dimercaptopropanol, 470 Dimethylamine, 483 Dimethylaminoethanol, 181 5,6-Dimethylbenzimidazole, 344 1 ,2-Dimethyl-4,5-diaminobenzene, 337, 343 Dimethylglycine, 181 Dimethylglyoxime, 442 Dimethylpyruvic acid, 135, 116 Dimethyl selenide, 182 Dimethyl sulfide, 181 Dimethyl telluride, 182 Dimorphic fungi, 10-11, 206, 327, 333 m-Dinitrobenzene, 245 3,5-Dinitro-o-cresol, 462 2,4-Dinitrophenol, 186, 210, 432, 443, 456, 462 Diosgenin, 157 Dipeptidases, 253 Diphenyl, 441 Diphenylamine, 160 Diphenylthiocarbazone, 315 Diphosphopyridine, nucleotidase, 275, 309, 393 Diphosphopyridine nucleotide, 122, 204, 213, 217, 223, 229, 338 Diploicin, 170 a.a'-Dipyridyl, 479, 480 SUBJECT INDEX 513 Dissociation, 454—455 Disulfide, 305, 393 Dithiazolidines, 472 Dithiocarbamates, 451, 454, 472, 481-484 diethyl-, 480 dimethyl-, 451, 480, 482 Diurnal cycles, 358 Dithionate, 305 Docosanoic acid, 48 Dodecane, 82, 163 Dodecanoic acid, 48, 154 Dormancy, 395-398 Dry weight, determination of, 11-12 Dulcitol, 64, 68-73 Dyes, 441, 455, 464 Eburicoic acid, 157, 158 Echinulin, 280 Ecology, and vitamin requirements, 346- 347 Economic coefficient, 89-90 Edestin, 251 Egg albumen, 251 Eicosanoic acid, 48 Embden-Meyerhof pathway, 212-217, 219 Emodin, 168 Endogenous respiration, 210-211, 463 Endopeptidases, 251-253 Enniatins, 255 Enolase, 213 Enzymes, adaptive, 75, 86-88, 207-208 inactivation of, 462-464, 470-472, 476- 477 induced, 75, 86-88, 207-208 Epoxides, 443 /rans-L-Epoxysuccinic acid, see Ethylene oxide a,/3-dicarboxylic acid Erdin, 183 Ergonovine, 279 Ergosterol, 49, 82, 155 pahnitate, 49 Ergot compounds, 266, 279-280 Ergothioneine, 273-274 Erythritol, 65, 68-73, 122 Erythrose-4-phosphate, 218 Erythrulose, 123 Esterases, 152 Ethanol, 57, 328, 329 formation of, 141, 213-216, 221 Ethanol, metabolism of, 83, 143, 214, 227 Ethanol dehydrogenase, 213, 309 Ethanolamine, 47 Ethyl acetate, 134, 143 Ethyl disulfide, 182 Ethylene, 163 Ethylene chlorhydrin, 178-179 Ethylenediaminetetraacetic acid, 265. 316, 467, 480, 481 Ethylene dn.sothiocyanate, 485 Ethylene glycol, 123 Ethylene oxide derivatives, 464 Ethylene oxide a,/3-dicarboxylic acid, 135, 146 Ethylene thiuram monosulfide, 485 Exopeptidases, 251-253 Expansin, see Patulin Fat coefficient, 150 Fats, formation of, 149-151 Fatty acid dehydrogenase, 152 Fatty acids, biosynthesis of, 151-152, 333, 462 oxidation of, 153-158 toxicity of, 455, 461 Ferguson's principle, 458-459 Fermentation, 202, 212-217, 221-222, 394 Fermentative assimilation, 212 Ferrichromes, 309, 344-345 Ferricyanide, 205, 463 Ferulic acid, 174 Fibrin, 251 Flavin adenine dinucleotide, 220, 231, 244-245, 261, 336 Flavin enzymes, 231, 337 Flavins, as photoreceptors, 361, 362 Flavocol, 283 Fluoride, 448, 449, 455 2-Fluoro-p-aminobenzoic acid, 340 Folic acid, see Pteroylglutamic acid Formic acid, formation of, 138, 143, 144 metabolism of, 181-182, 227, 337 utilization of, 68-73, 82 Formylkynurenine, 338 N-Formylpteroic acid, 342-343 Freezing injury, 425-427 Fructans, 76 Fructose, 60-62, 68-73, 122 Fructose- 1,6-diphosphate, 204, 213, 218 514 SUBJECT INDEX Fructose-6-phosphate, 213, 218 Fumarase, 223 Fumaric acid, biosynthesis of, 142, 227 formation of, 133, 141-143 metabolism of, 138, 223, 262, 203, 266, 269 utilization of, 68-73 Fumaryl-DL-alanine, 133, 112, 280 Fungicidal action, 447 Fungistatic action, 443-447 Fungisterol, 49 Fungitoxic action, and structure, 457- 465, 477 Fungitoxicants, effect of on mitosis, 441 effect of on morphology, 441 Furan derivatives, 180 Furfural, 397, 453 Fusaric acid, 280, 281, 283, 415 Fusarubin, 170 Galactocarolose, 42, 43 Galactogen, 119 Galactonic acid, 132, 135 i.-Galactono-7-lactone, 180 D-Galactose, utilization of, 61-62, 68-73 a-Galactosidase, 109, 110 £-Galactosidase, 87-88, 108 D-Galacturonic acid, 65 Gallic acid, 165, 175, 176 Gallium, 313 Gammexane, 340 Gangaleoidin, 170 Gelase, 120 Gelatin, 251 Gentianose, 109 Gentisyl alcohol, 165, 172-173 Geodin, 183 Geotropism, 357 Germ tube growth, 388, 391, 410 Gladiolic acid, 166, 172 Gliadin, 251 Gliotoxin, 280, 283, 472 Gliotoxin monoacetate, 280 Gluconic acid, formation of, 131-135, 137, 138, 141 2-keto-, 65, 132, 221 metabolism of, 208, 220-221 utilization of, 65, 68-73 D-Glucono-5-lactone, 220 Glucosamine, 121 Glucosamine-6-phosphate, 302 Glucose, effect of concentration, 85-86 metabolism of, 208, 213, 220 utilization of, 60, 68-73 Glucose aerodehydrogenase, 135, 220- 221, 229 Glucose dehydrogenases, 220 Glucose oxidase, see Glucose aerodehy- drogenase Glucose-6-phosphate, 204, 213, 217-218 Glucose-6-phosphate dehydrogenase, 204, 217, 218 /3-Glucosidases 108, 109, 110, 471 Glucosone, 121 Glucuronic acid, 65, 132, 135 Glutaconic acid, 135, 146 Glutamic acid, assimilation of, 259, 480 metabolism of, 225, 260, 262, 264, 266- 268 requirement for, 248 utilization of, 256, 257 Glutamic decarboxylase, 265 Glutamic dehydrogenase, 262, 266, 309 Glutamic-7-semialdehyde, 268 Glutamine, 258, 265, 268 ester of, 280 ■y-Glutamyl transferase, 256 Glutaric acid, 134, 146 Glutathione, 186, 253-254, 470, 484 Glyceraldehyde-3-phosphate, see Triose phosphates Glycerol, 123, 214, 469 utilization of, 65, 68-73 Glycerol phosphates, 123, 302 Glycerophosphatase, 153, 302 Glycine, copper complex of, 466-467 metabolism of, 143, 257, 260, 265, 269 toxicity of, 258 Glycogen, 42, 43, 121, 365 Glycogens, utilization of, 68-73, 76 Glycolaldehyde, 123 Glycolic acid, 138, 145, 269 Glycols, 123 Glycylglycine dipeptidase, 253 Glyoxylic acid, formation of, 134, 143, 145 metabolism of, 138, 145, 226, 227, 269, 278 Grifolin, 164 Griseofulvin, 183, 441 SUBJECT INDEX Growth, linear, 12-13 measurement of, 11-13, 321 rate of, 5-10, 102, 374 Guanidine, 278 Guanidinobutyramide, 264 Guanine, 275, 277, 337, 370 effect of on spore germination, 402 C.uanosine, 276 Guanylic acid, 276 Gyrophoric acid, 169 Hallochrome, 263 Heavy metals, toxicity of, 465-472 see also Copper, Mercury, Silver, Ura- nium Helvolic acid, 158 Heme pigments, 308 Hemicelluloses, 42, 76-77, 113-114 O-Heterobiotin, 334-335 Heterothallism, 374-375 7-Hexachlorocyclohexane, 340 Hexachlorophene, 464—165 Hexacosanoic acid, 48 Hexacosenoic acid, 48 n Hexadecane, 163 Hexadecanoic acid, hydroxy, 47 Hexadecenoic acid, 48 Hexanoic acids, 154 Hexokinase, 213 Hippuric acid, 265 Hippuricase, 253, 265 Histamine, 273. 370 Histidine, 313 accumulation of, 259 metabolism of, 257, 264, 271, 274 requirement for, 250-251, 259 i.-Histidinol phosphate, 271 Histidylhistidine, 250-251 Histozyme, 265 Homocysteine, 181, 270, 341 Homogentisic acid, 263 Homologous series and toxicity, 457-461 Homoprotocatechuic acid, 165 Homoserine, 270 Hormones, in reproduction, 373-375 Hughes press, 205 Humidity, effect of on growth, 25 effect of on reproduction, 364-365 effect of on spore germination, 25, 399-100, 404-406 515 H\drocarbons, metabolism of, 82, 163- 164 toxicity of, 457, 464 Hydrogen peroxide, 231 Hydrogen sulfide, 473-475 Hydrotropism, 364 Hydroxocobalamin, 343 Hydroxy acids, chelation by, 479 3-Hydroxyanthranilic acid, 338-339 p-Hydroxybenzoic acid, 444 o-Hydroxybutyric acid, 22 5-Hydroxy-N-dimethyl tryptamine, 274 2-Hydroxy-5-furan carboxylic acid, 181 3-Hydroxykynurenine, 263, 267, 338 Hydroxylamine, 245, 249 Hydroxvlamine reductase, 245 Hydroxymethylethylketone, 123 /3-Hydroxy-/3-methylglutaryl-coenzyme A, 161 p-Hydroxyphenylacetic acid, 186 2-Hydroxyphenylacetic acid, 174 p-Hydroxyphenylpyruvic acid, 263 Hydroxyproline, 257, 258, 268 8-Hydroxyquinoline, 315, 448 see also Oxine 8-Hydroxyquinoline-5-sulfonic acid, 481 5-Hydroxytryptophan, 263, 274 Hyponi trite, 245 Hypophosphite, 301 Hyposulfite, 305 Hvpoxanthine, 275, 276, 370, 397, 402 Imidazole acetol phosphate, 271 Imidazole glycerol phosphate, 271 Imidazoles, 484 Imidazolines, 447, 448, 451, 461, 462 Indigo, 280, 281 Indole, 263, 271-272, 280, 449 Indoleacetaldehyde, 264 Indoleacetic acid, 263-264, 361 Indolepyruvic acid, 264 Indole-serine ligase, 271, 309 Infrared radiation, 432, 435 Inoculum, 1, 3 Inosine, 276 Inosine-5-phosphate, 276 Inositol, 47, 323, 326, 329, 339-340 Inulase, 119 Inulin, 68-73, 76 Invertase, see Sucrase 516 Iodoacetamide, 391 Iodoacetic acid, 463 Iodosobenzoic acid, 463 Ionization, 454-457 Ionizing radiation, 433-435 /3-Ionone, 161-162 Iron, 38, 307-308, 313 physiological effects of, 139, 172-173, 307-308, 336-337, 370 Isoamyl alcohol, 262 Isoamylamine, 273 Isocitric acid, 138, 143, 145, 223, 225, 227 Isocitric dehydrogenase, 223, 226 Isocitritase, 226, 227 D-Isoleucine, 261 DL-Isoleucine, 257 L-Isoleucine, 270 Isomaltose, 102, 107 Isonicotinic hydrazide, 160 Isopropyl alcohol, 474 Itaconic acid, 134, 138, 144-145 Itatartaric acid, 134, 145 Itatartaric acid lactone, 145 Javanicin, 168, 170, 475 Junipal, 181 Keratin, 84, 251-252 /3-Keto acid decarboxylases, 154 a-Ketoadipic acid, 271 ^-Ketoadipic acid, 174 a-Ketobutyric acid, 262 2-Ketogluconic acid, 65, 132, 221 a-Ketoglutaric acid, metabolism of, 138, 223-225, 262, 266, 267, 377, 428 a-Ketoglutaric acid oxidase, 226 a-Ketoisocaproic acid, 270 a-Ketoisovaleric acid, 342 Ketones, a,/3-unsaturated, 477-478 5-Keto-octadecanoic acid, dodecyl ester, 47 6-Keto-octadecanoic acid, 47 Kojic acid, 178-179, 219 Kynurenic acid, 263 Kynurenine, 267, 338-339 Kynurenine transaminase, 267 Laccase, 175-176 Lachrymators, 478 Lactase, 87-88, 108 SUBJECT INDEX Lactic acid, biosynthesis of, 136, 215- 216, 228 formation of, 132, 136, 138, 185 utilization of, 68-73 Lactic dehydrogenase, 136, 230 Lactobacillus casei factor, 342-343 Lactobionic acid, 135 Lactose, metabolism of, 107, 108 utilization of, 56, 67, 68-73, 74, 185 Lag phase, 6 Lecanoric acid, 169 Lecithins, 47, 153 D-Leucine, 261 DL-Leucine, 256, 257 L-Leucine, 259, 260, 262, 270 L-Leucyl-L-proline anhydride, 255 Levansucrase, 120 Levomycin, 254 Lichenase, 114, 119 Light, 160, 357-362, 427-428 Lignin, 41, 83, 90 Lignoceric acid, 48 Lignosulfonic acid, 84 Lime sulfur, 472-473 Linoleic acid, 47, 48 Linolenic acid, 48 Lipases, 152-153 Lipids, 82, 374 metabolism of, 152-164 of cells, 35, 37, 41, 45-49, 392-393 see also Fats, Fatty acids, Phospholi- pids Lipositol, 340 Lipoxidase, 155 Luminescence, 26-27 Luteic acid, 43 Lycomarasmin, 254-255, 266 Lycopene, 158, 159, 161 Lycoxanthin, 159 Lyophilization, 4 Lysergic acid, 279, 280 a-D-Lysine, 261 a-L-Lysine, 257, 259, 261-262, 271 |3-Lysine, 266 Lysozyme, 453 Magnesium, of cells, 37, 38 physiological effects of, 19, 139, 306- 307, 313, 316, 371 requirement for, 304, 306-307 SUBJECT INDEX Malachite green, 475, 476 Malate synthetase, 227 Malic acid, 316, 466-467 formation of, 133, 142-143 metabolism of, 138, 222, 223, 225, 227 utilization of, 68-73, 81 Malic dehydrogenase, 223, 227 Malic enzyme, 142 Malonic acid, 81, 146, 224, 225, 276 Maltose, 66, 68-73, 107, 108 Manganese, 314, 315 physiological effects of, 139, 310, 370, 479, 480 requirement for, 310 Manganese dioxide, 205 Mannans, 43, 114 Mannitol, 64, 68-73, 122, 212 D-Mannoheptulose, 63 Mannonic acid, 132, 135 Mannose, 60-62, 68-73 Mannose-1 -phosphate, 62 a-Mannosidases, 110 Mannosidostreptomycin, 110 Mappin, 274 Marine fungi, 16, 26 Media, culture, 4 Melanins, 263, 377, 428 Melezitose, 109 Melibiase, 109 Melibiose, 67, 68-73, 109 Menadione, 344 Menadione reductase, 177 Mercaptide formation, 463, 470 Mercaptobenzothiazole, 472 Mercury, toxicity of, 307, 443, 446, 448, 463, 465^72 Metaphosphates, 38, 302 Methanol, 83 Methionine, metabolism of, 181, 186, 261, 270, 305-306, 327, 341 toxicity of, 258 utilization of, 13, 257 p-Methoxy-tetrachlorophenol, 1 83 4-Methoxy-2,5-£-toluquinone, 167 Methylamyl ketone, 154 Methylarsenate, sodium, 182 Methylation, 181-182, 341 2-Methyl-5-bromomethyl-6-aminopyri- dine, 330 2-Methylbutene-2, 163, 415 517 Methyl cinnamate, 165, 172 /3-Methylcrotanaldehyde, 161 /3-Methylcrotonyl-coenzyme A, 161 S-Methylcysteine, 270 5-Methylcytosine, 182 Methyldiethylarsine, 182 Methylene blue, 204, 205, 469, 475 2,2'-Methylenebis (3,4,6-trichlorophenol), 464-465 Methylethyl sulfide, 182 a-Methylglucoside, 65, 110 /3-Methylglucoside, 65 Methylheptenone, 161-162 4-Methyl-5-/3-hydroxyethylthiazole, 330 2-Methyl-3-hydroxy- ^naphthoquinone, 344 N-Methylisoleucine, 266 Methyl ketones, 154 a-Methylmannoside, 110 Methyl mercaptan, 182 Methyl-p-methoxybenzoic acid, 402 Methyl-p-methoxycinnamic acid, 172 2-Methyl-l,4-naphthoquinone, 344, 475 Methylnonyl ketone, 154 Methylpropyl ketone, 154 D-a-Methylserine, 266 7-Methyltetronic acid, 180 N-Methylvaline, 266 Mineoluteic acid, 47 Mineral oil, 4 Minerals, 37-38, 57, 300-322 and reproduction, 370-371 and spore germination, 401 Molybdenum, 244-245, 310-311, 314, 315 Monoallyl phosphoric acid, 123, 302 Monomethylamine, 466 Monomethylaminoethanol, 181 m-czs-Muconic acid, 174 Mucopolysaccharases, 119-120 Muscarine, 273 Mutarotase, 220 Mutation, 429, 432, 434-435, 443 Mycelianamide, 280 Mycelium, decomposition of in soil, 45 Mycodextran, 43 Mycogalactan, 43 Mycomycin, 164 Mycophenolic acid, 166 Mycorrhizal fungi, 91, 347 Myokinase, 203 518 Myristic acid, 48 Nalgiolaxin, 183 Nalgiovensin, 183 Naphthoquinone derivatives, 168, 170, 344, 475-477 w-(2-Naphthyloxy)-72-alkylcarboxylic acids, 461 Neopyrithiamine, 332 Neurosporene, 159, 161 Nicotinamide, 337 Nicotinic acid, 326, 327, 328, 337-339, 345 Nicotinic acid esters, 461 Nidulin, 169, 170, 183 Nigeran, 43 Nitrate, 242-245 Nitrate reductase, 244-245 Nitrite, metabolism of, 249, 335 physiological effects of, 215, 2-13, 246 utilization of, 246 Nitrite reductase, 245 m-Nitroaniline, 245 Nitroaryl reductase, 245 Nitrobenzene, 83 Nitrogen, total, 12, 36, 43-41 Nitrogen fixation, 249 Nitrogen mustard, 432, 443 Nitrogen supply, physiological effects of, 19, 41, 79, 139, 149, 178-179, 210, 247-248, 275, 369-370 Nitrophenols, 462-464 p-Nitrophenyl-/3-glucoside, 107 /7-Nitrophenyl phosphate, 302 ^-Nitropropionic acid, 280, 281 w-Nitrostyrene derivatives, 478 Notatin, see Glucose aerodehydrogenase Nuclei, of spores, 389-390 Nucleic acids, 45, 274-277 Nucleosides, 275-277 Nucleotides, 203, 275-277 Octadecadienoic acid, 48 Octadecanoic acid, 48 keto derivatives of, 47 Octadecatrienoic acid, 48 Octadecenoic acid, 48 Octanoic acid, 154, 155 Oleic acid, 48, 82, 333 SUBJECT INDEX Oligosaccharides, 66-75, 105-109, 368- 369 Oosporein, 167 Order of death at high temperature, 424-425 Ornithine, 261, 267, 268 Ornithine cycle, 268-269 Orotic acid, 277 Orotidine, 277 Orotidine-5'-phosphate, 277 Osmotic pressure, 25-26, 406 Oxalacetic acid, 138, 141-143, 223-229, 330, 333 Oxalacetic decarboxylase, 226 Oxalic acid, 228, 329 formation of, 134, 137, 138, 141, 144, 145, 180, 303 utilization of, 68-73, 81 Oxalic acid decarboxylase, 144 Oxalosuccinic acid, 223 Oxalosuccinic decarboxylase, 223, 226 /3-Oxidation, 153-155 Oxidation-reduction balance, 100-101 Oxidative assimilation, 212, 462-463 Oxidative phosphorylation, 203, 462 Oxine, 315, 478, 480-481 see also 8-Hydroxyquinoline d/-Oxybiotin, 334-335 Oxygen, determination of, 203 diffusion of, 205 physiological effects of, 23-24, 208- 209, 365-366, 394, 412-413, 435 Oxygen demand, 2-3 Oxyjavanicin, 170 Oxy tetracycline, 183 Ozone, 366 Palmitic acid, 48, 82 Pantethine, 161 Pantoic acid, 342 Pantothenic acid, 161, 329, 341-342, 345 Paraffins, 82 Partition coefficient, 458-459 Patulin, 172-173, 179-180, 477 Pectase, see Pectin-methylesterase Pectic materials, 41, 80, 114-119 Pectinase, see Pectin-polygalacturonase Pectin-depolymerase, 116, 118 Pectin-methylesterase, 115-119 Pectin-polygalacturonase, 115-119 SUBJECT INDEX 519 Penatin, 220 Penicillic acid, 180, 477 Penicillin, 15, 184-187, 308, 411 Penicillin amidase, 187 Penicillinase, 187 Penicillin B, 220 Penicilloic acids, 187 Pentaacetylglucose, 64 Pentonic acids, 222 Pentosans, 42, 43, 77, 113-114 Pentose phosphates, 217-218 Pentoses, metabolism of, 143, 208, 221- 222 Peptidases, 265, 479 see also Endopeptidases, Exopepti- dases Peptides, 250-251, 255-256, 479 Periodicity, 362 Permeability, 206-207, 397, 442-443, 449, 453-454, 458-460, 470 to acids, 80 to cations, 391 to metals, 468 to vitamins, 326, 340 Peroxidases, 232 Peroxides, 433, 443 Persulfate, 305 pH, effect of ammonium utilization on, 247-248 effect of organic acid utilization on, 80, 248 effect of on growth, 14-15, 19-23, 56 effect of on metabolism, 19, 141, 150, 178, 208, 337 effect of on mineral nutrition, 307, 312, 316 effect of on permeability, 80, 326 effect of on reproduction, 363, 366 effect of on spore germination, 403- 404, 409 effect of on substrate utilization, 79, 81, 244, 246 effect of on toxicity, 454-457, 468 effect of on vitamin requirements, 335, 337, 340 effect of on zonation, 359 internal, 22-23 Phallin, 255 Phalloidine, 255 Phenanthraquinones, 477 o-Phenanthroline, 479, 480 Phenazine derivatives, 205 Phenol, 83, 441 Phenols, oxidation of, 174-176 toxicity of, 449, 453, 455, 457, 463, 464 Phenolsulfatase, 306 Phenothiazone, 472 Phenylacetic acid, 173-174, 186, 208 Phenylalanine, 171, 257-259, 271-272 Phenylethylamine, 186, 273 a-Phenylglycine, 271 Phenylmercuric acetate, 467 Phenylpyruvic acid, 271-272 Phomazarin, 283 Phosphatases, 105, 123, 275, 302-303 Phosphate, 22, 249, 301-304 Phosphatides, 46, 47, 153 Phosphite, 301 Phosphoenolpyruvic transphosphorylase, 213 Phosphofructokinase, 213 Phosphogluconate oxidation pathway, 179, 217-220, 221-222 6-Phosphogluconic acid, 217 Phosphoglyceric acids, 213, 372 Phosphoglyceryl kinase, 213 Phosphoglyceryl mutase, 213 Phosphohexoisomerase, 213 Phospholipases, 153 Phospholipids, 152, 339 see also Phosphatides Phosphomonoesterases, 123 Phosphoproteins, 38, 44 Phosphorus, 8, 139, 149, 301-304, 469 bioassay for, 313, 314 of cells, 37, 38, 376 Phosphorylases, 121 Phosphorylation, oxidative, 213, 462 Photoreactivation, 433 Phototaxis, 360 Phototropism, 358, 360-362, 392 Phthalic acid, 22 Phycomycetes, physiological characteris- tics of, 78, 90, 215, 304-305, 347, 366 Phytic acid, 301, 302 Phytin, 339-340 Phytoene, 159, 161 Phytofluene, 159, 161 Picroroccelin, 280 Pimelic acid, 334 520 SUBJECT INDEX Pinselic acid, 168 Pipecolic acid, 262 Plant pathogenic fungi, 19, 21, 115, 347, 366, 377-378 Plasmoptysis, 405 Polyenes, 164 Polyenynes, 164 Polyfructoses of mycelium, 43 Polygalactoses of mycelium, 43 Polyglucoses of mycelium, 43 cz's- Polyisoprene, 164 Polymyxin, 453 Polyphenol oxidases, 84, 173, 174-176, 178, 263, 480 Polyphosphates, 302, 479 Polyporenic acids, 158 Polysaccharides, 42-43, 120-121, 368-369 Polyuronide of mycelium, 43 Porphyrins, 479 Potassium, 303-304, 316, 371, 468 bioassay for, 314 of cells, 37, 38 permeability to, 468 Powdery mildew fungi, spore germina- tion in, 396, 398, 399, 401, 405-406, 408, 412 Prephenic acid, 271-272 Preservation of cultures, 4-5 Pressure, 423, 433 Progesterone, 157 Proline, 257, 260, 265, 268 /3-Propiolactone, 454 Propionic acid, 68-73, 81, 462 Propylene oxide, 377-378 Proteases, 251-253 Protein, of cells, 37, 41, 44-45, 393 requirement for, 345 utilization of, 250-252 Proteinases, see Endopeptidases Protocatechuic acid, 171-172, 174 Protopectinase, 115-116 Pteroylglutamic acid, 341-343 Pthiocol, 344 Puberulonic acid, 169 Pulcherrimin, 283, 308 Purines, 275-278, 462 Putrescine, 273 Pyrazine derivatives, 283 Pyrazoles, 464 3-Pyridinethiol, 472 Pyridoxal, 335 Pyridoxal phosphate, 267, 271, 335 Pyridoxamine, 335 Pyridoxine, 326, 328, 329, 335, 345 Pyrimidine moiety of thiamine, 330-332 Pyrimidines, 275-277 Pyrithiamine, 332 Pyrone derivatives, 172, 178-180 Pyrophosphate, 38, 302 A'-Pyrroline-5-carboxylic acid, 268 Pyruvic acid, 329, 397 carboxylation of, 142, 228, 333 from serine, 143, 262 role of, in respiration, 138, 213-216, 218, 222-228 in valine synthesis, 270 Pyruvic carboxylase, 213, 216, 309 Pyruvic oxime, 249 Qio, of growth, 15 of respiration, 208 of thermal death, 425 Quinic acid, 171, 172 Quinoline derivatives, 283 Quinolinic acid, 338-339 Quinone reductase, 177 Quinones, in respiration, 177-178 toxicity of, 449, 463, 475-478 Raffinose, 67-73, 107, 109 Ravenelin, 168 Resistance, 378 Respiration, aerobic, 202 anaerobic, 202 endogenous, 210-211, 463 inhibition of, 442^143, 484 of spores, 393-395 terminal, 229-232 Respirometry, 203 Resting cells, 101 Resting spores, 396-398 Rhamnose, 63, 68-73 Rhizopterin, 342-343 Rhodoviolascin, 158, 159 Riboflavin, 307, 326, 328, 336-337, 345, 372, 402 Riboflavin monophosphate, 275 Ribonucleic acid, 45, 275 SUBJECT INDEX Ribose, 64, 68-73 Ribose-1 -phosphate, 276 Ribose-5-phosphate, 218 Ribulose-5-phosphate, 218 Rubber, 84-85 Rubidium, 303, 316 Rubrofusarin, 170 Rugulose, 43 Rust fungi, 26, 378, 426 spore germination in, 395, 397, 398, 399-401, 403, 405, 408, 412, 414 D-Saccharic acid, 65, 132, 135 Saponin, 57 Sarcosine, 266 Sawdust, as culture medium, 3 Scandium, 313 Sclerotia, 356-357 Sclerotiose, 43, 121 Sedoheptulose phosphates, 218 Selena te, sodium, 182 Selenide, sodium, 182 Selenium, 182, 475 Self-inhibition, 413^15 Serine, 47, 143, 257, 262, 269, 271 Serine dehydrase, 262 Shaker, 2 Shikimic acid, 171, 172, 271-272, 341 Silicon, 38, 313 Silver, toxicity of, 446, 447, 452, 453, 465-472 uptake of, 452, 468-472 Sodium, 38, 300, 303 Soil, as culture medium, 3 in preservation of cultures, 4 Soil fungi, 23, 247 Solanin, 441 Solanione, 170 Solubility, 458-459 Sonic oscillation, 205 Sorbitol, 68-73, 122 Sorbose, 122, 390 toxicity of, 10, 62-63 utilization of, 62, 68-73 Spectrophotometry, 204 Spiculisporic acid, 47 Spinulosin, 167 Spirilloxanthin, 159 Spore discharge, 364-365 521 Spore germination, effect of ionizing ra- diation on, 433-435 effect of light on, 428 effect of pH on, 22, 403-104 effect of temperature on, 18, 407-412 latent period of, 388, 391, 410-111 rate of, 388, 390-391, 395-396, 400, 410-411 "Spore starch," 42 Spores, maturation of, 395-398 nuclear number of, 389-390 respiration of, 393-395 swelling of, 391, 406, 413 Sporulation, effect of calcium on, 312 effect of zinc on, 309 inhibition of, 481 Squalene, 157 Stachyose, 109 Staling, 8, 249 Stannic chloride, 467 Stannous chloride, 467 Starch, 68-73, 75-76 Stearic acid, 48, 82 Sterilization, chemical, 377-378 Steroids, requirement for, 345 Sterols, 46, 49, 82, 155-157 Stipitatic acid, 169 Streptolin, 254 Streptomycin, 110, 160, 228, 308, 441, 452 Streptothricin, 254 Strontium, 311 Structure and fungitoxicity, 464-465 Styrene, 478 Succinate-ketoglutarate-isocitrate cycle, 224 Succinic acid, metabolism of, 142, 145, 146, 223, 225-228, 428 formation of, 133, 142-143 utilization of, 68-73, 81 Succinic dehydrogenase, 223 Succinoxidase, 230 Sucrase, 105-107, 374, 393, 471 Sucrose, 324 breakdown of, 105-107, 393 utilization of, 66-74, 369 Sucrose phosphorylase, 107 Sugar alcohols, 56, 122 "Sugar fungi," 91 Sugar phosphates, hydrolysis of, 105, 302 Sugars, decomposition of, 57-58 522 Sulfanilamide, 276, 442, 461 Sulfate, 182, 186, 305-306 Sulfhydryl compounds, 11, 393 Sulfhydryl enzymes, inhibition of, 462- 463, 470-472, 476-478, 485 SulBde, 305 Sulfite, 305 Sulfonamides, 341, 456, 461 Sulfur, 8, 139, 182 requirement for, 304-306 toxicity of, 450, 472-475 Sulfur mustard, 443 Sulochrin, 166 Surface action, 453-454 Surface tension, 458-459 Tannase, 176 Tannic acid, 83-84, 175 Tannins, 85 Tartaric acid, 68-73, 81, 134, 145 Taurocholate, sodium, 441 Taxonomy, 39-41, 79. 90-91, 103, 346- 347 Telemorphosis, 374 Tellurite, potassium, 182 Tellurium, 182, 475 Temperature, and vitamin require- ments, 326, 336 and zonation, 359 effect of on growth, 13-19 effect of on metabolism, 79, 208 effect of on pH response, 19 effect of on reproduction, 359, 362- 364 effect of on spore germination, 389, 397, 399, 407-112 lethal effects of, 423-427, 435 trophic effects of, 362 Terminal respiration, 229-232 Terrein, 183 Terrestric acid, 180 Tetrachloro-p-benzoquinone, 476 Tetrachloronitrobenzenes, 449 Tetracosanoic acid, 48 Tetracosenoic acid, 48 Tetracycline, 183 Tetradecane, 82, 163 Tetradecanoic acid, 48 Tetrahydrophytoene, 161 Tetramethylthiuram disulfide, 482-483 SUBJECT INDEX Tetramethylthiuram monosulfide, 483 Tetronic acid, 180 Thelcphoric acid, 167 Theophylline, 276 Thermal death point, 423-424 Thermal death time, 424 Thermophily, 18 Thiaminase, 329 Thiamine, 13, 215, 326, 328, 329-332, 335, 339, 345, 347, 371-372 Thiamine pyrophosphate, 329-330 Thiazole moiety of thiamine, 330-332 Thigmotropism, 392 Thiobisphenols, 472 Thiocyanate, 305 Thiocyanate derivatives, 472 Thioglycolic acid, 477 Thiol compounds, protection by, 463, 470, 476-477 Thiolutin, 280 Thionophosphate, 302 Thiosulfate, 305, 454 Thiourea, 184, 245, 304, 305, 472 D-Threitol, 122 Threonine, 257, 262, 264, 270 D-Threose, 64 Thujaplicins, 170, 475 Thunberg method, 202-203 Thunberg-Wieland reaction, 142-143, 227 Thymine, 275 Thymol, 441 Tin, 467 Tolerance, to toxicants, 448-449 Toluquinone, 173 Torularhodin, 159 Torulene, 159 Toxicity, apparent, 451 specific, 451 Transacetylase, 143 Transaldolase, 218 Transamination, 258, 267 Transfructosidase, 106-107 Transgalactosidase, 107, 108 Transglucosidase, 107, 108 Transglycosidases, 105-108 Transketolase, 218, 329 Transmethylation, 181-182 Trehalase, 108 Trehalose, 66, 68-73, 108-109 SUBJECT INDEX s-Triazines, 451-452, 453, 457, 463, 464 Tri-n-butyltin acetate, 467 N-(Trichloromethylthio)-4-cyclohexene- 1,2-dicarboxamide, 442 Trimethylamine, 273 Trimethylarsine, 182 Trimethylethylene, 163, 415 Triose phosphate dehydrogenase, 204, 213, 484 Triose phosphate isomerase, 213 Triose phosphates, 213, 218, 221 Tripalmitin, 82 Triphenylmethanes, 464, 475 Triphosphate, 302 Triphosphopyridine nucleotide, 204, 217-218, 223, 229, 245, 262, 338 Tristearin, 82 Triterpenes, 157-158 Tropisms, 391-392 Tropolone derivatives, 169, 170, 475 Trypsin, 252 Trypsinogen, 252 Tryptophan, 171, 256, 257, 259 metabolism of, 263, 271-272, 338-339 Tryptophan synthetase, 271, 309 Tungstate, sodium, 441 Turbidity, as measure of growth, 12 Tyramine, 263, 273 Tyrosinase, 175-176, 263, 376 Tyrosine, 171, 257, 259, 263, 271-272 Ultraviolet radiation, 429-433 effect of on reproduction, 357-358, 359, 376 Uracil, 275, 276 Uranium, 448, 454, 470 Urea, 45, 249, 277-279 metabolism of, 268-269, 276-279 Urease, 278, 477 L-Ureidosuccinic acid, 277 Urethane, 83, 249 7-(3,4-Ureylenecyclohexyl)butyric acid, 335 Uric acid, 275, 278-279 Uricase, 278 Uridine-5'-phosphate, 277 Urocanic acid, 264 Ustilagic acid, 47, 104, 219 Ustilic acids, 47 523 Utilization, definitions of, 55-56 Valine, 186, 257, 261, 270 Vanadium, 313 Vanillin, 174 Vapor pressure, 458-459 Varianose, 43 Variation, 3, 102 Versene, see Ethylenediaminetetraacetic acid Viomycin, 254 Viridicatin, 281, 283 Vitamin A, 345 Vitamin B1( see Thiamine Vitamin B2, see Riboflavin Vitamin B6, see Pyridoxine Vitamin D, 345 Vitamin E, 345 Vitamins B^, 343-344 Vitamins K, 344, 462 Vitamins, as impurities, 57 effect of on reproduction, 371-372 effect of on spore germination, 402 D-Volemitol, 122 Volutin, 38 Water, and growth, 25-26 see also Bound water, Humidity Water content of cells, 36 Wilt fungi, 18-19, 23 Wood-destroying fungi, 78, 83-84, 175, 347 Xanthates, 472 Xanthine, 275, 276, 278 Xanthine oxidase, 275, 278 Xanthones, 168, 170 Xanthophylls, 162 Xanthurenic acid, 263 X-rays, see Ionizing radiation Xylanases, 114 Xylans, 114 Xylobiose, 114 Xylonic acid, 132, 136 Xylose, 63, 68-73, 221 Xylulose-5-phosphate, 218 Yeast extract, 325 Zeaxanthin, 159 524 SUBJECT INDEX Zein, 251 173, 253, 275, 308-309, 325, 344, 371 Zinc, 38, 308-309 toxicity of, 446 induction of variants by, 443 Zonation, 18, 358-359 permeability to, 468 Zygotropism, 374 physiological effects of, 139, 142, 157, Zymonic acid, 181