g3 VITRO STUDIES OF COLLETOTRICHUM PHOMOIDES UNDER THE INFLUENCE OF SODIUM PYRIDINETHIONE AND OTHER ANTIFUNGAL MATERIALS By Samuel Morris Bingel AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology Year 1956 ngaJLLKIZ> Qdfi§JLL«L/€“C9 Approved Samuel Morris Ringel The following antifungal test compounds were screened in gitgg against Colletotrichum phomoides and Helminthosporium sativum employing Lilly and Barnett's glucose asparagine liquid medium: the sodium salt of l-hydroxy-2(1H) pyridine- thione, diaphine HCl, nystatin, compound M #575, rimocidin sulfate, thiolutin, candicidin and endomycin. Sodium 2-pyridinethione is moderately thermostable and breaks down slowly in aqueous solution at room.temperature. Endomycin was found to be strongly adsorbed onto the surface of asbestos Seitz filter pads. Sodium.2-pyridinethione is fungistatic at concentrations up to 0.12 Pg/ml. The fungicidal nature of the compound is exhibited when the spores of Q, phomoides remain in contact with the inhibitor for an hours at concentrations above 0.3 Pg/ml. Exposures for 20 minutes at concentrations higher than 100 Pg/ml. are also fungicidal. Physiological studies revealed that reducing sugars such as D-glucose decreased the antifungal activity of sodium 2-pyridinethione. It is suggested that the inacti- vation is due to the formation of a complex involving glucose and 2-pyridinethione. This is supported by ultra- violet absorption spectra studies. The complex is thought to be a thio hemi-acetal. The role of glucose in plants is discussed with relation to 2-pyridinethione inactiva- tiono Samuel Morris Ringel 2 The mode of action of 2-pyridinethione was not deter- mined. The inhibitor does not appear to act as a chelating agent for essential metals nor does it function as a sub- strate analogue for niacin. It is suggested that the sulfur portion of 2-pyridinethione is functional in causing in- hibition. IN VITRO STUDIES OF COLLETOTRICHUM PHOMOIDES UNDER THE INFLUENCE OF SODIUM PYRIDINETHIONE AND OTHER ANTIFUNGAL MATERIALS BY Samuel Morris Ringel A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements’ for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1956 masts ACKNOWLEDGMENTS The author wishes to express his sincere thanks to Dr. Everett S. Beneke, under whose constant interest, help- ful criticism and guidance this investigation was undertaken. The writer is also greatly indebted to Dr. Robert S. Ban- durski for his valuable suggestions and kind help in the chemical evaluations. To Mr. Phillip Coleman for aid in preparation of the photographic material. Sincere thanks are also due to the Squibb Institute for Medical Research at New Brunswick, New Jersey for providing prompt and help- ful suggestions via written communications. This work was made possible by a grant from the Horace H. Rackham.Research Endowment, for which the author expresses grateful appre- ciation. VITA Samuel Morris Ringel candidate for the degree of Doctor of Philosophy Final examination, February 21, 1956, 10 A.M., Botany and Plant Pathology Seminar Room Dissertation: 'Ig Vitro Studies of Colletotrichum.phomoides Under the Influence of Sodium Pyridinethione and Other Antifungal Materials Outline of Studies Major subject: Mycology Minor subjects: Bacteriology, Biochemistry Biographical Items Born, November 29, 192h, New York, New York Undergraduate Studies, Hunter College, l9ub-SO Graduate Studies, University of Michigan, 1950-51, Michigan State University, 1953-56 Experience: Jr. Microbiologist, Hoffmann-La Roche Inc., 1951-52, Special Graduate Research Assistant, Michigan State University, 1953-56 Member of Mycological Society of America, Torrey Botanical Club, Society of the Sigma Xi TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION . . . . . . . . . . . . . . . . . II. MATERIALS AND METHODS . . . . . . . . . . . . . A. Source and Maintenance of Materials . . . l. Antifungal Compounds . . . . . . . . \OCDODCIDH 2. Test Organisms . . . . . . . . . . . B. Techniques Employed . . . . . . . . . . . 10 1. Medium Used . . . . . . . . . . . . lO 2. Glassware Cleaning Procedure . . . . lO 3. Screening of Antifungal Materials . . 11 h. ”Shelf Life" Determinations of Antifungal Materials . . . . . . . 12 5. Spore Germination Tests . . . . . . . l3 6. Physiological Studies on Colletotrichum phomoides . . . . . . . . . . . . 13 a. Varying the medium with respect to the carbon source . . . . . 13 b. Varying the medium with respect to the nitrogen source . . . . 1H c. Preparation of inoculum . . . . 15 d. Incubation conditions . . . . . 18 e. Harvesting and data presentation 19 f. Size of inoculum. . . . . . . . 20 g. Concentration range of anti- fungal materials used in study. 23 TABLE OF CONTENTS (Cont.) CHAPTER 7. Additional Techniques Related to the Sodium Pyridinethione Study . . . . III 0 RESULTS AND OBSERVATIONS O O O O O O O O C O O A. Screening of Antifungal Materials . . . . 29 29 29 Be Stability TeStS e e e e e e e e e e e o e 1. Thermostability . . . . . . . . . . 2. "Shelf Life" 0 O O C O O O O O O O O a. Sodium pyridinethione and rimocidin via assay plate test. b. A more critical determination of the "shelf life" of sodium pyridinethione . . . . . . . . Co Endomy01n O I O '0 O O O I O O O 3. The Effects of Different Sterilization Treatments on the Potencies of Sodium Pyridinethione and Rimocidin . . . . C. Physiological Studies with Rimocidin . . . l. The Effects of Different Sugars on Antifungal Activity . . . . . . . . 2. The Protective Effect of D-Glucose and Sucrose on Rimocidin . . . . . . D. Antimicrobial Properties of Sodium Pyridinethione . . . . . . . . . . . . . l. Phenol Coefficient Against Micrococcus pyogenes var. aureus . . . . . . . . 2. Spore Germination Tests . . . . . . . 3. Fungicidal Property . . . . . . . . E. Physiological Studies with Sodium Pyridinethione ii 32 31+ 3h 37 38 38 38 39 h? CHAPTER TABLE OF CONTENTS (Cont.) Page The Effect of Varying the Carbohydrates H7 The Effect of Sugar Concentration on the Antifungal Property 0 e e e e o e o 0 SO Decreased Antifungal Activity in the Presence of Acetaldehyde . . . . . . 55 The Effect of Time on Mixtures of D- Glucose and Sodium Pyridinethione . . 57 a. Spectrophotometric determination . 58 b. Bioassay determination . . . . . . 62 The Effect of Varying the Nitrogen Source . . . . . . . . .‘. . . . . . 67 Growth Curves of Q. phomoides 080A in Response to Varying Cencentrations of Sodium Pyridinethione . . . . . . . . 70 The Effect of Adding Sodium Pyridinethione During Various Phases of the Growth Curve 0f 9-. Rh0m01des 080‘ e e e e e e e e 73 "Cd. Of Action 0 O O O O O O O O I O O 73 a. As a chelating agent . . . . . . 76 b. As a competitor for niacin or DPN. 79 DISCUSSION AND CONCLUSIONS . . . . . . . . . . . 80 SUMMARY eeeeeeeeeeeeeeeeee 93 LI MATIJ-RE CI TED . O O . O O O O O O O C O O O . 96 iii PLATE I. II. III. IV. V. VI. VII. VIII. LIST OF.PLATES Page Colletotrichum phomoides 080A conidial formation in liquid shake culture . . . . . . . . . . Concentration series of endomycin in ug/ml. against Colletotrichum phomoides C80A in liquid medium . . . . . . . . . . . . . Concentration series of endomycin in g/ml. atainst Helminthosporium sativum 925Pg liqu1d medium e e e e e e e e 0 Concentration series of endomycin in ug/ml. against Ustilag_ hordei R6 in liquid'1 medium. . Spore germination test. The effect of sodium pyridinethione on spores of Colletotrichum ph0m01d°8 080‘ O O O O O O O O O O O O O O 0 Spore germination test. The effect of sodium pyridinethione on spores of Helminthospgrium carbonum. . . . . . . . . . . . . . . . . . Spore germination test. The effect of sodium pyridinethione on uredospores of Puccinia floral O O O O O O O O O O O O O O O O O Fungicidal test for sodium pyridinethione. The inhibitor was removed after finite contact times and the treated spores of C. phomoides C8OA plated out on glucose asparagine agar . . Proposed scheme for the inactivation of pyridinethione by glucose . . . . . . . . . . iv 16 26 27 28 Al H2 #3 R6 88 TABLE I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. LIST OF TABLES Effect of spore concentration on agreement between replicate flasks treated with sodium pyridinethione . . . . . . . . . . . . . . . Activities of antifungal materials MIC in Pg/ml. glucose asparagine liquid medium . . . . . . . Thermostability of antifungal materials MIC in Pg/ml. glucose asparagine liquid medium . . Antifungal activity of stored sodium pyridinethione . . . . . . . . . . . . . . . Antifungal activity of autoclaved endomycin . . Antifungal activity of non-sterilized (unheated) endomycin . C C C C O O O O C C O C . 0 Response of‘Q. phomoides 080A to increasing concentrations of sodium pyridinethione in conjunction with different sugars . . . . . . The influence of D-glucose concentration on the antifungal activity of pyridinethione . . . . The influence of sucrose concentration on the antifungal activity of pyridinethione . . . . Sodium pyridinethione inactivation by acetaldehyde . . . . . . . . . . . . . . . . . Inactivating the antifungal property of sodium pyridinethione on prolonged contact with D'Slucose e e e e e e e e e e e e e e e e e Extent of inactivation when sodium pyridinethione is in contact with the complete medium . . . . Growth patterns of.Q, phomoides 080A subjected to different levels of sodium pyridinethione at the time of inoculation . . . . . . . . . Inactivation of sodium pyridinethione by IIiStidine O O O O O O O C O O O 0 O O O O O Page 21 25 3o 30 33 33 us 53 51+ 56 61L 66 71 77 (GRAPH I. II. III. IV. V. VII. VIII. LIST OF GRAPHS Page The amount of growth of Q. phomoides C8OA in the presence of Na pyridinethione or rimocidin which have been subjected to different sterilization treatments . . . . . . . . . . . . . . . . . . Response of C. _phomoides C8OA to increasing con- centrations_ of rimocidin with different sugars . Dosage response curves for sodium pyridinethione based on spore germination . . . . . . . . . . Response of Q, phomoides 080A to increasing con- centrations of sodium pyridinethione in conjunc- tion with different sugars . . . . . . . . . . Ultraviolet absorption spectra involving sodium pyridinethione and glucose . . . . . . . . . . Ultraviolet absorption spectra involving sodium pyridinethione and glucose . . . . . . . . . . Response of E. phomoides C8OA to increasing concentrations of sodium pyridinethione in conjunction with different nitrogen sources . . Growth curves of C. phomoides 080A subjected to different levels- of sodium pyridinethione at the time of inoculation . . . . . . . . . . . . Growth curves of C. phomoides C8OA in response to 0.12 pg/ml. sodium pyridinethione added at different times . . . . . . . . . . . . . . . vi 35 36 no A9 60 61 69 72 7A LIST OF TEXT FIGURES FIGURE ' Page 1. Protective effects of two sugars on the stability of rimocidin tested against'g. phomoides C8OA 37 Fungicidal properties of sodium pyridinethione on the spores of Q. phomoides C8OA . . . . . . . H5 Response of Q. phomoides C8OA to increasing concentrations of sodium pyridinethione in conjunction with different nitrogen sources . 68 vii CHAPTER I INTRODUCTION During the past decade, extensive efforts have been directed toward the development of antibacterial antibiotics for the control of human and animal diseases. Recently, this effort has extended into the field of phytOpathology. with the exception of actidione (cycloheximide) which is commercially available for agricultural use against phyto- pathogenic fungi, all other antifungal antibiotics are still at best in the experimental stage. Nystatin (9, 11) shows excellent promise ig‘zigg for the control of some of the systemic human mycoses such as histoplasmosis and meniliasis. Recently, a survey of the literature has been published con- cerning antibiotics (35) that may be of value in controlling plant diseases. Although chemosynthetic agents are the mainstay of the protective and erradicant spray programs, new synthetic materials are also constantly sought after from.the viewpoints of cheapness, ease of application, low mammalian toxicity and stability (when desired). In plant pathology, the biological evaluation of an antimicrobial compound is based on‘ig‘gitgg as well as $9 ZAZQ studies. Fortunately, such a joint programvwas made available by the Horace H. Rackham Research Endowment grant in the Department of Botany and Plant Pathology. This thesis is concerned exclusively with the in vitro phase of the physiological studies and trial application of antifungal materials for the control of plant diseases. At this point it seems appropriate to clarify some of the terms that are used in this study specifically with re- gard to the effects exhibited by materials antagonistic to fungi. McCallan and Hellman (38) stated that in the field of phytopathology ”A fungicide may be defined as an agent that kills or inhibits the development of fungus spores or mycelium." They further stated that in the restricted sense, however, fungicidal refers to the property of killing fungi and fungistatic, the property of inhibiting. In this study, the following terms were used in the restricted sense: Egggistatic: the growth-preventing or restricting effect on fungus spores or mycelium.by an agent, so long as it is in contact with the spores or the mycelium. Fungicidal action: the killing effect of an agent on fungus spores or mycelium.after contact for a lbmited time, making certain that the agent is comp pletely removed from.the organism following the contact time. Inhibition: see fungistatic. Antifuggal: the antagonistic effect of an agent on fungus spores or mycelium.but not specifying whether the effect is due to fungistatic or fungicidal action or a combination of both. The objectives of this study';g vitro were to evaluate: a. The minimal inhibition concentrations (MIC) of cer- tain test antifungal compounds against selected test phytopathogenic fungi in liquid medium. b. The stability of certain test antifungal compounds. c. The effect of four different carbohydrates and two nitrogen sources on the activities of rimocidin and pyridinethione when using Colletotrichum phomoides C8OA as the test organism. Due to the favorable screening results obtained with sodium pyridinethione and the fact that virtually no work had pre- viously been done with this compound, it was deemed advisable to investigate pyridinethione further, again using Colleto- trichum.phomoides 080A as the test organism, with respect to: d. Mode of action. e. Fungistatic-fungicidal properties. f. The effect upon the growth curve. 3. The inactivation of sodium pyridinethione by D-glucose and supported by spectrophotometric studies. ' Literature Review The basic moiety of pyridinethione is the heterocyclic nitrogen portion called pyridine. The first reported anti- microbial action by an analogue of pyridine was made by Browning 9_t_ _a_]_._. (10) in 1923. Bins and Bath (6) in 1928 reported pyridine arsonic acid to be effective against micrococci and streptococci in mice. ‘Uooley and his co- workers (59) in 1938 found pyridine-3-sulfonic acid caused nicotinic acid deficiencies in dogs. McIlwain (39) shortly thereafter reported that pyridine-3-sulfonic acid inhibited the growth of certain bacteria. The antibacterial and anti- fungal action of pyridine analogues of thiamine have received some attention (A7, 60, 62). Shaw gt 5;. in 1950 (52) syn- thesized a cyclic thiohydroxamic acid from 2-bromopyridine, N-oxide and reported a high in gitgg activity against a variety of microorganisms. Pansy gt 2;. (AZ) found this compound, l-hydroxy-2(1H) pyridinethione, to be extremely effective in controlling fungi in gitgg at concentrations of 0.0h - 1.0 Pg/ml. Allison and Barnes in 1956 (h) reported 2-pyridinethione to be moderately effective inflgizg against some phytopathogenic fungi and also gave an lg zitgg EDSO value of 0.03 - O.u ppm.against conidia of Monilinia fructicglg. Sander and Allison in 1956 (50) showed that l-hydroxy-2(1H) pyridinethione is translocated down in cucumber seedlings - but suggested that the material is inactivated in the young shoots and cotyledons. Soo-Hoo and Grunberg (53) conducted studies with various salts of 3-pyridinethiol with good re- sults against dermatophytic as well as phytopathogenic fungi mum. With regard to other antifungal materials used in this study, Hazen and Brown in 1951 (27) reported the isolation of a new antifungal antibiotic which they named fungicidin (now called nystatin). This material is effective i3 yitgg against medical and plant pathogenic fungi at concentrations up to 10 Pg/ml. Nystatin is reported to be quite effective in vivo against such systemic mycoses as histoplasmosis and moniliasis (11, 9). Compound M #575. an antibiotic, (55) has an _i_n_ mg minimal inhibition concentration of 6 - 12 pg/ml. Rimmcidin, first described by Davisson 23 5;. (15) in 1951, is an antibiotic inhibiting many of the human pathogenic fungi‘in‘ziggg at concentrations of one to five Pg/ml. Grosso (2k) in 195k tried rimocidin as a greenhouse spray for the control of tobacco blue mold. Thiolutin has been tried for the control of mycotic infections of man as well as in vivo screening against phytopathogenic bacteria and fungi (13, BO, 22); so far, the results have not been too promising. Candicidin, an antibiotic discovered by Lechevalier and his co-workers (3h) is reported to have an activity spectrum against yeast and yeast-like organisms but is only poorly active against the filamentous fungi and exhibits no activity against bacteria. The authors reported this compound to be very soluble in water but it is thermo- labile and showed a marked loss of potency after being kept in an aqueous solution for 2h hours at room temperature. Alcorn and Ark (2) reported good results using candicidin as a protectant spray as well as having some value as a protectant dip for peaches (3). Gottlieb gt g1. (23) in 1951 described endomycin as a broad spectrum.antifungal antibiotic which was also effective against some gram posi- tive and gram negative bacteria in vitro. Endomycin was reported to inhibit Colletotrichum phomoides at no Pg/ml. in broth and in addition, this antibiotic was also thermostable. Klomparens and Vaughn (33) found that endomycin showed in- creased antifungal activity when Fusarium.lycogersici was grown in liquid medium containing maltose as the carbon source. Many mdcrobial inhibiting agents act by virtue of being antimetabolites and several excellent reviews have been pub- lished on this matter (58, 61). Sulfanilamdde acts as a comp petitor for para-aminobenzoic acid which was found to be an essential metabolite for many microorganismsf126, 36). Zent- meyer (6h) in l9hh suggested that 8-hydroxyquinoline, an excellent fungistatic agent, functions by chelating zinc and thus depriving such fungi as Fusarium oxysporum var. lycoper- gig; of this essential metal. Reversal of inhibition was accomplished by adding excess zinc to the medium. Pyridine is the fundamental moiety of nicotinic acid and nicotinic acid amide which in turn are found in coenzyme I or II (diphosphopyridine nucleotide or triphosphopyridine nucleo- tide). Coenzymes I and II are required by many of the dehydrogenases. Various substrate analogues of nicotinic acid have been thus found to act as inhibitors, some examples being pyridine-3-su1fonic acid (20, 59), halogen substituted nicotinic acids (30, 31) and 2-sulfanilamido-S-nitropyridine (1h). Isonicotinic acid hydrazide (isoniazid) is bacteriostatic for the tubercle bacillus in gigg. The mode of action appears to be that of a competitive inhibitor for nicotinic acid (56) as well as an antimetabolite against pyridoxal synthesis (7, 63). From the preceding paragraph, it can be shown that by modifying the substrate in some way (be it in zitgg or in 1119), the activity of an antimicrobial agent can sometimes be altered. Thus, Horimoto and his co-workers (29) in 195k observed that the various B vitamins, nicotinic acid, DPN and some of the sugars are effective in reducing the bac- teriostatic action of isonicotinic acid hydrazide on tubercle bacilli. Di Raimondo (16) in 1953 also reported that the vitamin B complex group reduced the activities of the anti- biotics terramycin and tyrothricin against Micrococcus pyogenes var. aureus. According to Weinberg (57), the in- hibition of Mycobacterium avium by the antibiotics aureomycin and terramycin is significantly increased by the addition of phosphate to the medium. CHAPTER II MATERIALS AND METHODS A. Source and Maintenance of Materials for Study 1. Antifungal Compounds The following is a list of the compounds employed as anti- Ifiungal agents together with the methods for preparing the stock solutions: Sodium pyridinethione; (Squibb): very soluble in water. Diaphine Hg; (Pfizer): very soluble in water. Nystatin (Squibb): soluble in acid methanol. M 3575 (Squibb): raise to pH 11 with NaOH, solution then occurs; back titrate immediately with HCl. Rimocidin sulfate (Pfizer); soluble in.water, stock solutions of one mg/ml. will be slightly cloudy. Thiolutin (Pfizer): sparingly water soluble, 50 pg/ml., solubilIty can be increased to 150 Pg/ml. by using 70 percent methanol. Candicidin A (Dept. of Microbiology, Rutgers University): very soluble in water. Endomycin (Upjohn): soluble in water containing a little NaHCO . . 3 1 1-hydroxy-2(1H) pyridinethione (MC 3277), so designated ‘bF’ZE. R. Squibb and Sons. When a further supply of this material became necessary, lot no. Py-35h-75c was provided which Enrned out to be slightly more active than the previous material sed. With the exceptions of diaphine and pyridinethione, the hitherto listed compounds are antibiotic in source. All test materials were maintained in a dry state at 5° C. until removed for use. 2 . Te st Organisms The following fungi were employed as test organisms: Colletotrichum phomoideg (Sacc.) Chesterl, obtained from Indiana tomato fruits in the summer of 1953 and desig- nated as isolates 080A and 101. Helminthosporium sativum.Pam., King and Bakkez, isolated from.bar1ey in the "Thumb” area, Michigan, and designated as isolate 925. . Helminthosporium carbonum Ullstrupz. Ustilago hordei (Pers.) K.and SI, isolate obtained from Madison, Wisconsin and.designated as isolate R6. Puccinia sorghi Schw.2 Colletotrichum lagenarium (Pers.) E. and H.3 §;._;egenarium.was maintained on V-8 agar slants in order to infihice ample sporulation. The uredospores of P, sorghi were Stored at 5° C. All other organisms were routinely kept on Potato dextrose agar slants with the exception of Q. hordei whitch required a three percent glucose supplement. Cultures obtained from the following faculty members of the Dept. of Botany and Plant Pathology at Michigan State University, East Lansing, Michigan: 1Dr. E. S. Beneke 2Dr. R. L. Kiesling 3Dr. D. J. de Zeeuw 10 In order to minimize the possibility of biochemical variation, sub-culturing was kept to a minimum by allowing no more than five successive transfers from.the originally supplied cultures. B. Techniques Employed 1. Medium Used For the purposes of assay testing and physiological studies, Lilly and Barnett's (36) synthetic glucose aspara- gine medium was used. This medium, compounded on the liter basis, contained D-glucose, 10 g.; L-asparagine, 2 g.; MgSOu.7H20, 0.5 g.; KHZPOH’ 1.0 g.; Fe, 0.2 mg.; Zn, 0.2 mg.; Mn, 0.1 mg.; thiamine H011, 100 pg. and biotinl, 5 Pg. The pH was adjusted to 6.0 with a Beckman glass electrode pH meter. The medium was dispensed in various ways, as described later, and autoclaved at 2h0° F. for 15 minutes. The medium was removed from.the autoclave as soon as possible to offset caramelization. All materials used in the formulation of the medium were of reagent grade purity. 2. Glassware Cleaning Procedure All glassware employed in this study was previously cleaned in a synthetic detergent solution followed by a tap 1Kindly furnished by Hoffmann-La Roche Inc., Nutley. New Jersey. 11 water wash and a distilled water rinse. Pipettes were cleaned by soaking in chromic acid followed by seven rinses for each in tap and distilled water. 3. Screening of Antifungal Materials In all cases, solutions of the test antifungal compounds were freshly prepared prior to use. The compounds were sterilized in the following manner: one series via Seitz filtration and another series via autoclaving directly with the medium, thus affording information regarding thermo- stability. Lilly and Barnett's glucose asparagine liquid medium was used exclusively, intentionally omitting solidi- fying agents such as agar which may tie up some of the test compound by adsorption. For example, when nystatin was screened, the minimal inhibition concentration against 9. phomoides C8OA on agar was 100 Pg/ml. whereas in liquid, it was 10-50 Pg/ml. Spore inocula were used in the assays by preparing aqueous spore suspensions from two-week old culture slants. The spore suspensions were adjusted, using a Levy Hema- cytometer, so that a concentration of AGO-500 spores per m1. of medium resulted when one ml. was used as inoculum for 20 m1. of medium. Aseptic techniques were maintained throughout the procedure. One ml. of inoculum was pipetted into a sterile 12 Petri plate followed by the medium and then by the Seitz sterilized test compound (in the heat sterilized series, the test compound had already been added to the medium prior to autoclaving). The test compounds were serially diluted so as to give the desired concentration series for the assay. The plates were gently swirled after the addi- tion of each constituent to insure adequate mixing. All assay plates and controls were run in duplicate, incubated at 23. 25° C. for five days and the results recorded as plus or minus growth. A. "Shelf Life” Stability Determinations of Antifungal Materials In those instances where the test compounds showed ,good promise (minimal inhibition concentrations of 10 pg/ml. or less), further studies were conducted to determine the length of time that potency is retained in aqueous solutions at 10 Pg/ml. of materials at 5° C. and at room temperature. This is termed the ”shelf life” of the compound. In addi- tion, sodium pyridinethione was maintained at one pg/ml. under the same temperatures. Activity was determined by the Petri plate assay method but in one instance where sodium pyridinethione was kept at one Pg/ml., the evalua- tion was determined by weighing the recovered dry mycelium. This procedure is discussed in the section dealing with 13 techniques employed in the physiological studies on Q. phomoideg C8OA (page 19 ). 5. Spore Germination Tests Spore germination tests were set up for a 2h-hour period employing the glass slide technique with the test tube dilu- tion method as described by the American Phytopathological Society, Committee on Standardization of Fungicidal Tests (5). Distilled water was used as the suspending medium for the spores. 6. Physiological Studies on Colletotrichum phomoides C8OA a. Varying the medium with rggpect to the carbon source. Four different carbohydrates were investigated separately in conjunction with varying concentrations of the test antifungal compound. The basal medium employed was Lilly and Barnett's synthetic glucose asparagine, complete with the exception of the carbohydrate. The standard medium contained glucose equivalent to four g. carbon per liter; hence the following sugars were used in the quantities specified so as to give four g. carbon per liter: D-glucose, 10 g.; sucrose, 9.5 g;; D-xylose, 10 g. and alpha-lactose.H20, 10 g. The medium was compounded on the liter basis but made up to 960 m1. volume. This was done so that the subsequent addition of one ml. inoculum and one ml. of the antifungal 1h material to R8 m1. of medium would yield 50 ml. at the proper concentration of nutrients. Forty eight ml. of medium was dispensed into 250 ml. Erlenmeyer flasks which were then plugged with cotton (non-absorbent) and auto- claved at 2h0° F. for 15 minutes. The contents were removed from the autoclave as soon as possible to help offset cara- melization. One ml. of spore inoculum containing 150,000 spores (equivalent to 3,000 spores per ml. of medium) was aseptic- ally added to each flask followed by one ml. of a freshly prepared, unless otherwise specified, Seitz filtered solution of the antifungal material at that concentration designed to yield the desired final concentration in Pg/ml. The flasks were gently swirled after the addition of each con- stituent. All variations along with the controls were run in triplicate. b. Varying the medium.with respect to the nitrogen source. Ammonium.nitrogen as well as nitrate nitrogen sources were investigated separately in conjunction with varying concen- trations of the antifungal material. The standard Lilly and Barnett medium used was complete with the exception of the nitrogen source (asparagine). The standard medium con- tains asparagine equivalent to O.h2h g. N/liter; hence the following nitrogen compounds were used in the quantities specified so as to give the same amount of nitrogen (O.h2h 15 g./liter); sodium nitrate, 2.57 g.; and ammonium chloride, 1.62 g. The methods employed were the same as those pre- viously described for the carbohydrate studies. c. Preparation of inoculum. In the beginning, spore inocula for the physiological studies were routinely ob- tained from sporulating PDA slant cultures. Because of some unexplained factor 0E.combination of factors, cultures of Q. phomoides C8OA, incubated two weeks or longer at 25° C., produced decreasingly smaller crops of conidia or often none at all, although prolific quantities of acervulus-like bodies were apparent. This situation was detrimental in work re- quiring specific spore concentrations in the inocula.' Un- successful attempts were made to induce sporulation on agar slants by the use of such media as, Sabouraud, corn meal and yeast extract incubated over a temperature range of 16 to 30° C. When the physiological studies were first undertaken, it was noted that cultures incubated on the Kershaw rotary shaker produced a turbidity in the liquid medium. This was found to be due to prolific sporulation. These conidia were morphologically similar to those produced on semi-solid media. Acervuli do not form in the shake cultures, except on the aerial mycelial ring above the splash line. The typical.appearance of the organism in shake culture is pellet-like. Plate I shows that the conidia are borne directly on the hyphae or on short projecting conidiophores. 16 PLATE I Colletotrichum.phomoides 080A conidial formation.. Liquid shake culture, incubated four days at £5. 25° C. Fig. 1. A portion of a pellet showing some conidia. 9a.. 1 5250 Figs. 2 and 3. Enlargement showing conidia borne on short conidiophores or directly on the hyphae. Q3. x 1,000. 17 Some species of the genus Fusarium also produce abundant quantities of conidia in liquid shake cultures: Utilizing this information, it was possible to set up a simple pro- cedure to harvest the spores of Q. phomoidgg C8OA, wash and use them as inoculum. The following procedure was devised: (l) Fifty ml. of the standard glucose asparagine liquid medium was inoculated with a one ml. aqueous suspension of mycelial fragments obtained from a PDA slant culture. The flasks were incubated on a Kershaw rotary shaker at 1&0 rpm for four days at 23. 25° C. at the end of which time, large quantities of conidia were produced imparting a milky appearance to the medium. (2) Aspetically, a portion of the culture super-' natant, containing the suspended conidia, was decanted into a cotton plugged, sterilized 30 ml. pour-out-lip, roundebottomed centrifuge tube. (3) The material was centrifuged in an "International Centrifuge, Size 1, Model SBV" for one minute at an average Relative Centrifugal Force of 95 gravities. This forced down any pellets still present while the spores remained in suspension. (h) Aseptically, the spore suspension was decanted into another sterilized, cork-plugged, 50 ml. tapered- end centrifuge tube and recentrifuged for five minutes at 300 gravities. This served to centrifuge the spores down. The clear supernatant was rejected. 18 (5) The spore mass was washed twice with 10 ml. each of sterile distilled water, and each time step (A) was repeated. The spores were finally resuspended in 10 ml. sterile distilled water. (6) The spore count was determined with a Levy Hemacytometer and subsequently diluted to the desired concentration. Cultures inoculated with spores obtained in the above procedure gave results comparable with those obtained with inoculum secured from agar slants. Refrigerating the spore suspension at 5° C. for two days prior to use yielded like- wise comparable results. Using spore inoculum prepared in this manner has the following advantages over inoculum ob- tained from agar slant cultures: (1) Approximately 150-200 million spores can be recovered compared to 8-10 million spores from an ex- ceptionally heavy sporulating slant culture. (2) The obtained spore inoculum is free from hyphal and acervulal debris which can act as focal growth points. (3) The possibility of residual nutrient carry- over is eliminated. d. Incubation conditions. In all instances involving the carbon and nitrogen source variations, the flasks were prepared in two series, one to be incubated in a l9 stationary position and the other to be agitated on a Kershaw rotary shaker at lhO rpm. Since the results of a stationary culture series did not differ from those of the shake cul- tures, the former were omitted. Because of mechanical dif- ficulties involving the rotary shakers, some experiments have been run only in the stationary culture series. Occa- sional difficulties were encountered in trying to control the incubation temperature. This influenced the rate of growth of the organism under study and consequently made comparisons between experiments difficult. After the in- stallation of constant temperature devices by the Department of Botany and Plant Pathology, the temperature was held fairly uniformly at 23. 25° C. with the exception of a few weeks during the summer of 1955. The incubation period for shake cultures was six days and for stationary cultures, nine days. e. Harvesting and data presentation. At harvest time, the mycelium from each flask was filtered via suction on a Buchner funnel through a nylon filter. The mycelium was then washed three times with distilled water, placed in a num- bered, tared aluminum.weighing cup and dried overnight at 60° c. The cups with the dried mycelia were then kept in a dessicator until weighed. The pH of the filtrate was determined at harvest time. Each value reported, unless 20 otherwise indicated, is the average of three flasks and expressed as mg. dry weight. f. Size of inoculum. As previously stated, the standard spore concentration employed for the physiological studies is 3,000 spores per ml. of medium. In one experi- ment, the spore load was unintentionally adjusted to 130 spores/ml. medium instead of the standard concentration. At this low spore load and at varying concentrations of sodium pyridinethione, no agreement between replicate flasks of shake cultures was possible with the exception of the control flasks. All replicate treatments did agree however in the stationary incubated cultures. Table I shows the individual readings within given treatments for shake and stationary cultures at low spore loads as compared to the individual readings obtained for shake cultures under the same conditions but with the normal spore load inoculation of 3,000 spores per m1. of medium. At the end of six days, the shake culture flasks containing the low inoculum.a11 exhibited irregular sized pellets and often just one large mycelial clump per flask, with the exception of the control flasks which had good pellet growth of uniform small size. The series inoculated with the normal spore load, for the most part, appeared homogeneous, macroscopically, and ir- regular mycelial clumps were observed only when the organism 21 TABLE I EFFECT OF SPORE CONCENTRATION ON AGREEMENT BETWEEN REPLICATE FLASKS TREATED WITH SODIUM PYRIDINETHIONEs Na Pyridinethione Concentration of Inoculum / 13o Spores/ml.Medium 3,000 Spores/m1.Medium ml. Pg Shake Cult. Stat. Cult. Shake Cult. ms- me. me- o.oo rep 1 205 190 1ho rep 2 192 196 15 rep 3 19k 183 1h 0.0h rep 1 188 190 220 rep 2 118 18 215 rep 3 207 16 202 0.08 rep 1 88 lh5 195 rep 2 1H5 1&5 192 rep 3 202 1H8 --- 0.12 rep 1 109 79 6 rep 2 139 79 9 rep 3 188 71 35 0.32 rep 1 no growth A no growth rep 2 w n 2 n a rep 3 N H 2 II n *0. phomoides C80A cultured in glucose asparagine medium. Incubated for six days at ca. 25° C. and expressed as mg. dry mycelium per fifty ml. medium. 22 was greatly retarded at the higher concentrations of sodium pyridinethione. This appearance of irregular mycelial clumps at the higher concentrations agree with the results of Oster and Golden (hl) who stated that the greatest vari- ations were exhibited at either very low or very high con- centrations of test antifungal substances. Duckworth and Harris (18) found that spores of Penicillium chgysogenum at one million spores per ml. of medium gave the best non- anastamosed hyphal growth in submerged cultures. In addition, Foster (19) stated that the uneven large clump- 1ike colonies in submerged cultures are mostly due to an inadequate amount of viable inoculum. In the previously described observations, an inoculum size of 130 spores per ml. of medium.epparently was adequate for the control flasks in submerged culture since a good uniform growth was ob- tained. Under the influence of even minute concentrations of sodium.pyridinethione (0.02 ug/ml.), it was to be ex- pected that some of the spores would be inhibited. The remaining number of Spores, which were able to germinate, probably was at a sub-optimal level and thus caused the irregular mass like growths. At the higher spore loads of 3,000 per ml., the number of spores unaffected by the in- hibitor was perhaps still large enough to induce the uniform pellet like growth and hence, good reproducibility between replicate flasks. 23 g. Concentration range of antifungal materials used in study. A preliminary investigation showed the working range of the antifungal materials employed in the physio- logical studies to be: Rimocidin: one to ten Pg/ml. Sodium pyridinethione: 0.02 to 0. 32 lug/nu. 7. Additional Techniques Related to the Sodium Pyridinethione Study Additional methods and techniques pertaining to the physiological study with sodium pyridinethione will be dis- cussed in Chapter III, entitled RESULTS AND OBSERVATIONS. 2h CHAPTER III RESULTS AND OBSERVATIONS A. Screening of Antifungal Materials The results presented in Table II indicated that sodium pyridinethione, rimocidin and endomycin were most effective against the organisms tested. It was due to these results that sodium pyridinethione and rimocidin were selected for some of the physiological studies. Endomycin, although showing good potential, was not included in the physiological studies because it was screened some time after the project was in progress. Seitz filtered endomycin preparations showed no activity against the test organisms at concentrations up to 200 Pg/ml. It was noted that the normal yellow color of the endomycin stock solution was colorless after passing through the Seitz filter. The antibiotic had apparently been adsorbed onto the asbestos filter pad of the Seitz apparatus which in turn was colored yellow. A duplicate experiment yielded the same results. Because endomycin has been reported to be thermo- stable (23), it was sterilized by autoclaving directly with the medium. Plates II, III and IV show the difference between the two treatments of endomycin. 25 TABLE II ACTIVITIES OF ANTIFUNGAL MATERIALS MICl IN PG/ML. GLUCOSE ASPARAGINE LIQUID MEDIUM Material 9. phomoides Q. phomoides H. sativum C 0A 101 925 Na pyridinethione <'1 ('1 < 0.1 Diaphine HCl - 100-200 50 Nystatin 1>10 50 >50 M 11575 > 200 >50 >200 Rimocidin SOL)» ‘ 10 ‘ 10 < 10 Thiolutin > 15 > 15 > 15 Candicidin A - 200 200 Endomycin3 4 10 < 10 < 10 1Minima1 inhibition concentration. 2Incubated for four days at 23. 25° C. 3Sterilized via autoclaving directly with the medium; all other materials Seitz-filter sterilized. 26 Concentration. series of endomycin in rig/ml. against Colletotrichum.phomoideg C8OA in liquid :medium. Incubated for 35 days at 23. 25° C. Upper: Control plate and plates containing autoclaved endomycin. Lower: Seitz-filtered endomycin. .- PLATE III 27 CONTROL Concentration series of endomycin in.Pg/ml. against Helminthosporium sativum.925 in liquid medium. Incubated for 35 days at 25. 25° 0. Upper: Control plate and plates containing autoclaved endomycin. Lower: Seitz filtered endomycin. PLATE IV 28 7-__.. CONTROL . 99—} ._ h___ Concentration series of endomycin in pg/ml. against Egtilagp hordei R6 in liquid medium. Incubated for 35 days at 25. 25° C. Left to right: Control, autoclaved and Seitz-filtered endomycin. 29 B. Stability Tests Since the antifungal compounds sodium pyridinethione, rimocidin and endomycin exhibited relatively high 13 thgg activities, they were further evaluated with regard to various stability aspects. Although diaphine did not show very good screening results, it was included in these tests by way of comparison. 1. Thermostability Losses in potency were observed when the compounds sodium pyridinethione, rimocidin, diaphine and endomycin were subjected to autoclaving with the medium for 15 minutes at 2h0° F. Diaphine exhibited the greatest loss due to heat inactivation whereas the other compounds re- mained fairly stable. In the case of sodium pyridinethione, the medium darkened somewhat upon autoclaving. The results are shown in Table III. 2. "Shelf Life" a. Sodium pyridinethione and rimocidin via assaygplate test. Using H. sativum 925 and Q. phomoides C80A as the test organisms, these compounds when stored at 10 Pg/ml. in glucose asparagine medium retained their activity for: TABLE III THERMOSTABILITY OF ANTIFUNGAL MATERIALS MIC IN GLUCOSE ASPARAGINE LIQUID MEDIUM* PG/ML. 30 Method of Sterilization Autoclaving With °°mp°un° Seitz Filtration the Medium ‘9. phomoides 73- sativum. Q. phomoides ‘fl. sativum C OA 92 C 0A 925 Na Pyridinethione < 1 <~O.1 10 5-10 Rimocidin < 10 < 10 < 10 < 10 Diaphine 100-200 50 200 200 Endomycin '< 10** <'10** 10 10 *Cultures incubated for four days at 25. 25° C. ‘**Non-sterilized (unheatedL. TABLE IV ANTIFUNGAL ACTIVITY OF STORED SODIUM PYRIDINETHIONE TESTED AT 0. 12 pG/ML. GLUCOSE ASPARAGINE LIQUID MEDIUM AGAINST C. PHOMOIDES 080A AND REPORTED AS MG. DRY MICELIUM PRODUCED IN SIX DAYS AT‘QA. 25° C. Tested Na Pyridinethione Immediately After After 25 Days 5 1/2 Months Freshly prepared 50 -- -- Stored at 5° C. -- NS 133 Stored at room.temperature -- no 203 Q, phomoides C80A in the control yields 200 mg. dry mycelium. 31 sodium pyridinethione: six months at 5° C. six months at room temperature. rimocidin: 2 l/2 months at 5° C. l l/2 months at room temperature. b. A more critical determination of the "shelf life" of sodium pyridinethione. Since most of the physiological work was to be concerned with this compound, it was deemed advisable to determine whether this compound underwent de- gradation during an incubation period at those concentrations 0.02 to 0.32 pg/ml., used in the physiological study. Aque- ous, Seitz filtered solutions of sodium pyridinethione at a concentration of 10 pg/ml. were maintained at both 5° C. and at room temperature for varying periods of time. At appropriate intervals, portions were removed, diluted to a final concentration of 0.1 Pg/ml. medium and shake flasks were inoculated with spore suspensions of Q. phomoides CBOA, incubated on Kershaw rotary shakers and the mycelium pro- cessed according to the method described in Chapter II. Thus, at the end of a 25-day storage period, the material kept at either room temperature or at 5° 0. exhibited no loss of potency. 0n the other hand, at the end of 5 1/2 months, there was virtuallyem.100 percent loss in potency at both room temperature and 5° C. These results are pre- sented in Table IV. Since the incubation periods do not exceed nine days in the physiological studies, no problem was presented with respect to the self-degradation of sodium 32 pyridinethione on the basis of this information. On the con- trary, sodium pyridinethione can be considered a fairly stable antifungal material. 0. Endomycin. Six concentrations ranging from 0.1 to 200 Pg/ml. were tested against three organisms. Non- sterilized (unheated) endomycin, together with a combination of 20 unitspenicillin and ho units streptomycin per ml. of medium to keep down bacterial contamination, retained full potency even after an incubation period of 35 days. The autoclaved endomycin lost a little of its potency during heating since some growth was apparent at the end of 35 days (Tables V and VI). The "shelf life" of endomycin is good, as can be seen in Plates II, III and IV. 3. The Effects of Different Sterilization Treatments on the Potencies of Sodium Pyridinethione and Rimocidin Because of bacterial contamination which would other- wise be present, the antifungal materials had to be sterilized prior to being brought into contact with the test organism(s). The selected method used during the physiological studies was Seitz filtration since it was shown that autoclaving directly with the medium altered the activity level of the anti— fungal materials. In order to determine whether any material was lost in the process of Seitz filtration, by adsorption, 33 TABLE V ANTIFUNGAL ACTIVITY OF AUTOCLAVED ENDOMYCIN AT VARYING CONCENTRATIONS IN GLUCOSE ASPARAGINE LIQUID MEDIUM CULTURES INCUBATED AT 9;. 25° c. game. of Q. ghgglgigfl fl. sativum _U_. hordei domycin C 0A _§§5_—— -R€——— in pg/ml. h da. 35 da. , h da. 35 da. h da. 35 da. 0.0 ++ +++ ++ +++ + +++ 0.1 ++ +++ ++ +++ + +++ 1.0 ++ ++ ++ +++ + +++ 10.0 - + - +++ _ _ TABLE VI ANTIFUNGAL ACTIVITY OF NON-STERILIZED (UNIIEATED) ENDOMYCIN AT VARYING CONCENTRATIONS IN GLUCOSE ASPARAGINE LIQUID MEDIUM CULTURES INCUBATED AT 93. 25° c. Cone. of _C_. ghomoides _H_. sativum Endomycin 680A 92 in Pg/ml. u da. 35 da. h da. 35 da. 0.0 ++ . +++ ++ +++ 0.1 ++ +++ ++ +++ 1.0 ++ ++ ++ ++ 10.0 "' " " - 31; it was deemed advisable to compare the activities of materials sterilized by Seitz filtration to those sterilized via sintered glass filtration. An ultra-fine (bacterial) sintered glass funnel was employed. As indicated in Graph I, some activity is lost when sodium.pyridinethione is Seitz-filtered and that there is a complete loss upon auto- claving (compare this to the results obtained in the section on thermostability). In the case of rimocidin however, a negligible amount of activity is lost upon Seitz filtration and it is only moderately inactivated by autoclaving. C. Physiological Studies with.Rimocidin l. The Effects of Different Sugars on Antifungal Activity The results whereby varying concentrations of the anti- biotic rimocidin were used in conjunction with four'different sugars are presented in Graph II. The antibiotic activity rate was markedly increased when D-glucose or sucrose was used in comparison to D-xylose or alpha-lactose. The differ- ences in incubation times were due to temperature difficul- ties. The glucose and sucrose phases were conducted at the same time under temperatures of 28°-30° C. and therefore were harvested at the end of four days which was the original schedule. In the case of the xylose and lactose run, also conducted simultaneously, the temperature in the incubation 3S GRAPH I THE AMOUNT OF GROWTH OF _C_. PHOMOIDES 080A IN THE PRESENCE OF NA PYRIDINETHIONE OR RIMOCIDIN WHICH HAVE BEEN SUBJECTED To DIFFERENT STERILIZATION TREATMENTS* 160 O \ Sterilized via: L \ n— -D sintered glass filtr. é 120‘ \ O——-o Seitz filtration v3! \ .——O autoclaving O E 1‘ \\ fl- Shake cultures grown h ’ in glucose asparagine ,3 80. \K medium for 6 days at \ £_a_. 25° C. .5 \ I: \ \ 1+0-i \ ‘D O ~”-—r‘=:~u “ i“‘”“‘ ’Iu“ .2 0.14. 0.8 0.12 Pg/ml Na pyridinethione MOT M g . dry mycelium T T 3 * fig/ml Rimoci in Mg . dry mycelium 36 GRAPH II RESPONSE 0F 9. PHOMOIDES 080A TO INCREASING CONCENTRATIONS 0F RIMOCIDIN IN CONJUNCTION WITH DIFFERENT SUGARS SHAKE CULTURES1 2001 l 1801' . A ‘ 4. -------- D-glucose La. days 160 1&0'1 -------- sucrose h days 120' 100‘ 80-1 60-1 4 -------- D-xylose 6 days to J 20" L”- alpha-lactose 6 days a “1 I A'l’ w‘l' ‘1 7 ' 10 \0-4 I 0 1 2 3 h ‘5 6 7 8 Rimocidin Fg/ml 1Inoculum obtained from FDA slant cultureg. D-Slucose and sucrose incubated at 28-30 C. D-xylose and alpha-lactose incubated at temperatures ranging up to 33° C. 37 room rose to 33° C.‘ At this high temperature, the organism was retarded and hence required a longer incubation period. The harvest time of six days was arbitrarily set in the lat- ter case. 2. The Protective Effect of D-Glucose and Sucrose on Rimocidin Shake flask cultures of Q. phomoideg 080A involving a sucrose and a D-glucose series at 6 and 10 Pg/ml. of rimocidin each were maintained over a 38-day period. From.the data presented in Text Figure 1, it is seen that when the organism was incubated in a glucose medium, growth was evident in nine days when the concentration of rimocidin was six fig/ml. and at 15 days when at 10 pg/ml. In the sucrose medium, highly retarded growth was observed at the end of 27 days at six Pg/ml. and total inhibition was still maintained at the 10 Pg/ml. level at the end of 38 days at which time the experi- ment was terminated. PROTECTIVE EFFECTS OF Two SUGARS ON THE STABILITY OF RIMOCIDIN TESTED AGAINST _C_. PHOMOIDES C8OA1 Rimocidin Sugar Incubation in.Day§i_ in PB/ml- 9 15 22 27 38 6 D-glucose growth 10 D-glucose - growth 6 Sucrose - - ' SPOWPh 10 Sucrose - - ' ‘ ' 1Recorded as the incubation time required for the appear- ance of growth. Text Figure 1 38 D. Antimicrobial Properties of Sodium Pyridinethione l. Phenol Coefficient Against Micrococcus pyogenes var. aureus The phenol coefficient for an aqueous solution of sodium pyridinethione was evaluated against Micrococcus pyogenes var. aureus in conformance with the U. S. Food and Drug Administra- tion procedure (M9). In addition, a Shippen's modification was run whereby the test compound was removed from the bac- teria via dilution. This was done to determine whether the inhibition was due to bacteriostatic or bacteriocidal proper- ties of the sodium pyridinethione. The phenol coefficient of sodium.pyridinethione for Micrococcus pyogeneg var. aureus was found to be M3 but the Shippen's modification showed the compound to be bacteriostatic, since growth ensued after re- moval of the test compound. 2. Spore Germination Tests* Spore germination tests were set up for a 2h-hour period employing the glass slide technique (5) in which the test tube dilution method was used with distilled water as the suspending medium. Colletotrichum.phomoid§§ C80A, Colleto- trichum.lagenarium, Helminthosporium.carbonum.and Puccinia Eggghi were used as the test organisms because of the differ- ence of cell size and wall thickness between species. ¥ “The Spore germination studies were conducted ointly with Mr. 01 e B. Kena a of the De t. of Botan and Plan Pathology Michiggfi State UnIversity, Eagt Lansing, Nichigan. ’ 39 Dosage response curves produced by sodium.pyridinethione against three organisms are presented in Graph III. The dosage response curves for Q. phomoides 080A and 9, lggenarium were closely parallel to one another, as might be expected with two similar organisms. Plates V, VI and VII show the effect of sodium pyridine- thione on the spores of Q. phomoidgg C80A,‘§. carbonum and 2. sorghi. Increasing sub-lethal concentrations of the test material induced marked changes in germ.tube formation. This was especially evident in the case of g. carbonum, where 0.05 ug/ml. of the inhibitor caused vesicle formation at the end of the germ.tube. The response of Q, phomoides 080A was quite dramatic, since as little as 0.005 Pg/ml. of sodium sodium.pyridinethione induced retardation in germ tube develop- ment and total inhibition occurred at 0.05 Pg/ml. To sum up, the concentrations of sodium.pyridinethione in.Fg/m1 required to completely inhibit spore germination for 2h hours in distilled water at‘gg. 25° C. were as follows: 9, phomoideg C80A, 0.05; 9, lagenarium, 0.05; g. carbonum1 0.10 and nggigig sor hi, 0.05. 3. Fungicidal Property The preceding spore germination tests yielded an index of inhibitory activity on the part of sodium pyridhnethione but the results are limited in value. Although inhibition Percent inhibition hO GRAPH III DOSAGE RESPONSE CURVES FOR SODIUM PYRIDINETHIONE BASED ON SPORE GERMINATION 1 1 1 l l I [ALL .001 .005 .01 .05 Sodium pyridinethione pg/ml Logarithmic: three by three inch cycles .1 PLATE V Spore germination test. The effect of sodium pyridinethione on spores of Colletotrichum phomoides C80A. Twenty-four hour incubation at 25° C. Fig. 1. Control. 9g. x 675. Fig. 2. 0.005Pg/UILSOdium pyridinethione. 9g. x 675. Fig. 3. 0.01pgflvd. " " " Fig. ii. 0.05M1. " " " PLATE V \s f __. _.. _.r... - Qt '1”! a: WWII- ‘- "I.-'. .h 5.5.. aunt-l n... . '.L.' n. . ..0 II“..1 IL PLATE VI Spore germination test. The effect of sodium pyridinethione on spores of Helminthosporium carbonum. Twenty-four hour incubation at 25° C. Fig. 1. Control. Cg. x 525. Fig. 2. 0.05pgmfl.sodium pyridinethione. 19g. x 1,000. Fig. 3. 0.1Pgfinl. " " " Fig. h. 1.0}nyh1. ” " Cg. x 525. 14.2 PLATE VI - .lv .Iv C ’ 25. PLATE VII Spore germination test. The effect of sodium pyridinethione on uredospores of Puccinia sorghi. Twenty- four hour incubation at 25° C. Fig. 1. Control. 92, x 150. Fig. 2. 0.01}mManodium pyridinethione. 93. x 150. Fig. 3. Control. 'Qg. x 6h0. Fig. 11,. 0.01 rig/ml. sodium pyridinethione. 93.. x 6’10. ’4 occurred at very low concentrations, it was not possible to deduce whether the action was fUngistatic or whether the spores were actually killed. Therefore, in order to test the fungicidal capacity of sodium pyridinethione, an experiment was designed whereby the spores of Q, phomoides C80A were allowed to remain in contact with varying amounts of the test compound for two periods of time after which, the via- bility was determined. The following procedure was employed: a. Seitz filtered aqueous dilution series of sodium pyridinethione were prepared ranging in concentra- tion from.0.1-1,000 Pg/ml. b. Washed spores of Q. phomoides C80A were standardized so that there would be 300,000 spores per m1. of test compound (this is 100 times greater than the concentration of spores used as inoculum.in the physiological studies). c. After exposure times of 20 minutes and 2h hours at 26° C., three m1. portions of the sodium.pyridine- thione and spore mixtures were aseptically removed and centrifuged at 3001 gravity in an International Centrifuge, Size 1, Model SBV for ten minutes. The supernatant was discarded and the remaining spores washed three times with five m1. portions of sterile distilled water and centrifuged each time. The final centrifuged spore mass was suspended in three ml. sterile distilled water, mixed and one drop deposited on a Petri plate containing the standard glucose asparagine agar medium; McCallan and Hellman (38) used a similar technique, the major difference being that they employed a 20-second, cen- trifuged wash period. Since the conidia of Q. phomoides are small in mass, longer centrifuge times were used in order to bring down the spores. The results as tabulated in Text Figure 2 indicate that after a 20 minute exposure, fungicidal #5 action occurred at greater than 100 but less than 1,000 Pg/ml. 0n the other hand, after a 2h-hour exposure, the spores of Q. phomoides C80A were virtually all killed at the lowest concentration tested namely 0.1 Fg/ml. and 100 percent kill took place between 1.0 and 10.0 Fg/ml. Plate VIII shows the comparison of growth between the controls and the spores treated at sub-lethal concentrations. These results confirm the observations made in shake cultures from which only a few fragmented spores were recoverable at the 0.32 Pg/ml concentration level after a two-day period. In this experiment, all the spores were killed upon a 20 minute exposure to concentrations of sodium pyridinethione greater than 100 Pg/ml. These spores were still intact however, when examined microscopically. 0n the other hand, at the end of the 2h hour exposure time, spores in contact with as little as 0.1 Pg/ml. of sodium pyridinethione were seen microscopically to be in a state of disintegration. At concentrations above 10 Pg/ml., no spores were visible al- though much debris was seen microscopically. FUNGICIDAL PROPERTIES OF SODIUM PYRIDINE HIONE ON THE SPORES OF 9. PHOMOIDES CBOA Ifii‘gfitggnigrggved Sodium Pyridine thi one gig/ml Time For 0 0.1 1.0 10 100 1,000 20 minutes +++ +++ +++ +++ +++ - 2h hours +++ + + - - - 1Inhibitor removed by washing the spores three times with distilled water. One drop of washed spore sus ension was lgced on glucose asparagine agar and incubated for f ve days at C. Growth recorded as +. Text Figure 2 PLATE VIII ' “6 Fungicidal test for sodium pyridinethione. The inhibitor was removed after finite contact times and the treated spores of Q. phomoides 080A plated out on glucose asparagine agar for five days at 26° 0. Top row: 20 minute exposure left to right: control, 100 lug/m1. and 1,000 Pg/ml. Bottom row: 2h hour exposure left to right: control, 1.0 g/ml. and 10 pg/ml. At 1.0 pg/ml., only a few spores germinated, to yield 2 small colonies. #7 E. Physiological Studies with Sodium Pyridinethione l. The Effect of Varying the Carbohydrates Four different carbon sources were investigated separately in conjunction with varying concentrations of the test compound to determdne how nutritive environmental con- ditions might influence antifungal activity towards a phyto- pathogenic fungus such as‘g. phomoidqg 080A. These results are presented in Table VII. In addition, the data have been plotted with the weight of the dry mycelium against the con- centration of sodium pyridinethione. Graph.IV shows that sucrose was the most effective sugar in enhancing the anti-6 fungal preperty of the test compound. At 0.02 pg/ml., growth was inhibited 82 percent and total inhibition took place at slightly over 0.03 Pg/ml. This corresponds quite well with the spore germination tests wherein the spores of Q, phomoides C80A were totally inhibited at 0.05 Pg/ml. of an aqueous solution of sodium pyridinethione. Q, phomoides C8OA does not utilize alpha-lactose to any extent1 and was not markedly affected by increasing concentrations of sodium pyridinethione. The general shapes of the curves for both D-glucose and D-xylose are similar at the lower portion with respect to the slopes. A sharp drop in growth is evident up to 0.12 Pg/ml. and thereafter, a more gradual decrease 1This agrees with the results obtained by Lilly and Barnett TABLE VII h8 RESPONSE OF C. PHOMOIDES C80A T0 INCREASING CONCENTRATIONS OF SODIUM PYRIDINETEIONE‘IN CONJUNCTION WITH DIFFE T SUGARS SHAKE CULTURES INCUBATED FOR SIX DAYS Pyridgfiethione D:glucose Sucrose D-XyloseZ ¢><-Lactose2 in.Pg/m1. mg? palL mg. pH mg. pH mg. pH 0.00 200 7.8 1&5 7.6 220 7.h 39 8.5 0.02 199 7.7 27 6.1 - - i - - 0.0h 212 7.6 +1 6.1 170 7.0 10 7.7 0.08 193 7.u O 6.1 105 6.8 3 7.0 0.12 50 6.1 O 6.1 55 6.5 +1 6.0 0.32 0 6.1 0 6.1 9 5.6 0 6.0 1 D-glucose and sucrose incubated at 22-28° C. D-xylose and alpha-lactose incubated at ca. 25° C. 2Spore inoculum obtained from.PDA slant cultures. 3Weight of dry mycelium. upH of filtrate at the time of mycelial recovery. 1+9 GRAPH IV RESPONSE OF 2. PHOMOIDES C8OA TO INCREASING CONCENTRATIONS OF SODIUM PYRIDINETHIONE IN CONJUNCTION WITH DIFFEREN SUGARS SHAKE CULTURES INCUBATED FOR SIX DAYS 220 200 ISO /D-qlucose IZO IOO 80 Mg. dry mycelium 60 \ O, *— 0.02 0.04 0.08 0J2 0.32 ALaJml. Sodium pyridinethione 2.x alpha - lactose ‘0<’ D-glucose and sucrose incubated at 22-28° C. D-xylose and alpha-lactose incubated at 33. 25° C. spore inoculum obtained from.PDA slant cultures. 50 occurred until at 0.32 hg/m1., the organism was totally inhibited. In the glucose series, the cultures in the control flasks and those containing 0.02 Pg/ml. sodium pyridinethione showed signs of autolysis. ‘This was evi- denced by a darkening of the medium and the mycelial pel- lets. The latter also appeared to be partially fragmented. Autolysis of Q. phomoides C8OA is accompanied by an increase in pH which is evident in Table VII and is more noticeable in Table XIII. No autolysis was observed in the other flasks in the glucose series containing concentrations of sodium pyridinethione greater than 0.02 rig/ml. Thus, the apparent stimulation in the glucose curve in Graph IV is erroneous . 2. The Effect of Sugar Concentration on the Antifungal Property of Sodium.Pyridinethione Medium constituents in some way influence the antifungal activity of sodium pyridinethione. This is based on the ob- servation that in the presence of the Lilly and Barnett basal medium to which sucrose has been added as the carbon source, the test compound was very effective causing complete inhibition at slightly over 0.0h Pg/ml. This is the same MIC obtained in germination tests of spores of Q. phomoides 080A, with the washed spores, sodium pyridine- thione and distilled water as the only materials present. 51 The sugars D-glucose, D-xylose and alpha-lactose, on the other hand apparently had some effect on the activity of the compound since 0.32 pg/ml. was required to induce total inhibition. Further investigations were carried out by adding increasing concentrations of either D-glucose or sucrose to the medium containing a standard concentration of sodium pyridinethione. This was done to see whether an in- creased concentration of these two sugars may have any addi- tional effect on the antifungal activity of sodium.pyridine- thione. The standard Lilly and Barnett glucose asparagine medium. contains 10 g. glucose/liter which is equivalent to h 3. available carbon. The concentrations of D-glucose and of sucrose used in this experiment were equal to 1/2, one, two and three times the amount of available carbon found in the standard medium. Employing routine procedures, the inoculated flasks were incubated in a stationary position fer nine days at 2h° C. and the mycelial dry weights evaluated. Percent in- hibition by sodium.pyridinethione was calculated the following way: 1 - g x 100 = percent inhibition where A is the dry mycelial wt. with Na pyridinethione and B is " " " " without " In order better to evaluate the sugar to inhibitor relatiOnship, molar ratios were employed. The empirical formula of the sodium salt of l-hydroxy-2(1H) pyridinethione 52 is CSHHNNaOS (5h) and hence the molecular weight (MW), excluding degree of purity, is 1h9. Disregarding the salt formation by sodium, the MN is 127, thus involving a factor Of 1.2 in determining molar concentrations of pyridinethione when the sodium salt is used. Since the variation of sugar concentration is based on the standard concentration of four g. of carbon per liter, the amount of sodium pyridinethione to be used was chosen by the inspection of Graph IV. De- siring a concentration that falls in a good working range, the following amounts of sodium.pyridinethionelwere chosen: For glucose: 0.12 pg/ml. me um equivalent to 0.1 ml. medium.(8 x 10’ M) pyridinethione. For sucrose: 0.02 pg/ml. medium, equivalent to 0.017 Pg/ml. medium.(1.3h x 10' M) pyridinethione. The results as tabulated (Tables VIII and IX) show that increasing the concentration of D-glucose while maintaining the same concentration of the test compound served to inac- tivate progressively the antifungal capacity of the sodium pyridinethione. 0n the other hand, increasing the concen- tration of sucrose had no effect on either diminishing or increasing the antifungal properties of sodiwm pyridinethione. Not related to the pyridinethione study but interesting nevertheless, was the observation that in the control flasks, g, phomoides C8OA grew best when the sugar concen- trations were between one and two percent (D-glucose 11.2 -2 x 10 M and sucrose 5.6 x 10"2 M). At sugar concentrations 1Lot no. Py-35h-75c, for explanation see page 8. 53 .AEDHODS ocflmwawdme omoodaw oapompamw m.ppoaamm one mHHHA Ea new: .ocoo camommpm ompv aopaa sou sonamo mo .m Loom op umoam>H5UM3 .oCoanpoCHoHama aopfinfincfi amp moNHHonshm Hm .hao>oooa Hefiaoohs no asap on» no opeapaam no ma N .o aim on when a oopmnsocfl moaspaso hamcoapmpm .dowo moofioeomd am pmafiomo pounced om ouoa K on.: m.m :hm m.m mom m.oH o: o-oa n sa.e a.m nod m.m own N.HH on muoa a ::.H m.: ms o.o Hos so.m do. m-oa a om.m m.m . a v a.a so w.m swim 0? soapanamaH oauom undo: de Edaaoomz man mma Edaaeohz ham 2 mica K 0H N my oaoanpoadoaahm manaz noose omoo: A. a a a m Ho\H osoasoomaoaaaa nea3 .ocoo omoosao HMZOHmBmszHmHm a0 MBH>HBO< QchDmHBz< Ewe zo 20H84mazmozoo HMOUDQOIQ mo HUZMDHEZH Ema HHH> mqmHDGMJ .odoaneoaaoaaaa separates oar nonaaonsan Hm .hao>oooa Headache no easy on» no opeapaflm no mo N .o odm as when m .osoaH moadeSO ELEQOHpmpm .4omo maoaoaomm .m.pmaamme coumoafl on onoa a o.H m.m mm m.m Ham m.m Ha o-oa A :.m o.o an o.o mam o.m mm ouoa a m.: m.m mm m.a omm :m.m on ouoa w o.o H.o mm o.m cos :.H 0% ewflm doananasdH oarnm anaos mma seaflooaz nan mma saaaooaz has 2 OH R paooaom memoaodm\H NI odour» A: -oa a :m.av mdaoaaam and: odoadnodaoaaaa oases .onoo omvosm HmZOHmBMZHQHmMm mo NBH>H80¢ ddeDbHBz¢ HEB zo ZOHBHao<2H EZOHmamzHonwm season x mumda 57 in the medium containing an acetaldehyde-sucrose mixture, the antifungal activity of sodium pyridinethione was dimin- ished, whereas a high level of activity was maintained in the sucrose medium alone. Thus, acetaldehyde is also capable of reacting in some way with pyridinethione to inactivate its antifungal mechanism. Apparently, this is due to the aldehyde group of the compound, since in this respect, it is the same as the reducing sugars. These results also showed that, in the presence of D- glucose, Q. phomoides C8OA was able to overcome the inhibitory effects of sodium pyridinethione. Therefore, the compound is fungistatic and not fungicidal at least under the condi- tions tested. h. The Effect of Time on Mixtures of D-Glucose and Sodium Pyridinethione It is evident from the preceding observations that the antifungal capacity of sodium pyridinethione was reduced when the material was in contact with reducing sugars, specifically D-glucose, or acetaldehyde. It was not known however, whether the fungus itself played any part in this inactiva- tion (or reversal of activity) or if the inactivation of pyridinethione was due solely to exogenous interactions with the medium constituents. In order to shed light on this problem, a series of’experiments was designed to determine 58 the ultraviolet absorption spectra for mixtures of sodium pyridinethione and D-glucose. In another series, various contact times were allowed, between sodium pyridinethione and certain medium constituents. For the assay of anti- fungal potency that remained after the various contact periods, the test organism.g, phomoideg 080A was used. a. Spectrophotometric determdnation. Since most materials eXhibit definite absorption spectra patterns, a resulting chemical reaction between two materials may often be observed spectrOphotometrically by a comparison of the ultraviolet absorption spectra of the reactants and their mixture. Sodium pyridinethione and D-glucose were thus pre- pared in the same fashion employed in the microbiological studies. The ultraviolet absorption spectra were then de- termined at varying time intervals for the components as well as for the mixture in an effort to determine any change(s). The materials and techniques employed.were as follows: (1) Sodium pyridinethione1 at a concentration of h ug/ml. in distilled water (the maximum concentration of 0.32 pg/ml. as used in the physiological studies was too low to be de- termined spectrophotometrically) was Seitz filtered in order to duplicate the routine procedure. Graph V [AI shows a slightly different pattern for Seitz filtered material as compared to sodium pyridinethione which was not Seitz filtered. The latter absorp- tion spectrum curve more closely duplicates the results obtained by the Squibb Labora- tories (5h). The Seitz filtered material 1Lot no. Py-35h-75c, for explanation see page 8 . (2) (3) (h) (5) (6) (7) 59 behaved as if some acid was present (h8) but the pH of the two preparations were about the same since the pH of the distilled water used was 5.7, the pH of sodium pyridinethione (h ug/ml.) was 6.1 and that of the Seitz filtzrzd sodium.pyridinethione (h Pg/ml.) was . D-glucose at a concentration of nine mg/ml. in distilled water and autoclaved at 2h0° F. for 15 minutes. Mixtures of (l) and (2) at double strength of ingredients so as to give the same final concentration as the ingredients alone. The ultraviolet absorption spectra were deter- mined with the aid of a Beckman Quartz Spec- trophotometer, Model DU, employing quartz cuvettes whicn.were calibrated for correction. The solutions of D-glucose, sodium pyridine- thione and the mixtures of the two were stored at 26° C. for varying periods of time before analysis. The ultraviolet absorption spectra curves were plotted from the data obtained as, Optical density (0D) against the wavelength 7\ (in mp). The criterion for determining whether any reaction took place was by comparing the observed ultraviolet absorption spectra of the mixture to the one calculated by adding the spectra of D-glucoSE—and sodium pyridine- thione together. If no reaction occurred, then the curves for the observed and the calculated mixtures should be the same. The results as shown in Graphs V and VI indicated that a reaction occurred in a mixture of sodium pyridinethione and D-glucose. In addition, the reaction was dependent 'on time. In the instance where the mixture was prepared just at the time of taking the readings, no reaction took place since the observed and the calculated mixture curves 60 EEVK in; K can on 00m omN omN OoN CNN O can ONm oom omN omN OVN CNN 0 q q _ q a a a _ a L a _ _ _| d a 4 a a _ 4 a a a L _ g; i P 6.0.056 lmo. \YII lmo dddtqatgda l 9c“. nO-oMy l o 5.. a“ -m- OIO e 1 CO \‘OCOOV .\1 I. *N. /MO°| 00 \ 1 cm . i . i . \ . .. Nm 0 e 1 Nm 0 1 \ I sad. i oe. " ..o..o. 1 cc. «V .. m o ..o m + < do cozoEEam £1 a ..1 6.53:. 6203260 I fi .M . ._E can mc Ev 202352.18 Eaton ollo 1 .01». + 3320-0 .9: m . do 8256 62,330 on. ommm.—.:u Ntwm 1mm. .2: .01 v o ._E\.oE m 63020.0 nlo < ww. 1 cm. mmbom NH mom .0 com ad ammfi mazuHaflmmzH mmDQm NH mom .0 com 84 HQMM cued: nmanamHn .AZ\6L.: man mam 61 2:54 225‘ con CNN 8N O¢N ONN own ONn oon CNN OwN owN ONN — H _ H H H H o H u H q H m H o DIDIUd 6.0-odd. .. odd-0.0.0 mo. .46.. 1cm. .3. .0 1 .0 10¢. v. n+4 3:06.58 633% 38.3.51 . mm. ace. .3 "3....“ . .2... _ . 61 up... 3%0206 .9: m ollo do 9536 32390 cm. Saxon». {3.52.2.8 Eaton 4.5.4 m . .2566 a .33290 allu < 9N5 when .5on mom .o .8 a4 Ems 253505 mmpom ma mom .b .em .3 Ens mafiafimefi 55.33”! in SSH 559tz gem NH 33.3mm ZOHHAHmommd aflqOH> mac 62 were the same (Graph V [B]). On the other hand, when sodium pyridinethione and D-glucose were in contact with one another for a l2-hour period at 26° C., the observed and the cal- culated mixture curves were not the same (Graph VI [A]). This observed mixture curve was almost exactly the same as the curve for sodium pyridinethione, indicating that the mixture curve was predominantly influenced by the pyridine moiety. An observed eight-day mixture curve (Graph VI EH) was not significantly different from the 12-hour mixture 6 except that the extinction coefficient has been somewhat further reduced. In addition, it was observed that some change occurred in the pyridinethione solution at the end of eight days since its ultraviolet absorption spectrum curve became somewhat less pronounced when compared to the 12-hour sample of sodium pyridinethione. b. Bioaggay determination. This method, involving the use of Q. phomoides 080A, was used to assay the anti- fungal potency remaining after various contact periods be- tween sodium pyridinethione and certain medium constituents. The standard Lilly and Barnett synthetic glucose asparagine inedium.was used containing one percent D-glucose (5.6 x 10"2 M and.equivalent to four g. C per liter). By basal medium, it is understood that all the constituents were present with 'the exception of D-glucose. The complete medium indicates ‘the basal plus D-glucose. Four series were set up as follows: 63 (1) Complete medium (control) (2) Complete medium plus sodium pyridinethione (3) Basal medium plus sodium pyridinethione. The D-glucose is added at inoculation time. (u) Basal medium. Glucose and sodium pyridinethione . mixture is added at inoculation time. Each series was prepared in sufficient replication, each replicate stored in 300 m1. soft glass media bottles with screw caps to offset evaporation, so that three replicates of each series would be inoculated with‘g.‘phomoide§ 080A at the end of the following contact periods: 0, one week, two weeks, three weeks and four weeks. Inocula were prepared in the routine manner employed for the other physiological studies and concentrations of 3,000 spores/ml. medium were used. All medium constituents were autoclaved at 2h0° F. for 15 minutes except sodium pyridinethione, which was sterilized via Seitz filtration. 1 was employed at a concentration of Sodium pyridinethione 0.32 ug/ml. medium. All the series, prior to and after inoculation, were maintained at 26° C. The inoculated bottles were incubated for nine days in a stationary posi- tion and the results evaluated on the basis of thedry :mycelial weights obtained in the routine manner. The results as presented in Table XI indicate that when sodium pyridinethione was added to the complete glucose asparagine medium, the antifungal activity diminished with 1Lot no. Py-3Sh-75c, for explanation see page 8. 6h Jim; was N .0 new no ammo scan you oceansoca mondpazo mammoapmpmn o 0 mad 05H 2 o 0 cos «NH m o 0 mo mwa m o o a: mod H o o H v mod 0 mafia .oofiH pa vooc< coaumH500cH Mo mafia moccaspocaeasmm «z as eoee<_oaoosam-n mosoanpocaeashm one o oonac-n no oedema: moccaspmsaeashm «2 gas: «2 gas: flamenco Edaooz Hmmwm JissHeos spasmsoo _Edfiaoowz man .mz Amxooz Gav :oHpmHSOOGH OB nodam cede pompcoo some mmoHozomm..m.emzHHBUmbo 3830mm 'mnxteofim flap '3“ 75 but more specifically from the standpoint of basic physiological research, especially if it acts as an antimetabolite. Sodium pyridinethione while fungicidal at higher concen- trations, exerts a fungistatic effect at the low concentra- tions (0.02 - 0.32 Pg/ml.) used in this study. In one of the few papers published to date dealing with this new com- pound, Donovick 23.2l- in 1952 (17) reported that the pyri- dinethione as well as the 8-hydroxyquinoline activity against E. Mycobacterium tuberculosis var. Eggig ECG was reversed by El histidine. Since 8-hydroxyquinoline was known to be an ex- b cellent chelating agent (64), these workers suggested the possibility that iron chelation by these two compounds was reversibly offset by the competitive action of histidine for the iron in the medium. It was, therefore, considered ad- visable to pursue further this concept in the current inves- tigation. In addition, the possibility that the mode of action of pyridinethione.might be due to the competition for a substrate analogue such as niacin was also investigated. This latter suggested role for the activity of pyridinethione was based on the fact that such known pyridine compounds as pyridine-3-sulfonic acid (39) and isoniazid (56) do act by virtue of their competitive and, therefore, reversible in- hibition for niacin and niacin-containing compounds of meta- bolic importance. 76 a. As a chelating agent. First in order to duplicate the findings of Donovick and his co-workers (17) under con- ditions applicable to this investigation, varied concentra— tions of histidine were added to the sucrose asparagine medium containing sodium pyridinethione. In addition, a comparisori was made employing the same medium that was used by the above investigators, a modified Kirchner's medium, except that Tween 80 was substituted for Triton A-20. The next step in this investigation was to increase the trace metals concentrations of the sucrose asparagine medium in the presence of 0.32 Pg/ml. sodium pyridinethione and the test fungus‘Q, phomoides 080A to see whether in- hibition could be reversed. The complete sucrose asparagine medium contained the standard concentrations of the follow- ing metals: 0.2 mg. Fe, 0.2 mg. Zn and 0.1 mg. Mn. The basal medium was complete with the exception of the metal under investigation which was added at one, ten and 100 fold concentrations. Moreover, all the metals in the medium were simultaneously increased according to the previously listed concentrations. The shake cultures were incubated for six days at 23. 25° C. Table XIV is a compilation of the results indicating that histidine inactivated the inhibitory properties of pyridinethione when tested against 9, phomoides C8OA in a sucrose asparagine as well as a modified Kirchner's f2. 1" 1:1,. .73"? .' 7.5“7‘ 77 r I .in I C. II. I...“ I'm. .oeoaeooeaeanao nonessena one nonaaonsan He 0 cos 0 cos m.o smooo.o snoa a o.H am ma on so Hooo.o N.H nuoa a m no a so ea moooo.o o.o muoa a s nooanaesa nopanaesa A: as a m no donnooom soaoanaeeH do Hoono>om nosusnaneH aunao ozv .ds\ws undo: pcooaom paooaom udooaom paooaom adaooa m.aoaneawm canon undo: suave: .nooo4 .nosm aosaeanosm\H noauwnunoonoo oaHoHpmHm HBOHN mamda 78 medium. Donovick (17) reported an 83 percent reversal of the inhibitor pyridinethione when using M. tuberculosis var. bgzig BCG. This corresponds to the results obtained by this author, since the same histidine to pyridinethione ratio, an 84 percent reversal was obtained in the sucrose asparagine medium and a 93 percent reversal in the modified Kirchner's medium. It is to be noted here that, on a molar basis, an histidine to pyridinethione ratio of l0,000:l was necessary for an inhibition reversal of 36 percent when tested against E, phomoides C80A in a sucrose asparagine medium. No reversal of inhibition was noted when as much as one hundred times the standard concentrations of zinc, iron and manganese were added to the glucose asparagine medium.con- taining 0.32 Pg/ml. sodium pyridinethione and inoculated with the spores of‘Q. phomoides 080A. These results suggest that the inhibitory action of pyridinethione is not due to the inactivation of metals by chelation. With the exception of iron, the excessive amounts of the metals used neither enhanced nor decreased the amount of growth in the control cultures. In the case of iron, at concentrations of 20 Pg/ml., a slight toxic effect was evidenced by somewhat decreased mycelial weights. 79 b. As a competitor for niacin or DPN (diphosphopyridine nucleotide). Niacin1 1 , nicotinamide and.DPN were separately added to flasks containing glucose asparagine medium and 0.32 Fg/ml. (2.15 x 10..6 M) sodium pyridinethione. Spores of the organisms‘g. phomoides 080A andlg, carbonum were tested separately. Since DPN is thermolabile, it was sterilized via Seitz filtration and added to the autoclaved medium at the time of incorporating the inhibitor and the spore inocula. The concentrations of niacin and nicotinamide employed were, on a molar basis, as much as 100 times greater than that of the inhibitor. DPN was used at the same molar concentration as the inhibitor. The results were all negative, since neither niacin, nicotinamide nor DPN reversed the inhibitory action of pyridinethione. This indicates that pyridinethione does not act as an antimetabolite for niacin or DPN. 1Kindly furnished by Hoffmann-LaRoche,Inc., Nutley, New Jersey. 80 CHAPTER IV DISCUSSION AND CONCLUSIONS Screening of Antifungal Compounds In Vitro 0f the six antibiotic and two synthetic materials screened for antifungal activity against two isolates of Colletotrichum.phomoides (080A and lOI) and Helminthggporium sativum, the compounds sodium pyridinethione, rimocidin and encomycin exhibited the best activities with minimal inhibi- tion concentrations of less than 10 Pg/ml. 0n the basis of this investigation, candicidin A showed poor antifungal activity against the test organisms; in addi- tion, the material was degraded rapidly. While this informa- tion corresponds to that obtained by Lechevalier (3h), Alcorn and Ark (2, 3) indicated candicidin to be a good protectant dip and spray as well as a fairly stable compound. This apparent conflict of results may be due to the different sources of the materials. In this investigation, the mater- ial was obtained directly from the laboratory wherein the compound was first described (3k) and perhaps from the first batch produced. The candicidin which Alcorn and Ark used was supplied by a pharmaceutical house and presumably could have been a more active preparation. 81 Aside from.the preliminary screening conducted at the outset of this investigation, no attention was paid to nystatin from the phytopathological viewpoint since three other materials exhibited higher igpgitgg activities. Cur- rently however, nystatin shows excellent promise of being the first successful antifungal antibiotic in the field of medicine. Hazen and Brown (27), the discoverers of this antibiotic, report that it is not inactivated by either blood or horse serum. Campbell 33 3;. (11) demonstrated good mammalian tolerance to high dosages in addition to its effect against histoplasnosis, and Brown 33 3;. (9) showed nystatin to be quite effective in gggg for the control of moniliasis. This investigation confirmed the report made by Gott- lieb gt_§l, (23) that endomycin is thermostable. The anti- biotic however is strongly adsorbed. The implication of this latter observation could be its impracticability as a soil drench for the control of such diseases as damping off. However, since endomycin is active at fairly low con- centrations and does not deteriorate either upon autoclaving or prolonged contact with the medium used, it should merit further investigation. The stability of sodium pyridinethione was investigated. Glucose asparagine medium containing 10 Pg/ml. sodium pyri- dinethione was still capable of completely inhibiting the test fungi after a storage period of six months at room 82 temperature. This however did not indicate the amount of degradation that took place, since if even 75 percent loss occurred, there would still be 2.5 Pg/ml. sodium pyridinethione present in the medium which would be more than sufficient to inhibit‘g. phomoides C80A whose MIC is 0.32 Fg/ml. in glucose asparagine medium. Hence, when an aqueous solution of the inhibitor was stored at 10 Pg/ml. and subsequently tested at 0.1.Pg/ml., it was found that full potency was retained for 25 days at room.temperature. 0n the other hand, the sodium pyridinethione was completely inactivated after storage at room temperature for 5 1/2 months. Sixty-six percent of the activity was lost when the inhibitor was kept at 5° C. for the same period of time. Thus with respect to time, aqueous solutions of sodium pyridinethione were slowly in: activated. This supports the earlier data obtained by the Squibb Institute for Medical Research wherein physical techniques were employed in the stability tests of this compound (5b). Physiological Studies with Rimocidin Rimocidin is quite effective against Q,phomoide§ 080A in the presence of sucrose or glucose as compared to xylose or lactose. The observation that controls grew :much better when cultured with glucose or sucrose than when cultured with xylose or lactose indicates a higher metabolic 83 rate for the organism in the presence of the former sugars. This seems to correlate well with the antifungal activity. Highly active tissues such as meristematic growing points in plants are usually more susceptible to phytotoxic effects when sprayed with antifungal compounds such as rimocidin (NS). When flasks containing glucose asparagine medium and rimocidin at a concentration of 10 Pg/ml. were inoculated with a spore suspension of Q. phomoides C8OA, a fungistatic effect was observed for a period of 15 days after which time, growth became apparent. If sucrose was substituted, all other conditions being equal, the fungistatic effect was pro- longed considerably, in this case for 38 days, at which time the experiment was terminated. A possible explanation for this might be attributed to the reducing property of glucose which may serve to break down the rimocidin over a period of time thus allowing the spores which were held in check, to germinate. Sucrose on the other hand, apparently does not contribute to the inactivation of rimocidin. Antimicrobial Properties of Sodium Pyridinethione According to the results based on the Shippen's modi- fication of the phenol coefficient test, sodium pyridinethione appears to be bacteriostatic. The results of the spore ger- mination tests indicate that 0.05 Fg/ml. causes 100 percent inhibition for the spores of‘g. phomoides C80A at the end 81+ of 2h hours. This, however, does not tell us whether the compound is fungistatic or fungicidal. At very low concentrations, in the presence of glucose asparagine medium, sodium pyridinethione is fungistatic, since increasing the concentration from.0.02 - 0.12 Pg/ml. merely induced greater lag periods in the growth curves of'g. phomoideg 080A. Hartelius (26) obtained similar results for Aspergillus giggg using increasing concentra- tions of sulfanilamide. The fungicidal nature of sodium “n' If“ g..- mrgeu-u- “P . pyridinethione became apparent when its concentration was increased beyond 0.3 Pg/ml. and the spores of‘Q.phomoides C80A remained in contact with the inhibitor for 2h hours. Short time exposures for 20 minutes at concentrations of over 100 Fg/ml. were also fungicidal. Physiological Studies with Sodium Pyridinethione Sodium pyridinethione was most effective againstig. phomoides C8OA in the presence of sucrose; total inhibition occurred at 0.0h pg/ml. Sucrose does not hinder the effi— ciency of sodium pyridinethione since the spore germination test for this fungus in distilled water showed total inhibition to be at 0.05 Pg/ml. The sugars D-glucose, D-xylose and alpha-lactose, on the other hand, apparently had some effect on the activity of the compound. In the presence of these sugars, 0.32 Pg/ml. was required to induce total inhibition 85 (N6). At this point, an explanation is in order to account for the differences obtained when the fungus was grown in the presence of xylose (compare Graph II to Graph IV). In the rimocidin series, the xylose controls grew poorly, whereas in the pyridinethione series, the xylose controls grew very well. Early stationary culture work confirmed the observations made by Lilly and Barnett (37). that Colletotrichum phomoides grows poorly on xylose at first but after a lag period, is capable of utilizing this sugar as well as glucose. Since the xylose run in the rimocidin series was made at uncontrollably high temperatures, the non-utilization phase was probably extended, so that even at the end of the six-day incubation period, growth was still poor. In the case of the sodium pyridinethione series how- ever, the lag phase for xylose was considerably shortened since the 25° C. incubation temperature was more favorable for the organism. The sugar studies done on rimocidin, indicated that fast growing vigorous mycelium is rendered more susceptible to the antifungal action of rimocidin. There seems to be no such relationship between growth rate and susceptibility to sodium pyridinethione. The difference of antifungal acti- vity when tested with different sugars in this latter case might be explained in another way. Because the carbohydrates employed, with the exception 0f sucrose, are reducing sugars, it was suspected that their 86 free carbonyl groups may have something to do with inac- tivating, at least in part, the antifungal properties of sodium pyridinethione. This is supported by the results with acetaldehyde which, when added to the medium contain- ing sucrose and pyridinethione also diminished the antifun- gal activity. In addition, increasing the concentration of glucose inactivated the inhibitor progressively, whereas the I) same level of activity was maintained at various sucrose concentrations. The inactivation of pyridinethione in a i glucose asparagine medium is also dependent on time. The presence of glucose alone is not sufficient to completely inactivate the inhibitor but requires some additional con- stituent(s) of the medium. According to Agren (l), the presence of phosphate accelerated a complex formation in- volving glucose and cysteine. The medium used in this study also contained phosphate in the form.of KH2P0h° It is therefore suggested that the constituents of the medium be investigated separately by addition to a glucose pyridinethione mixture and the results determined via bioassay. The suggestion is hereby made that a definite chemical reaction occurs between pyridinethione and glucose. This is substantiated by the ultraviolet absorption spectra curves of glucose and pyridinethione mixtures. Should spectrOphoto- metric studies be conducted with the complete medium con- taining pyridinethione, it could be expected that much more pronounced results will be obtained. 87 Some speculation as to the chemical nature of this glucose (or other carbonyl containing compound) and pyri- dinethione interaction seems to be in order at this point. The reader is referred to Plate IX for the proposed chemi- cal scheme concerning the inactivation of pyridinethione by glucose. The Squibb research group (5k) state that pyridinethione 5 exists in equilibrium with its tautomeric form.pyridinethiol. ) .Employing the nitroprusside test (25), this author was un- i able to detect the presence of sulfhydryl groups in fresh aqueous solutions of either pyridinethione or its sodium salt. Accordingly, Brewster (8) stated that equilibrium often favors the aldo or keto form.rather than the enol form.of a tautomeric mixture. Thus, Plate IX (A) shows the structural formulas of both pyridinethionefi and pyri- dinethiole with the tautomeric equilibrium far to the left in favor of pyridinethione. Plate IX (B) shows that a ‘possible reaction between glucose and pyridinethiol is the .formation of a thio hemi-acetal. A literature review in- (iicated that such a reaction is chemically feasible. Holleman, in his organic chemistry text (28). stated that alpha-picoline (2 methyl pyridine) undergoes a condensation IJIth acetaldehyde to form a hemi-acetal. Cavallini (12) supported Schubert’s concept (51) that thio hemi-acetals are formed between thiols (glutathione, thioglycollic acid) —_———v *See bibliographical reference (5h). 88 PLATE IX PROPOSED SCHEME FOR THE INACTIVATION OF PYRIDINETHIONE BI GLUCOSE 0' 0" ' ' L1 8+ w' 8 _..; \ SH ; (A) V z/’ a// [ l-hydroxy-2(1H)-pyridinethione 2-pyridinethiol-l-oxide (keto form) (enol form) i” 0‘ H I‘ f \ as I; N: l (B) + ___\ —s-c-n Osc-R I z// = //' 0H carbonyl cpd (zig. glucose) thio hemi-acetal ‘3' °‘ a I If H u? l \\ sn ' JL \\ -S-C-R (C) ( ~4- o:c-n + x ‘— I ' / / -OH 89 and acetaldehyde, pyruvate and glucose. In their studies on glyoxalase activity, thio hemi-acetal formation was supported by Jowett and Quastel (32), Platt and Schroeder (N3) and Racker (NN). While the ultraviolet absorption spectra curves help support the possibility of a glucose- pyridinethiol interaction, bioassay results indicate some additional factor(s), as yet undetermined by this inves- L tigator, is required to shift the equilibrium of the reac- tion to the right, so that most of the pyridinethiol be- comes tied up in the thio hemiacetal complex. This is ex- pressed in Plate IX (C). The overall reaction whereby the antifungal activity of pyridinethione becomes inactivated, is a slow process. A possible explanation might be attributed to the rela- tionship of the tautomeric equilibrium between pyridine- thione and pyridinethiol. Since it has already been sug- gested that veny little pyridinethiol is present at any one time, the pyridinethione is only gradually depleted to maintain the tautomeric equilibrium.as the pyridinethiol becomes tied up with the sugar. Providing that a radioactively tagged preparation of pyridinethione can be obtained, it is suggested that fur- ther substantiation of the pyridinethiol-glucose complex be investigated via autoradiography. In short, this involves comparing the Rf values of glucose and the glucose-pyridinethiol 9O complex by paper chromatography. If the mixture spot con- tains glucose but has a non-glucose Rf value and also ex- hibits radioactivity upon exposure to an X-ray plate, the implications suggest a union of glucose and pyridinethiol. The explanations given for the inactivation of pyridine- thione have been made on the basis of indirect association. Since the training of this investigator has been in the field of microbiology, he feels that elucidation concerning the verification and elaboration of the thio hemi-acetal formation between glucose and pyridinethiol belongs to the realm of the chemist. Mode of Action of Pyridinethione The author does not at this time know why histidine is able to inactivate the antifungal effects of pyridine- thione. Histidine does not appear to act as a competitor with the inhibitor for iron since excess iron does not re- verse the inhibition by pyridinethione. The results indi- cate therefore that pyridinethione does not act as a chelating agent to deprive the fungus of essential metals. In addition, it does not appear to be a substrate competi- tor for niacin or DPN. The mode of action has not yet been determined by this investigator but he believes that the sulfur containing portion of the pyridinethione is important in the mechanism of inhibition. This assumption 1-.--w-nu-tlpi ‘. .- 91 is based on the previous suggestion of a thio hemi-acetal formation between the carbonyl group of glucose and the sulfhydryl group of pyridinethiol which is concomitant with inactivation. Other Significant Conclusions For the purpose of replication between laboratories, a description of the test medium and the concentration of its constituents can be quite important in reporting activity levels of an antimicrobial agent. The results with sodium pyridinethione can be cited as an example. In sucrose asparagine medium (the sucrose concentration is immaterial), the MIC for g. phomoides C80A is 0.01. fig/m1. If in place of sucrose, one percent glucose is used, the MIC increases to 0.32 Pg/ml. Hence eight times more sodium pyridinethione was required in the latter test. Moreover, if a higher glucose concentration, say two percent, were used in the same basal medium, a much greater concentration of the in- hibitor would have been required. Quite often antifungal test compounds require much greater concentrations to control phytopathogens igiygyg as compared to the ig.git£g control of the same organisms (N5). These larger levels required i§.y;19 may be due to factors or combination of factors which serve to decrease the efficiency of the compound. Some of these factors _fl _— , we.“ —_‘ 92 may be lack of penetration, sensitivity to sunlight (ultra- violet light), thermolability, self degradation in solution and chemical inactivation by other sprays or plant exudates. Since pyridinethione is inactivated by reducing sugars, it might be conceivable that if used as a protectant systemic spray, the antifungal agent may become inactivated, at least in part, by the glucose present in the plant sap. Thus, Sander and Allison (50) recently reported that 2- pyridinethiol-l-oxide while translocated downward in cucum- ber seedlings, nevertheless was found to be inactivated when extracts of the cotyledons were assayed. This investigator suggests that the inactivation might have been due to the large concentrations of reducing sugars found in the cotyle- dons of such plants as cucumber at the time of germination. The manner in which carbohydrates influence activity appears to differ between antimicrobial materials. In the caseiof pyridinethione, reducing sugars serve to inactivate the compound by combining with it. When the metabolic rate of a fungus is enhanced by certain sugars, its susceptibility to rimocidin is increased. 0n the other hand, this correla- tion seems to be reversed in the case of sulfonamides, since Gerundo (21) reported that bacterial inhibition occurred if no sugars were added to the medium.or if the sugar was not one usually fermented by the bacterial organism. 93 CHAPTER V SUMMARY 1. 0f eight antifungal test compounds screened $3 ‘31359 against Colletotrichum phomoides (080A and 101) and _Iie‘lminthosporium sativum, the following compounds exhibited minimal inhibition concentrations of less than 10 Pg/ml.: sodium pyridinethione, rimocidin and endomycin. 2. Sodium.pyridinethione is moderately thermostable and breaks down only slowly in aqueous solution at room temperature. 3. Endomycin was found to be strongly adsorbed onto' the surface of asbestos Seitz filter pads. N. Using Colletotrichum.phomoides 080A as the test organism, the $2;!$E£2 antifungal effect of rimocidin was markedly increased in the presence of D-glucose or sucrose as compared to D-xylose or alpha-lactose. Rimocidin at a level of 10 Pg/ml. in the presence of glucose asparagine medium maintained fungistatic activity for a period of 15 days. On the other hand, when sucrose was substituted, the fungistatic effect was increased to over 38 days. 5. The concentrations of sodium pyridinethione in pg/ml. required to completely inhibit spore germination for 2N hours in distilled water at 23, 25° C. were as follows: 9h Colletotrichum.phomoida§ C80A, 0.05; Colletotrichum lagenarium, 0.05; Helminthosporium.carbonum, 0.10 and Puccinia sor hi, 0.05. At 0.05 Pg/ml. sodium.pyridinethione, abnormal vesicle formation was noted at the end of the germ tubes of'H. carbonum. As little as 0.005 Pg/ml. was needed to induce retardation in germ.tube development of Q. phomoides 080A. 6. Sodium pyridinethione is fungistatic at concen- trations up to 0.12 Pg/ml. The fungicidal nature of the compound is exhibited when the spores of Q. phomoides 080A remain in contact with the inhibitor for 2N hours at con- centrations above 0.3 Pg/ml. Short time exposures for 20 minutes at concentrations higher than 100 Pg/ml. are also fungicidal. 7. Increasing concentrations of sodium pyridinethione at fUngistatic levels progressively retard the growth curves of Q. phomoides C80A. 8. In the presence of sucrose asparagine medium, sodium pyridinethione totally inhibited Q. phomoides 080A at 0.0N Pg/ml. When the reducing sugars D-glucose, D-xylose or alpha-lactose were substituted, 0.32 pg/ml. of the in- hibitor was required to produce the same effects. 9. Asparagine medium containing increasing concentra- tions of D-glucose progressively inactivated sodium pyri- dinethione. This inactivation is also dependent on time. Sucrose had no effect on the antifungal inactivation of the compound. 95 10. It is suggested that glucose inactivates pyridine- thione by the formation of a thio hemi—acetal complex. Other carbonyl containing chemicals such as acetaldehyde also inactivate pyridinethione. The postulation of an inter- action between glucose and the inhibitor is supported by ultraviolet absorption spectra data. The role of glucose in plants is discussed with relation to pyridinethione inactivation. 11. The mode of action of sodium pyridinethione remains unknown to this investigator. The inhibitor does not appear to act as a chelating agent for essential metals nor does it function as a substrate analogue for niacin. It is sug- gested that the sulfur portion of pyridinethione is func- tional in causing inhibition. ‘1— . 1. 2. 3. 7. 9. 10. 96 LITERATURE CITED Agren, G. 19N1. The interaction of amino nitrogen and carbohydrates. Enzymolgia 2; 321-328. Alcorn, S. M. and P. A. Ark. 195N. The effect of candicidin in plant pathogenic fungi. Plant Dis. Reptr. 38: 705-709. Alcorn, S. M. and P. A. Ark. 1955. The antibiotic candicidin, a protectant dip against brown rot in— fection of peach fruit. Plant Dis. Reptr. 32: 210-212. Allison, Patricia and G. L. Barnes. 1956. Plant disease control by a new class of chemicals, 2- pyridinethiol and derivatives. Phytopath. N6: 6. American Phytopathological Society. Committee on Standardization of Fungicidal Tests. 19N7. Test tube dilution technique for use with slide germination method of evaluating protectant fungicides. Phytopath. 31; BSA-356. Binz, A. and C. Bath. 1928. Biochemical preperties of pyridine and quinoline derivatives. Bioch. Z. 20}: 218-222. Boone, Irene U. and K. T. WOodward. 1953. Relation of pyridoxine and its derivatives to the mechanism of action of isoniazid. Proc. Soc. Exptl. Biol. and Med. 8N: 292-296. Brewster, R. Q. 19N8. Organic Chemistry. Prentice- Hall Inc., New'York. 8N0 pp. Brown, Rachel, Elizabeth L. Hazen and Alice Mason. 1953. Effect of fungicidin (nystatin) in mice in- Jected with lethal mixtures of aureomycin and Candida albicans. Science 111: 609-610. Browning, C. H., J. B. Cohen, S. Ellingworth and R. Gulbransen. 1923. Antiseptic action of the styryl- pyridines and styryl-quinolines. Brit. Med. J. II: 326. 97 11. Campbell, C. C., E. P. Hodges and G. B. Hill. 195k. Therapeutic effect of nystatin (fungicidin) in mice experimentally infected with Histoplasma gapsulatum. Antibiotics and Chemotherapy E: NOEJNIO. 12. Cavallini, D. 1951. The coupled oxidation of pyruvate with glutathione and cysteine. Biochem. J. N2: 1—5. 13. Cohen, R. 1951. Four new fungicides for Coccidiodes immitis. 1. sodium caprylate. 2. ethyl vanillate. 3éuFradicidin. N. Thiolutin. Arch. Pediat. 68: 259- 2 0 IN. Cote, L., J. J. Oleson and J. H. Williams. 1952. Nicotinamide inhibitors. Proc. Soc. Exptl. Biol. and Med. g9; N3N-N36. 15. Davisson, J. W., F. W. Tanner, Jr., A. C. Finlay and I. A. Solomons. 1951. Rhmocidin a new antibiotic. Antibiotics and Chemotherapy';: 289-290. 16. D1 Raimondo, F., N. Mannino and M. L. Orabona. 1953. Inhibition of antibiosis by vitamins through meta- bolic antagonism. Research in vitro on Mécrococcug pyogenes var. aureus with Terramycin and Tyrothricin. Boll. ist. sieroterap. milan.gérfi NS-Sl. (Seen in abstract only. C. A. 195N. 7933.) 17. Donovick, H., A. Bayan and D. Hambre. 1952. The reversal of the activity of antituberculous compounds in vitro. Am. Rev. Tuberc. 69: 219-227. 18. Duckworth, R. B. and G. c. M. Harris. 19N9. The morphology of Penicillium chrysogenum in submerged fermentations. Trans. Brit. Mycol. Soc.‘3g: 22N-225. 19. Foster, J. W. 19N9. Chemical Activities of Fungi. Academic Press, New'York. 6N8 pp. 20. Gaebler, 0. H. and W. T. Beher. 1951. Antimetabolites. I. Pyridine-3-sulfonic acid and 3-acetylpyridine. J. Biol. Chem. 188: 3N3-3N9. 21. Gerundo, M. 19N9. Antagonistic influence of carbo- hydrates upon bacterial inhibition by sulfonamides in vitro. Texas Repts. Biol. and Med.‘1: 58-68. 22. Cepalkrishnan, K. S. and J. A. Jump. 1952. The anti- biotic activity of thiolutin in the chemotherap of the fusarium wilt of tomato. Phytopath. N2: 33 -339, "T... a i '- - In. _ 23. 2N. 25. 26. 27. 28. 29. 30. 31. 32. 33- 98 Gottlieb, D., P. K. Bhattacharyya, H. E. Carter and H. W. Anderson. 1951. Endomycin, a new antibiotic. PhytOpath. N_: 393-NOO. Grosso, J. J. 195N. Control of tobacco blue mold by antibiotics. Plant Dis. Reptr. 36: 333. Grunert, R. R. and P. H. Phillips. 1951. A modifica- tion of the nitrOprusside method of analysis for glutathione. Arch. Bioch. 36: 217-225. Hartelius, V. 19N6. Antivitamin effect of sulfanila- mide on‘A. ni er. Compt. rend. trav. lab. Carlsberg, Ser. physiol. : 178-18N. -—. _a— -.v‘ V“ *- - ‘— Hazen, Elizabeth L. and R. Brown. 1951. Fungicidin, an antibiotic produced by a soil actinomycete. Proc. Soc. Exptl. Biol. and Med. 16: 93. —-——— Holleman, A. F. 1951. Organic Chemistry, by J. P. Wibaut. Translated from.the l6th.Dutch ed. by S. Coffey. Elsevier Pub. Co., New York. 660 pp. Horimoto, S., F. Ito, M. Yamamoto, I. Inoue, J. Sakai and T. Sugano. 195N. The influence of various vitamins, sugars and ketones on the bacteriostatic action of isonicotinic acid hydrazide (INAH) on tubercle bacilli. Kekkaku (Tuberculosis) 2 : 166- 167. (Seen in abstract only. C. A. 195N. 8: 9N6Nd.) Hughes, D. E. 1952. The mechanism of the inhibition of growth and cozymase synthesis in bacteria by 5-f1uorenico- tinic acid. Cong. intern. biochim., Resumes communs., 2° Congr. Paris, 1952 103-10N. (Seen in abstract only. C. A. 195,40 AL: 711611). Hughes, D. E. 195N. Inhibition of growth and cozymase synthesis in bacteria bg halogen substituted nicotinic acids. Bioch. J. 51: N 541,95. Jowett, M. and J. H. Quastel. 1933. LXX. The gly- oxalase activity of the red blood cell. The function of glutathione. Bioch. J. 21: N86-N98. Klomparens, W., and J. R. Vaughn. 1953. Studies of the properties of antibiotics -- translocation and physio- logical studies with cyclohexamide (acti-dione), endomycin and streptomycin sulfate. Michigan State University. (Unpublished manuscript). 3h- 35- 36. 37. 38. 39- N0. N1. N2. AB. NSo no. 99 Lechevalier, H., R. F. Acker, C. T. Corke, C. M. Haenseler and S. A. Waksman. 1953. Candicidin, a new antifungal antibiotic. Mycologia Ni: 155-171. Lechevalier, H. 195N. Antifungal Antibiotics. Congr. igterg. bot., Paris. Rapp. et communs. 8, sect. 21-27. 9 ‘10 e Lilly, V. G. and H. L. Barnett. 1951. Physiology of the fungi. McGraw Hill, New York. N6N pp. Lilly, v. G. and H. L. Barnett. '1953. The utilization .m of sugars by fungi. W. Va. Univ. Agric. Expt. Sta., . Bull. 362T. A McCallan, S. E. A. and R. H. Wellman. 19N2. Fungicidal L: versus fungistatic. Contribs. Boyce Thompson Inst. lg: u51-u630 McIlwain, H. 19N0. Pyridine-3-sulphonic acid and its amide as inhibitors of bacterial growth. Brit. J. Exper. Path. 21: 136-1N7. Murneek, A. E. 1952. Thiolutin as a possible inhibitor of fire blight. PhytOpath. 6;: 57. Oster, K. A. and M. J. Golden. 19N7. Evaluation of fungistatic laboratory test methods. J. Amer. Pharm. Assno, 50. Ed. Jé: 283-288. \ \“‘ . . Pansy, F. E., H. Stander, W. L. Koerber and R. Donovick. 1953. EB vitro studies with 1-hydroxy-2(1H) pyridine- thione. Soc. Exptl. Biol. and Med. 6gt.122512N. Platt, M. E. and E. F. Schroeder. 193N. The applica- bility of the manometric method to the study of glyoxalase. J. Biol. Chem. 10g: 281-297. . Hacker, E. 1951. The mechanism of action of glyoxalase. J. Biol. Chem. 120: 685-696. Ringel, S. M., E. S. Beneke, C. Kenaga and R. Kiesling. 195N. Physiological studies and trial application of antifungal antibiotics for the control of plant diseases. Michigan State University. (Unpublished manuscript.) Ringel, S. M. and E. S. Beneke. 1956. The influence of certain sugars on the antifungal activity of sodiwm pyridinethione. Mycologia (in press). N7. N8. N9. 50. 51. 52. 53. SN. 55. 56. 57. 58. 59. IOO Robbins, W. J. 19Nl. The pyridine analog of thiamin and the growth of fungi. Proc. Nat. Acad. Sci. 21: h19’h220 Ross, I. G. 1951. Structure of N-pyridinethione. J. Chem. SOQe, Pt. 2. 137(4-‘13750 Ruehle, G. L. A. 1937. U. S. Food and Drug Adminis- tration methods of testing antiseptics and disinfectants. U. S. D. A. Circ. 198. Sander, Evamarie and Patricia Allison. 1956. Bioassay of the translocated fungicide, 2-pyridinethiol-l-oxide in cucumber seedlings. Phytopath. N6: 25. Schubert, M. P. 1936. Compounds of thiol acids with aldehydes. J. Biol. Chem. 116: 3N1-350. Shaw, E., J. Bernstein, K. Losee and W. A. Lott. 1950. Analogs of aspergillic acid. IV. Substituted 2- bromopyridine-N-oxides and their conversion to cyclic thiohydroxamic acids. J. Am. Chem. Soc. 1g: N362-N36N. Soo-Hoo, G. and E. Grunberg. 1950. The antifungal activity of metal derivatives of 3-pyridinethiol. J. Invest. Dermat. 16: 169-172. The Squibb Institute for Medical Research, New Brunswick, New Jersey. 2-pyridinethiol, 1-oxide, a brochure (per- sonal communication). The Squibb Institute for Medical Research, New Brunswick, New Jersey (written communication). van Rey, W. 195N. The question of biological antagonism between nicotinic acid and isonicotinic acid hydrazide. Klin. WOchschr. 2: 229-230. (Seen in abstract only. c. A. 195N. N6: 685h.) Weinberg, E. D. 1953. The influences of various sources of N on the activity of antimicrobial drugs. II. Mycobacterium.avium. Am. Rev. Tuberc. 61: 503-508. welch, A. D. 19N5. Interferences with biological processes through the use of analogs of essential metabolites. Physiol. Revs. 25: 6 7-715. wooley, D. W., F. M. Strong, R. J. Madden and C. A. Elvehjem. 1938. Antiblack tongue activity of various pyridine derivatives. J. Biol. Chem. 126: 715-723. 60. 61. 62. 63. 6N. lOl Wooley, D. W. and A. G. C. White. 19N3. Selective reversible inhibition of microbial growth with pyri- thiamine. J. Exptl. Med. 16: N89-li97. Neoley, D. W. 1952. A study of antimetabolites. Wiley and Sons Inc., New York. 269 pp. Wyss, 0. 19N3. Antibacterial action of a yridine analogue of thiamine. J. Bact. N6: N83-N8N. Yoneda, M., N. Kato and M. Okajima. 1952. Competitive action of isonicotinic acid hydrazide and vitamin B6 in the formation of indole bng. coli. Nature 170: 803. Zentmeyer, G. A. 19NN. Inhibition of metal catalysis as a fungistatic mechanism. Science 100: 29N-295. '2) {.3 All. 1": k; fir .13 a U 1' Date Due .- CHLY Borneo-293