STUDIES ON THE FUNGICIDAL ACTIVITIES AND PHYTOTOXIC PROPERTIES OF SEVERAL ANTIFUNGAL COMPOUNDS By Clare Burton Kenaga 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 1957 ProQuest Number: 10008504 All rights reserved INFORMATION TO ALL USERS The quality o f this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete m anuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008504 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 ACKNOWLEDGMENTS The author extends his sincere appreciation to Dr. R. L. Kiesling under whose direction and assist­ ance this work was undertaken, and to the Horace H. Rackham Research Endowment for the grant which made this work possible. Sincere thanks are also expressed to Mr. Philip Coleman, for the preparation and pre­ sentation of the photographic material. The author especially wishes to express his thanks and gratitude to his wife Doris for her constant encouragement, aid and patience during the course of this study and the writing of this thesis. STUDIES ON THE FUNGICIDAL ACTIVITIES AND PHYTOTOXIC PROPERTIES OF SEVERAL ANTIFUNGAL COMPOUNDS By Clare Burton Kenaga 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 1957 Approved ABSTRACT Acti-dione, Panogen 15, hexachlorobenzene and the Omadine salts of copper, zinc, manganese and sodium when applied as foliar sprays controlled Phoma 1ingam on cabbage, Colletotrichum lindemuthianum on bean and Puccinia sorghi on corn. The best overall control was ob­ tained with sodium Omadine, copper Omadine and Panogen 15* Concentrations of sodium Omadine necessary to produce total inhibition of the spores of Colletotrichum lagenarium, Helminthosporium carbonum in distilled water were respectively. phomoides and 0 .0 5 , 0 . 0 5 and 0.10 ppm Vesicles or swellings were occasionally produced at the tips of the germ tubes of H. carbonum at concentrations of 0.05 ppm. The lethal threshold for growth of Lemna minor under the influence of sodium Omadine was between 0.5 and 0.1 ppm. 0,005 Concentrations as low as ppm produced a marked decrease in growth. Sodium Omadine does not act as a systemic fungicide, and up to 50 ppm of the compound is inactivated immediately upon contact with extracts from radish and tomato. The ultraviolet absorption spectra of guttation water of cucumber seedlings grown at temperatures of 68° to 70° F in sand treated with sodium Omadine indicated a lower amount of inorganic substances were probably present than from control plants. At higher temperatures these differences were not present, and the absorption spectra curves for control plants from both high and low ranges of temperatures were entirely different. iv The hypothesis was made that limited amounts of -SH groups were present in aqueous solutions of* sodium Omadine, and that therefore several chemical reactions characteristic of the mercaptans were possible. Circumstantial evidence was presented as the basis for the hypothesis, and evidence is presented to show that some of the reactions characteristic of mercaptans do take place. Evidence was presented to the effect that in a solution containing water-soluble metal salts there is a replacement of the sodium ion attached to the Omadine molecule by the metal element in solution. A second hypothesis was made that the Omadine salts act as chelators in which the preformed metal chelates, possessing a fat-water solubility balance, permeate the cellular membranes and preform a toxic reaction within the cell. Evidence is given in support of this hypothesis. A third hypothesis was made that once the Omadine molecule enters the cell it undergoes reduction, leaving the pyridine ring which is the toxic property. Circumstantial evidence is presented in support of this hypothesis. v TABLE OF CONTENTS CHAPTER II INTRODUCTION............................................ 1 LITERATURE REVIEW........................... 1 MATERIALS AND METHODS................................... 6 Materials Employed................................... Compounds: Their Source and Maintenance........... Fungal Organisms and Their Culture................. Methods Employed..................... Studies of the Various Test Compounds.............. 1. _In vivo screening of test compounds.......... 2 . Phytotoxicity............................... 3. Experiments on the systemic activity of several antifungal compounds in the control of CL lindemuthianum on bean.................... a. Via detached leaf culture.......... 10 b. Via hydroponics....... Studies of Sodium Omadine. ..................... 1. Spore germination tests...................... 2. Stability of sodium Omadine on bean foliage.... 3. Growth response of Lemna minor L. to sodium Omadine............ U . Translocation studies of sodium Omadine... 13 a. Via hydroponics................... 13 b . Via bioassay of guttation and stem exudate from plants grownin treated sand c. Via bioassay of split-stern sections of tomato seedlings grown in treatedsand... d. Via bioassay of expressed juice from tomato and cucumber seedlings grown in treated sand....................... 15 e . Via bioassay of expressed juice from foliar treated plants............ 5. Physiological studies on the influence of sodium Omadine on host plants............ 16 a. Ultraviolet spectrophotometric determination...................... 16 b . Free and combined amino acid studies of treated plants..................... 17 c . Reducing sugar studies of treatedplants. . I Page vi 6 6 7 9 9 9 10 10 11 12 12 12 12 lU 15 16 17 TABLE OF CONTENTS - Continued CHAPTER Page 6. Inactivation of sodiumOmadine................ a . Guttation water....................... b. Expressed juiceofplant parts........... c. D-Glucose............................. Studies Concerning Mode of Action of the Omadine Compounds...................................... 1. Purification and investigation of chemical composition................................. a. Studies of technical grade sodium Omadine by paper chromatography................ b . Purification. ...................... c. Chemical tests.... *................... 2. Location of the action of the Omadine compounds Additional techniques related to this study........ 1. Filter-paper disc bioassay technique......... 2, Paper chromatography: procedures and tech­ niques a. Extraction and preparation of samples.... b . Application of samples................. c. Further techniques..................... 3- Test for reducing sugars..................... 6 ' Precipitate test for aqueous solution of sodium Omadine .................................... III RESULTS................................................ Studies of the Various Test Compounds.............. 1. In vivo screening of test compounds.......... 2. Phytotoxicity............................... 3. Experiments on the systemic activity of several antifungal compounds in the control of C. lindemuthianum on bean. . ................. Studies of Sodium Omadine..................... 1. Spore germination tests.......... 2. Stability of sodium Omadine on bean foliage .... 3. Growth response of L_. minor to sodium Omadine.. 6. Translocation studies of sodium Omadine...... 5. Physiological studies on the influence of sodium Omadine on host plants................ a. Ultraviolet spectrophotometric determination.................. b . Free and combined amino acid studies of treated plants ................... c. Reducing sugar studies of treated plants. vii 18 18 18 20 20 20 20 21 22 23 26 26 25 25 26 26 27 27 29 29 29 36 62 63 63 55 65 68 56 56 61 61 TABLE OF CONTENTS - Continued CHAPTER Page 6. Inactivation of sodium Omadine............... Studies Concerning Mode of Action of the Omadine Compounds............................... 1. Purification and investigation of chemical composition.......... ....................... a. Studies of technical grade sodium Omadine by paper chromatography..... b. Purification.......................... c. Chemical tests..... ,.................. 2. Location of the action of the Omadine compounds IVDISCUSSION AND CONCLUSIONS................................ In Vivo Screening of Antifungal Compounds............. Phytotoxicity........................................ Systemic Activity.................................... Stability of Sodium Omadine on Host Plants............ Physiological Studies on the Influence of Sodium Omadine on Host Plants.................................... Inactivation of Sodium Omadine........................ Chemical Reactions of the Omadine Derivatives......... Mode of Action of the Omadine Derivatives............. 66 66 66 66 72 76 76 82 82 86 85 88 89 90 91 95 VSUMMARY.................................................. 102 LITERATURE CITED.............................................. 105 APPENDIX.................... ................................. Ill Multiple Spotting Apparatus for Paper Chromatography.... Ill LIST OF TABLES TABLE II III IV V Test organisms and common names ofdiseases theyproduce... 8 Influence of various antifungal materialson disease incidence of Phoma lingam..................... . 30 Influence of various antifungal materials on disease incidence of Colietotrichum lindemuthianum on beans....... 33 Influence of various antifungal materials on disease incidence of Puccinia sorghi on corn..... .................. . I Page Influence of various antifungal materials via systemic action on disease incidence of Colletotrichum lindemuthianum on bean leaflets in detached leaf culture............. U3 Bioassay of moisture from sodium Omadine treated and non­ treated sand in which tomato seedlings were grown: sixteen days after drench treatment............................... 52 VII Bioassay of distilled water dilutions of sodium Omadine.... 52 VIII Bioassay experiments on juice expressed from roots, stems and leaves of tomato seedlings sprayed with sodium Omadine. 53 Bioassay experiments on juice expressed from roots, hypocotyls and leaves of radish seedlings sprayed with sodium Omadine................................................... 53 VI IX X XI XII Reducing sugar determinations of extracts from control and sodium Omadine treated plants as ascertained via the Somogyi method . Foliar spray treated plants harvested 2U hours after 200 ppm sodium Omadine treatment. Root drench treat­ ed plants harvested H days after application of 5 0 ppm sodium Omadine. Determinations are based on an average of eight plants per treatment................................ 63 Bioassay experiments on sodium Omadine treated extract expressed from radish seedlings.................... 65 Bioassay experiments on sodium Omadine treated extract expressed from tomato seedlings . ............ 65 ix 39 LIST OF TABLES - Continued TABLE XIII Page Percent inhibition of spores of Glomerella cingulata after 2b hours in 2 percent sucrose, after prior 2U hour exposure to treatments............. 76 A Composition of basal medium used in culture of Lemna minor. 119 B HoaglandTs complete nutrient solution.................... 119 x LIST OF GRAPHS GRAPH I II III IV V VI VII VIII Page Dosage responsecurves of the spores of three fungal organisms under influence of sodium Omadine at several concentrations.......................................... I4I4 Influence of sodium Omadine at seven concentrations on the growth of Lemna minor given in percent change from control on wet weightbasis (after U weeks)............ .......... 67 Influence of 0.05 ppm sodium Omadine on the growth of Lemna minor.................................................. 50 Ultraviolet absorption spectra for guttation water from 50 ppm sodium Omadine drench and non-treated cucumber seed­ lings grown in greenhouse temperatures of 68-70 F ....... 55 Ultraviolet absorption spectra of water of guttation 36 hours and 60 hours after treatment. Guttation water collected from cucumber seedlings.................. 56 Identical curves of the ultraviolet absorption spectra for undiluted guttation water from 50 ppm sodium Omadine., and water drench treated cucumber seedlings grown in greenhouse temperatures of 76-90° F.,.............................. 58 Concentrations of 0.05 - 1 percent D-glucose as determined via the Somogyt method and read in a Klett-Summerson Photo­ electric Colorimeter.................................... 62 Ultraviolet absorption spectra of sodium Omadine samples at 10 73 xi LIST OF FIGURES FIGURE Page I Chromatograms of UO jj.g of sodium Omadine and disulfide Omadine showing "quench” spots as observed under fluores­ cent light........................................... 68 II III Autobiographs of UO ^ig of technical grade and "purified” sodium Omadine . ....................................... 70 Structural formulas of sodium Omadine and the parent Omadine formsand two possible reactions of sodium Omadine ^2 xii LIST OF PLATES PLATE I Page Spray trials for control of Phoma 1ingam on Chinese cabbage (experiment 10). (hexachlorobenzene, Panogen 15 and zinc Omadine).......... 31 Spray trials for control of Phoma 1ingam on Chinese cabbage (experiment 10). (manganese, copper and sodium Omadine).................... 32 Spray trials for control of Colletotrichum lindemuthianum on bean (experiment 7). (Acti-dione and hexachlorobenzene)....... 35 Spray trials for control of Colletotrichum lind emuthianum on bean (experiment 7). (Panogen 15 and zinc Omadine).................... 36 Spray trials for control of Colletotrichum lindemuthianum on bean (experiment 7)* (manganese and copper Omadine)...... 37 Spray trials for control of Colletotrichum lindemuthianum on bean (experiment 7)• (sodium Omadine, Acti-dione, Panogen 15 and hexachloro-' benzene)................................................. 38 Phytotoxicity caused by one foliar spray of diaphine HC1 at 200 ppm on cucumber-Phytotoxicity caused by one foliar spray of diaphine HC1 and sodium Omadine at 200 ppm on bean J4I Spore germination test. The effect of sodium Omadine on spores of Helminthosporium carbonum. Twenty-four hour incubation at 25 C ...... ............................ 56 IX Growth of Lemna minor after a four-week period............ 59 X Cucumber seedlings grown in pot saucers in sand to which two drench treatments 5 days apart of sodium Omadine were applied. Photographed 8 days after the first treatment.... 60 Bioassay plates of sodium Omadine at 100 p.g/ml alone and in combination with D-glucose at)fmg/ml. Solutions bioassayed after standing at room temperature for 10 days........... 67 II III IV V VI VII VIII XI xiii LIST OF PLATES - Continued PLATE XII Page Bioassay from fluorescent "quench1’ spots of chromatograms of technical grade sodium Omadine, developed in watersaturated n-butanol, The discs in the two bioassay plates are from corresponding spots from two different chromatograms 69 XIII Growth of spores of Glomerella cingulata which had been washed, brought in contact with various compounds for 12 hours, rewashed and a drop of spore suspension from each treatment placed on potato dextrose agar plates for four days. Each compound in the treatments was present at 1 ppm 78 XIV Growth of spores of Glomerella cingulata which had been washed, brought in contact with various compounds for 2h hours, rewashed and a drop of spore suspension from each treatment placed on potato dextrose agar plates for four days. Each compound in the treatments was present at 1 ppm 79 XV Growth of spores of Glomerella cingulata which has been washed, brought in contact with various compounds for 2U hours, rewashed and a drop of spore suspension from each treatment placed on potato dextrose agar plates for four days. Each compound In the treatments was present at 1 ppm 80 XVI Growth of spores of Glomerella cingulata which had been washed, brought in contact with various compounds for 2U hours, rewashed and a drop of spore suspension from each treatment placed on potato dextrose agar plates for four days. Each compound in the treatments was present at 1 ppm 8l A Multiple sample spotter for paper chromatography....... 115 B Adjustable spotting rack set up for one- and two-dimen­ sional chromatograms................................. 116 C Multiple sample spotter in position in front of the adjust­ able spotting rack which is set up for two-dimensional chromatograms......... 117 D Sample spots at various percentage timer settings...... 118 xiv CHAPTER I INTRODUCTION A study of the influence of several antibiotic and synthetic organic materials upon certain plant pathogens and their hosts was initiated in 19£U- These compounds were screened against several organisms causing foliar diseases and formed the basis for further selection of materials employed in physiological studies of host reaction, possible systemic action, inactivation and mode of action. The derivatives of l-hydroxy-2(lH) pyridinethione (Omadine) were selected from a group of artifungal materials for physiological studies on the basis of the screening trials since they exhibited low phytotoxicity and excellent antifungal properties. The sodium salt of 1hydroxy-2(lH) pyridinethione was selected for most of these studies since it was water soluble. These compounds represent a new group of anti- fungal matei-ials with which little work had previously been done. LITERATURE REVIEW The antibiotic thiolutin was screened for chemotherapeutic activity in 195>2 by Gopalkrishnan and Jump (17). They found thiolutin to be more active in vivo than in vitro, in the control of Fusarium wilt of tomato, although higher concentrations of this antibiotic produced a stunting accompanied by marked epinasty of the leaves. Nickell and Finlay (U3) found that 1 ppm thiolutin produced some stimulation of growth of 1 2 Lemna minor. Although many accounts of this antibiotic have appeared in the literature concerning its antibacterial and antifungal activity of human and plant pathogenp, thiolutin has met with very limited success (17,67,8,141,22). Rimocidin, an antibiotic, was first described by Davisson et_ a l . (11) in 1951 "who found it inhibited many of the human pathogenic fungi in vitro at low concentrations. Malcolmson and Bonde (36) found that rimocidin sulphate alone and together with Agrimycin■gave good control of the bacterial rots, Erwinia atroseptica and Pseudomonas fluorescens, on potato seed pieces. Vaartaja (6 7 ) reported that rimocidin at a wide range of concentrations controlled Rhizoctonia solani and P.ythium debaryanum on pine and birch seed in Petri dishes without marked phyto toxicity. Antibiotic XG (later called fungistatin XG) was found by Hobby et al. (23) in 19U9 to be effective against the growth of many human pathogenic fungi in vitro, while Wallen and Skolko (71) in 1950 found it to be effective as a seed treatment for the control of Ascochyta pisi on peas. Hazen and Brown in 1951 (21) reported that fungicidin (later called nystatin) was effective in vitro against human and plant patho­ gens at concentrations of 10 ppm and lower. Acti-dione (cycloheximide) was isolated from Streptomyces griseus by Whiffen et al. in 19U6 (7U)• Considerable material exists in the literature concerning this antibiotic. Whiffen in 19U8 (73) found it highly active in vitro against human pathogenic fungi. Felber and Hamner in 19U8 (15) found that this antibiotic controlled mildew on bean. 3 Wallen e^b al. (72) reported it had a high phytotoxicity against pea seed. Gottlieb et al. (18) reported that Acti-dione when applied as foliar sprays was very phytotoxic. Cation (7 ) and Hamilton and Szkolnik (20) reported control of cherry leaf spot with foliar sprays of 1 to 2 ppm. Vaughn (6 8 ) found this antibiotic effective against turf diseases. Hexachlorobenzene (perchlorobenzene) was found effective in con­ trolling dwarf bunt as well as common bunt of wheat by Siang and Holton (£8 ) in 1953 y who attributed the control to inhibitory action of vapor on spore germination. Purdy in 1956 (5l) found that in five Pacific Northwest states, hexachlorobenzene was the most effective chemical for wheat smut control, Martin (38) described Panogen (methylmercurie dicyandiamide ) as a fungicide for use on seed of cereals, flax, sorghums, cotton and sugar beet. He stated further that it was used in Sweden since 1938 and in North America since 19h9 as a seed disinfectant. Leukel (3U) reported Panogen to be effective in controlling bunt on wheat, loose and covered smut on oats and stripe disease on barley. The prototype for a new class of compounds possessing antifungal activity, a cyclic thiohydroxamic acid, was synthesized by Shaw et al. in 1950 (57). This compound exists in two tautomeric forms and as such may be chemically described as l-hydroxy-2(lH) pyridinethione, or 2 -pyridinethiol, 1 -oxide ( 6 2 ^U5) * P ^ s y et al. in 1953 (50) conducted screening studies against numerous microorganisms in vitro by this compound and found it to exhibit antifungal as low as O.0 I4. ppm. activity at concentrations Norman (UU) reported that low concentrations of h this compound and several of its analogs inhibited root elongation and dry matter increase in cucumber and barley, but foliar applications at comparable or higher concentrations did not produce noticeable responses in top growth or development. However, Kenaga and Kiesling (29) found that repeated application of the sodium derivative as foliar sprays and root drenches caused chlorosis and stunting. Allison and Barnes in 1956 (l) evaluated 2-pyridinethiol, 1-oxide and several derivatives and found them to be promising as foliage and soil fungicides, and also claimed that the copper and zinc derivatives had a high residual activity on tomato foliage. Couch and Cole (9) reported that the disulfide provided a limited amount of control on rust of Merion bluegrass, and in 1957 (10) these authors reported that it provided significant re­ duction of melting-out of Kentucky bluegrass. Disulfide, manganese and thio-urea derivatives of 2-pyridinethiol, 1-oxide were found effective in reducing the disease incidence of Alternaria blight when tested in mist stream propagation of carnations in the greenhouse in 1956 by Skiver (59). Sander and Allison (56) reported that 2-pyridinethiol, 1-oxide was absorbed through the leaves of cucumber seedlings and trans­ located downward, and suggested that the chemical was inactivated in extracts of young shoots and cotyledons. Ringel and Beneke (5U) showed that the sodium derivative was most effective in inhibiting the growth of Colletotrichum phomoides in liquid culture when in the presence of sucrose, and that the reducing sugars D-glucose, D-xylose and alphalactose reduced the inhibitory effect of the compound. Ringel (53) suggested that the sodium derivative was not a competitor for the 5 substrate analogues niacin, nicotinamide and diphosphopyridine nucleotide. This writer also reported that histidine caused a 36 percent inactivation of the sodium derivative when present at a molar ratio of 10,000 to 1 respectively, and that an excess of zinc, iron and manganese failed to reverse the inactivation. He therefore, concluded that the inhibitory action of this compound was not due to chelation of metals. One of the antifungal compounds reported in the literature and closely related to 2-pyridinethiol, 1-oxide was the compound 3-pyridinethiol. The disulfide and metal derivatives of this latter compound were studied for anti­ fungal activity by Soo-Hoo and Grunberg in 195>0 (6l), who found them to possess a high antifungal activity. CHAPTER II MATERIALS AND METHODS Materials Employed Compounds: Their Source and Maintenance The following is a list of antifungal materials employed in this study and their source: Thiolutin, diaphine HC1, rimocidin sulfate and fungistatin X G : furnished by Charles Pfizer and Company, Brooklyn, New York. Panogen l£ , (2.2% methyl mercury dicyandiamide in liquid carrier) and hexachlorobenzene, (h0% furnished by Panogen, Inc., Ringwood, Illinois. Acti-dione (cycloheximide): Kalamazoo, Michigan. furnished by the Upjohn Company, Nystatin (fungicidin): furnished by the Squibb Institute for Medical Research, New Brunswick, New Jersey. „ Sodium, zinc, manganese and copper Omadine, (salts of 1 -hydroxy-2 (1H) pyridinethione); and Omadine disulfide, (2,2I-dithiodipyridine1,1*-dioxide): the sodium salt was originally furnished by the Squibb Institute for Medical Research, New Brunswick, New Jersey, now a division of Olin Mathieson Chemical Company, Baltimore, Maryland, who furnished succeeding lots of this and remaining Omadine compounds. Acti-dione, thiolutin, fungistatin XG, nystatin and rimocidin sulfate were antibiotic materials. Diaphine HC1, Panogen If? and the Omadine compounds were synthetic organic compounds. Diaphine HC1, rimocidin sulfate, Panogen Acti-dione and di­ sulfide and sodium Omadine were soluble in water at the concentrations used, and fresh stock solutions of these materials were prepared before ■use. The remaining compounds were not soluble at this stock 6 concentration, and an emulsifying agent, Tergitol No. 7 (38), was employed to keep them in suspension. All of the test materials were maintained in a dry state at room temperature until used. Throughout this study each concentration of a test compound in solution or otherwise is given as concentration of active ingredients. Fungal Organisms and Their Culture The fungal organisms employed in this study together with the common names of diseases they produce are given in Table I. C_. lind emuthianum was maintained on bean-pod agar slants. To obtain abundant sporulation, flasks containing sterilized barley were inoculated with a spore suspension and allowed to grow for four days after which spores were collected by rinsing with sterile water and straining through cheesecloth. C. lagenarium was maintained on V-8 agar to induce abundant sporu­ lation. Spores of P_. sorghi and P_. graminis tritici were collected via cyclone spore-collector from the diseased leaves of their respective host plants and stored under refrigeration. The remaining fungi were maintained on potato dextrose agar. Host Plants The following varieties of economic plants obtained from FerryMorse Seed Co., Detroit, Michigan, were used in this study: Golden Cross Bantam corn National Pickling cucumber Michelite bean Marglobe tomato1 1 Marglobe variety of tomato was used in initial screening trials, but was replaced with Bonny Best tomato in all later runs and physiological studies 8 TABLE I. Test organisms and common names of diseases they produce. Organism Alternaria solani (Ell. and G. Martin) L , R . Jones and Grout4 ............. ...... Disease Early Blight of Tomato Colletotrichum lagenarium (Pers.) E. and H.3 .... Cucumber Anthracnose C. lindemuthianum (Sacc. and Magn.) Briosi and Cav. alpha strain1............. Bean Anthracnose C. phomoides (Sacc.) Chester2.................. Tomato Anthracnose G1omer ella cingulata (S tan.) Spauld . and Schrenk4 ......... ............ Bitter Rot of Apple Helminthosporium sativum (Pam.) King and Bakke4 ........... .......... . Spot Blotch of Barley H. carbonum Ullstrup4 .......................... Leaf Spot of Corn Phoma lingam (Tode and Fr.) Desm.4 ............. Blackleg of Crucifers Puccinia graminis tritici Eriks, and E. Hemm.4 .. Stem Rust of 'Wheat P . sorghi Schw.4 ..... ......................... Corn Rust Fungal organisms in the form of cultures or spores were obtained from the following faculty members of the Department of Botany and Plant Pathology at Michigan State University, East Lansing, Michigan: xDr. 2Dr. 3Dr. 4Dr. A. E. D. R. Andersen S. Beneke J. de Zeeuw L. Kiesling. 9 Bonny Best tomato Scarlet Globe radish Mandarin Chinese cabbage Marion Market cabbage Method s Employed Studies of the Various Test Compounds vivo screening of test compounds Spores of test organisms were suspended in distilled water, sprayed by atomizer onto the foliage of their respective hosts, and allowed to dry for U5 minutes. Test compounds were then applied by atomizer to rows of inoculated plants until the point of run-off (UO). Cardboard strips set upright between rows protected plants on either side from drift or accidental spray contamination. The plants were then kept in a moist chamber for three days and removed to a bench. Readings were taken after lesions appeared on the untreated inoculated checks and were based on a numerical gradation of 1 to 10; 1 indicating no visible symptoms, and 10 indicating death of the plant. At least seven repli­ cations were used in each experiment. Cabbage plants 3~U weeks old were used in the initial trials involv­ ing P. lingam. Since it was necessary to remove the bloom from this type of cabbage by rubbing the leaves before inoculation, subsequent trials were run using Chinese cabbage which was susceptible to this test organism and free from bloom. Beans in the first to third tri- foliate-leaf stage were used in the C. lindemuthianum trials; and corn in the it-5 leaf stage, in the P_. sorghi trials. A. solani and C. phomoides on tomato proved unsuccessful as test organisms due to failure to obtain constant and uniform infection. 10 KunkelTs method (31) of increasing sporulation in culture by scraping the surface of the culture and using fluorescent light was employed (52). Spore production was greatly increased, but infection either because of the strain or because of the conditions, was not materially increased. Marglobe variety of tomato was replaced with a more susceptible variety, Bonny Best (UO), with little or no increase of infection. Therefore, these fungal organisms were dropped from the screening program. In initial trials all compounds were applied at 200 ppm, with the exception of Acti-dione and Panogen 15* These latter compounds served as checks, and concentrations of 3 ppm a-nd bM ppm respectively were employed unless otherwise stated. 2. Phytotoxicity Phytotoxicity tests were run for all compounds tested at 200 ppm with the exception of Acti-dione and Panogen 15, see above. HC1 was tested further at 100, 50, and 25 ppm. Diaphine Compounds were applied as foliar sprays. 3. Experiments on the systemic activity of several antifungal compounds in the control of _C. lind emuthianum on bean a. Via detached leaf culture This study was designed to evaluate the materials under test for systemic action (25,37>?6). Acti-dione, Panogen 15, hexa­ chlorobenzene and the four Omadine salts were tested. Mature and near mature leaflets were cut from bean plants, immediately dipped into a spore suspension of £. lindemuthianum, and allowed to dry for a period of five minutes. They were then placed petiole 11 down in glass jars (l*^* inches in diameter by 3 inches tall) con­ taining a 10 percent sucrose solution to which one of the antifungal compounds had been added. Acti-dione was tested at 1 ppm, Panogen 15 at 0.11 ppm and the other compounds at 5 ppm. of each treatment was run. Eight replications Control runs of inoculated and uninocu­ lated leaves were placed in a 10 percent sucrose solution with eosine. The jars were covered with Petri dish tops and placed under continuous light. Readings were taken after 5 days and based on numerical gradation of 1 to 10 as previously defined. b . Via hydroponics Test compounds were added to Hoagland’s nutrient solution (16, Table B, p. 119) as follows: Acti-dione at 3 ppm* Panogen 15 at 0.11 ppm, hexachlorobenzene at 5 ppm and the four Omadine salts at 5 ppm respectively. An untreated nutrient check was also run. Pint jars covered with brown wrapping paper and dipped in paraffin served as containers. Bean seedlings germinated in paper toweling were placed in the jars and supported by l/U inch wire mesh screen cut into squares and fitted over the jar tops. Each test compound was replicated five times. Distilled water was added as needed to maintain the solution level and prevent concentration of salts. After four days the plants were inoculated with a spore suspension of C. lindemuthianum and left in the inoculation chamber for two days, then removed to a greenhouse bench. previously described. Readings were taken as 12 Studies of Sodium Omadine 1. Spore germination tests1 The glass slide technique with the test tube dilution method was employed as described by the American Phytopathological Society, Committee on Standardization of Fungicidal Tests (2,3). as the suspending medium for the spores. Distilled water was used CL phomoides, CL lagenarium, H. carbonum and P. sorghi were used as the test organisms and provided a wide range of difference of cell size and wall thickness. 2. Stability of sodium Omadine on bean foliage Sodium Omadine at 200 ppm was sprayed on 12 bean plants to test its stability on foliage without washing. Sprayed plants were held for two hours, two days and four days at 85° to 90° F daytime temperature. Leaf punches (33) from treated and untreated plants were bioassayed together with filter-paper discs which were run as bioassay controls.2 3. Growth response of Lemna minor L . to sodium Omadine (2?) Microorganism-free stock cultures of L. minor were obtained as follows: roots were removed and rosettes pulled apart into separate fronds by use of fine forceps. The fronds were wrapped in several folds of cheesecloth and weighed down in a large beaker, then washed with running tap water for 12 hours . After a momentary dipping in mild sodium hypochlorite solution* followed by rinsing in sterile distilled 1The spore germination studies were conducted jointly with Dr. Samuel M. Ringel, former Graduate Research Assistant of the Botany and Plant Pathology Department, Michigan State University, East Lansing, Michigan. 2 This procedure is discussed under section entitled "Filter-paper disc bioassay technique,” page 2Lp 13 water, each frond was placed in a separate bottle containing sterile liquid medium (U3, Table A, p. 119 )* The bottles were laid at an angle under continuous light in an air conditioned culture room with controlled temperature of 22° C . Forty-eight ml of liquid medium were poured in culture bottles and sterilized via autoclave for 20 minutes at 19 pounds pressure. Two ml of sterile distilled water were added to each of seven bottles which served as checks. Four concentrations of sodium Omadine were sterilized via sintered-glass filter, and two ml of each concentration were added aseptically to each of seven bottles. 9, 1, 0.9, and 0.1 ppm. to each bottle. The final concentrations were Rosettes of L. minor were transferred immediately Experimental transfers for each experiment were accomp­ lished by placing microorganism-free rosettes of four fronds in individual bottles. All transfers for a given experiment came from one stock flask. Wet weight determinations were made after a growth period of four weeks . A second experiment was run using concentrations of 0.1, 0.09, 0.01, and 0.009 ppm sodium Omadine. A six-week growth response study was also made with sodium Omadine at 0.09 ppm. harvested every week for U. six weeks. Controls and treatments were Seven replications were used. Translocation studies of sodium Omadine a. Via hydroponics Bean plants in the fifth trifoliate-leaf stage were placed in HoaglandTs nutrient solution (16, Table B, p. ppm and 29 ppm sodium Omadine. 119) containing 9 The plants were placed under continuous light for four days at 78° F, after which they were u removed, washed and the secondary roots and leaves cut from the stem. Nine root and stem sections l/8 inch in length were cut from each of* the plants as follows: the first at root tip, the next two at l/2 inch intervals up the root, and the last six at 1 inch intervals, The sections were placed on bioassay plates with the cut side down. The plates were incubated for [4.8 hours and examined for antifungal activity by the presence of zones of inhibition. b . Via bioassay of guttation and stem exudate from plants grown in treated sand Tomato, corn and cucumber seedlings respectively were grown in sand in six-inch pot saucers. The seedlings were allowed to wilt slightly, then one drench application of sodium Omadine at 50 ppm was applied until an excess of liquid was in evidence at the surface of the sand. Care was taken not to splash any compound on the seedlings. of control plants. Distilled water drench was applied to saucers Bell-jars were placed over the pot saucers, and water of guttation was collected with a syringe and blunt needle. Collections were made each evening for three successive evenings following treatment. Each collection was bulked as to treatment and control. The cotyledons were cut from the stems of cucumber seedlings four days after treatment. The sap which was exuded at the cut surface of each stem was collected 12 hours after cutting. The water of guttation and stem exudate was immediately examined for biological activity by means of bioassay. Sodium Omadine at 15 concent-rati ons or 1, 5 and 25 ppm in distilled water and in guttation water were used as bioassay checks. To determine the extent of* the activity present in the sand of the treated pot saucers, the tomato seedlings were removed from their saucers but the bell-jars were left covering the saucers. No water was added to these saucers, After sixteen days the moisture from the treated pot saucers was examined by bioassay to determine the presence of biological activity. c. Via bioassay of split-stern sections of tomato seedlings grown in treated sand Tomato seedlings were grown in treated sand in pot saucers as above. After three days several plants from both control and treated groups were cut off at the soil line, the leaves removed, and the stems split lengthwise and plated cut-side down on bioassay plates. d. Via bioassay of expressed juice from tomato and cucumber seed­ lings grown in treated sand Cucumber and tomato seedlings were grown in sand to which sodium Omadine was applied as a drench. The aerial portions of the plants were removed, washed and blotted dry three days following treatment. Stems and leaves of both tomato and cucumber seedlings were ground in a mortar and the juice expressed through cheesecloth. Water-drenched plants served as controls. Each sample was examined for biological activity by means of bioassay. 16 e. Via bioassay of expressed juice from foliar treated plants Foliar applications of 100 and 200 ppm sodium Omadine re­ spectively were sprayed on two lots of 3-inch high tomato seedlings grown in sand. The sand was flushed from the roots of the plants 12 hours after treatment, and the plants were washed in running tap water followed by a distilled water rinse and then blotted dry. The plants were then separated into roots, stems and leaves. Each of these separations was ground in a mortar and the juice expressed through cheesecloth. Each lot of juice was immediately bioassayed against G. cingulata, with juice expressed from untreated plants serving as controls. The leaves of two lots of radish seedlings were sprayed with 100 ppm and 200 ppm sodium Omadine respectively, and a set of untreated seedlings served as controls. This experi­ ment -was performed in the same manner as the experiment just described, with the exception that juice was expressed from roots, hypocotyls and leaves. 5- Physiological studies on the influence of sodium Omadine on host plants a. Ultraviolet spectrophotometric determination Cucumber seedlings grown in sand in pot saucers were treated with a root drench of 50 ppm sodium Omadine. Water of guttation from treated plants was collected after each of four succeeding 12-hour periods. Samples from the untreated control plants were collected after the first and the last periods only. under refrigeration. All samples were stored The samples were diluted 1 to b with 17 glass-distilled water and the ultraviolet absorption spectra for each sample determined by use of a Beckman Model DU spectrophotometer. A glass-distilled water solution of sodium Omadine at U ppm was prepared and the ultraviolet absorption spectra obtained. Three other experiments were run similarly, except that water of guttation was collected from both treated and untreated seed­ lings at the end of each 12-hour period for seven periods, encompass­ ing 8U hours from the time of original drench application. b . Free and combined amino acid studies of treated plants Foliage of Chinese cabbage, tomato, cucumber and bean plants was sprayed with one application of sodium Omadine at 200 ppm. After 20 hours the foliage from treated and untreated check plants was removed, washed, and the free amino acid samples extracted and examined by means of paper chromatography for quantitative difi ferences. Combined amino acid samples were extracted from tomato and cucumber plants and similarly examined. Cucumber and bean seedlings were grown in pot saucers and treated with one drench application of sodium Omadine at 100 ppm. After three days the foliage was harvested and free amino acid samples extracted and examined as above. c . Reducing sugar studies of treated plants 2 Somogyi’s test 1 Paper chromatography: for reducing sugars was run on cucumber, . procedures and techniques, section 2, page 25. 2 Test for reducing sugars, section 3, page 27* 18 tomato, bean and Chinese cabbage, all of which had been treated with one foliar spray of sodium Omadine at 200 ppm. was removed for sample extraction 21+ The foliage hours after treatment. These same types of plants were also grown in six-inch pot saucers in sand to which one application of sodium Omadine at 50 ppm had been added. The foliage was removed for sample extraction after four days . Concentrations of D-glucose at 0.05 to 1 percent were examined by this method and plotted on logarithmic scale graph paper. The sample readings of reducing sugars were thus measured in equivalent amounts of D-glucose. 6. Inactivation of sodium Omadine a . Guttation water Guttation water from corn and cucumber seedlings was examined to determine if any sodium Omadine inactivation properties were present. One part sodium Omadine was added to four parts of both boiled and unboiled guttation water from corn and cucumber re­ spectively. The final concentrations of the compound were 1, 5* 12.5* 25* and 50 ppm in each trial. The solutions were allowed to stand 0 and 12 hours, then bioassayed against G. cingulata. Distilled water concentrations of sodium Omadine were used as checks. b . Expressed juice of plant parts The leaves and hypocotyls of three-week old radish seedlings were harvested separately, washed, blotted dry, ground in a mortar 19 and the juices of each expressed through cheesecloth. Each lot of juice was separated into 2 ml samples and placed in capped vials. One-half ml amounts of sodium Omadine at five concentrations were pipetted into the samples of leaf and hypocotyl juice respectively. The final solutions of juice contained 5, 10, 25, 50 and 100 ppm sodium Omadine. These vials of treated juice were allowed to stand for 12 hours at room temperature and then bioassayed. Samples of leaf and hypocotyl juice containing concentrations of 10 and 50 ppm of the inhibitor as well as distilled water checks were bioassayed immediately after preparation. The leaves and stems of three-week old tomato seedlings were harvested separately, washed, blotted dry, ground in a mortar and the juices expressed through cheesecloth. The juices were separated into eight 2 ml samples and prepared and bioasaayed similarly to the previously discussed experiment on radish juice. Radish leaves and hypocotyls were harvested, washed, blotted dry and ground separately in a mortar. The juice was expressed through cheesecloth and divided into samples of 0.9 mm each. Four samples each from leaves and hypocotyls were boiled for 5 minutes in a water bath, removed and allowed to cool. One-tenth ml of sodium Omadine at two concentrations was added to each of the boiled and unboiled samples so that the final concentrations were 50 ppm and 100 ppm. trols. Distilled water was added to other samples as con­ After 2 hours the samples were bioassayed, and the unboiled samples were examined for differences in free amino acids by means 20 of chromatography 1 and reducing sugars via Somogyi's method. 2 Leaves from cucumber and tomato seedlings were harvested and treated as just described for radish. c. D-Glucose An aqueous solution of sodium Omadine at 100 pg/ml and D-glucose at 225 mg/ml was prepared. The solution was bioassayed after stand- at room temperature for 10 days. Separate solutions of sodium Omadine and D-glucose were used as bioassay controls. Studies Concerning Mode of Action of the Omadine Compounds 1. Purification and investigation of chemical composition a. Studies of technical grade sodium Omadine by paper chromatography Four-tenths ml samples of sodium Omadine at 100 p.g/ml in glassdistilled water were chromato grained . Glass-distilled water satu­ rated n-butanol was employed as the solvent. of 6.7 and the sodium Omadine a pH of 6.5. The solvent had a pH Descending chromatography with a development time of 15 hours was employed. The resulting 3 spots on developed chromatograms, visible under fluorescent light were outlined in pencil. Discs 11 mm in diameter were cut from the center of the outlined spots, and bioassayed. Vaper chromatography: procedures and techniques, section 2, page 25. 2 Test for reducing sugars, section 3* page 27. Sodium Omadine was detected on paper chromatograms as a "quench" spot under fluorescent light at quantities of 10 pg or greater. 3 21 An t-obiograms of "the above chroma to grams were made as follows: the edges of two 18 inch square glass panes were covered with Scotch masking tape. One-quarter inch wide cardboard strips were inserted under the masking tape along the edges of the glass on one side of each pane. Potato dextrose agar seeded with spores of G. cingulata was poured onto the surface of one of the glass plates. The chromatogram strips were placed on the surface of the solidified agar. The second glass pane was positioned over the first, and the outline of the chromatogram strips and the pencil outlines of the ,Tquench1' spots on the chromatograms were outlined on the surface of the upper glass pane. The chromatogram strips were removed after 5 minutes and the upper glass pane sealed to the first with masking tape. The assay plate was allowed to stand for 36 hours at room temperature. The outlines of the zones of inhibi­ tion were marked on the surface of the upper glass pane and also on the respective chromatograms. b . Purification All lots of the Omadine compounds were supplied as technical grade compounds. Attempts to obtain chemically pure compounds from the manufacturer were unsuccessful. Communications from Squibb Institute for Medical Research (62) and Olin Mathieson Chemical Corporation (U5) stated that the sodium salt was insoluble in the common organic solvents, and that it could contain as much as two moles of water of crystallization depending upon its method of 22 preparation. However, if a solvent were found in which sodium Omadine were soluble at room temperature or slightly higher, purifi­ cation of the inhibitor could be readily accomplished by recrysta'llization. Therefore, over thirty organic solvents were used in an attempt to find one in which sodium Omadine was soluble. None was found; however, it was noted that in several solvents there was a color change of the solvent while the sodium Omadine appeared to remain unchanged. It was hypothesized that impurities were soluble and could therefore be removed by several successive exposures to fresh solvent followed by filtration. Iso-propyl alcohol, tertiary- amyl alcohol, and secondary-butyl alcohol were selected as solvents. Technical grade sodium Omadine Id. JIP-8Cr-113U was exposed to four washings of each of these solvents respectively. After the final filtration, the filtrates were dried for 12 hours under vacuum. Melting points of the technical grade compound and the compound as extracted by each of the three solvents were determined. The ultra­ violet absorption spectra for 10 pg/ml of the compound from each extraction method was determined and compared to that presented in a brochure by the Squibb Institute for Medical Research (62). The extracted compounds were also bioassayed and chromatogramed, and compared to technical grade sodium Omadine. c . Chemical tests The sodium azide:iodine reaction (5), the sodium nitroprusside test (66), the platinic iodide test (75), and the bromine 23 reaction (6U) were used to determine the presence of -SS and -SH groups of technical grade and "purified" sodium Omadine in paper chromatograms. Omadine disulfide, methionine and cysteine were run as checks for all chemical tests employed. 2. Location of the action of the Omadine compounds Spores of G. cingulata were suspended in sterile glass-distilled water, centrifuged and resuspended in sterile glass-distilled water five successive times. Washed spores were standardized so that there would be 600,000 spores per ml. Seven 2 ml samples of the spore suspension were pipetted into separate test tubes. Disulfide, sodium, ferric and copper Omadine solutions, as well as sodium Omadine:cupric chloride, and cupric chloride solutions were prepared at 2 ppm in sterile glass-dis­ tilled water. Two ml samples of each of the above solutions were added to the 2 ml samples of spore suspensions. The final spore suspension was 300,000 spores per ml, and the final concentrations of metal salts and Omadine compounds were all 1 ppm. check was also used. A sterile glass-distilled water The spore suspensions were allowed to stand for 12 hours and 2h hours, after which they were centrifuged and resuspended in U ml sterile glass-distilled water five successive times. One drop of the final spore suspensions was deposited on a Petri plate containing potato dextrose agar. After three days the plates were examined for growth. In a second experiment, spores were washed as before and standardized so that there were 1,000,000 spores/ml. The final spore suspension in 2L the treatments was 500,000 spores/ml. Ferric chloride, zinc chloride, manganous sulfate and cupric chloride were added respectively to a sterile glass-distilled water solution of sodium Omadine. The final solutions contained 2 ppm of the metal salt plus 2 ppm sodium Omadine. Check solutions of sodium Omadine and each of the four metal salts respectively were prepared at 2 ppm. also included. A glass-distilled water check was Drops of the final spore suspensions were placed on Petri plates containing potato dextrose agar as before. The remaining spore suspensions were centrifuged and the spores resuspended in Ij ml of 2 percent sucrose. One drop of the spore suspension in sucrose was deposited on microscope slides which were inverted and suspended in a moist chamber for 2U hours, after which time a spore germination count was made. Additional techniqu.es related to this study 1. Filter-paper disc bioassay technique The filter-paper technique proved excellent for assay of water soluble inhibitors, but was of little value for assay of insoluble com­ pounds (30). Many modifications of this technique have been published (19,32,35,^5,70). follows: The method employed throughout this study is as potato dextrose agar was melted and allowed to cool to approxi­ mately U2° C, after which it was seeded with spores of G. cingulata and poured into Petri dishes. Each Petri dish contained about 10 ml of media spread thinly over the bottom of the dish. Discs 11 mm in diameter were cut from large sheets of Whatman filter paper No. 1 by means of a cork 25 borer. The discs were saturated with samples ho be examined and the excess removed by momentary blotting on clean filter paper by means of forceps. After two days zones of inhibition (clear zones) were measured in mm from the edges of the discs to the rim of growth. 2. Paper chromatography: procedures and techniques a. Extraction and preparation of samples Free amino acids were extracted by a modification of the method of Dent et al. (13)* Composite samples of fresh leaves were pul­ verized in a blender with sufficient 95 percent ethanol so that the final concentration of ethanol was approximately 80 percent. The mash was evaporated to dryness in a 60° C oven, ground in a mortar, passed through a fine wire mesh screen, placed in a capped bottle and stored at room temperature. Two ml of 80 percent ethanol were added to every 10 mgm of dried sample. This was placed in a refrig­ erator for 2U hours, and shaken at intervals. The insoluble material was removed by filtration and washed with 80 percent ethanol (0.5 ml per 10 mgm original dried material). The ethanol extracts were bulked and 3 volumes of chloroform added to each volume of ethanol extract. After shaking, the resulting aqueous layer was removed and stored under refrigeration (U)■ The extraction with ethanol diminished interference by substances such as inorganic ions and certain proteins (26), while chloroform extraction further removed many fatty materials. The chloroform remaining in the aqueous layer aided in preserving the amino acids and sugars present (63). 26 Combined amino acids were extracted by the method employed by Hrushovetz (26). The residue was treated with the ethanol and chloroform extraction method as employed for free amino acids. The resulting aqueous layer containing the amino acid hydrolysates was exposed to 6N NH4OH vapors under a bell jar, until pH 7 was reached. The removal of inorganic ions and fatty substances via these methods was excellent. When placed in a Reco Electric Desalter, Model R1500, samples repeatedly drew less than 0.2 amperes at 30 volts. This is as good or better ion extraction than could be obtained by use of such an electrolytic desalting apparatus alone. b . Application of samples Samples were applied to Whatman N o . 1 filter paper strips and sheets via a multiple spotting apparatus designed and built by the writer (28, pp. 115-17). Sample volumes of 0.1-0.2 ml of free amino acids and 0 .05-0.1 ml of combined amino acids were spotted. c . Further techniques The one-dimensional descending technique (5) proved expedient, with n-butanol : acetic acid : water = 250:60:250 v/v/v, the solvent. The chromatograms were developed by spraying with 0.25 percent w/v ninhydrin in acetone. To fully develop the color, the sprayed chromatograms were dried in a hood then placed in a moist atmosphere at 90° C 10 minutes. 27 Semi-quantitative determinations of samples from treated and untreated plants were made by use of a Photovolt Densitometer Model U25 (5). 3. Test for reducing sugars The samples were prepared using the methods of extraction employed in preparation of samples for paper chromatography. Two ml of Somogyi’s reagent (60) were added to 2 ml of the sample and the mixture placed in a boiling water bath for 10 minutes. After cooling, 2 ml of Nelson’s reagent (U2) were added and the resultant mixture shaken and allowed to stand for 20 minutes. The sample was then brought up to 100 ml with glass-distilled water and read in a Klett-Summerson Photoelectric Colorimeter, Model 800-3, using a blue filter No. 1±2. U. Precipitate test for aqueous solutions of sodium Omadine In a brochure from the Squibb Institute for Medical Research (62), it was stated that the water-insoluble Omadine salts such as the copper or the zinc salt may be prepared by the treatment of an aqueous solution of the sodium salt with an aqueous solution of a water-soluble metal salt. Since the resulting reaction caused a precipitate to form, it was reasoned that this could be employed as a test for sodium Omadine. It was found that the resulting water-insoluble salts if present at concen­ trations of 100 pg/ml or above readily formed a visible precipitate. If present at lower concentrations, the precipitate settled out upon standing, and as low as 10 pg were detected by filtration since the precipitate imparted a slight discoloration to the filter paper. 28 No other Omadine derivatives reacted with these water-soluble metal salts. This test was employed for aqueous solutions of sodium Omadine. CHAPTER III RESULTS Studies of the Various Test Compounds 1. In vivo screening of test compounds Thiolutin, diaphine HC1, rimocidin sulfate, fungistatin XG and nystatin failed to control P_. lingam on cabbage, and A. solani and C_. phomoides on tomato (55)- Sodium Omadine, Panogen 15 and Acti-dione gave excellent control of all three diseases at concentrations of U.U and 3 ppm respectively. 200, In the P. lingam trials (Table II), hexa- chlorobenzene, Panogen 15 and zinc, manganese, copper and sodium Omadine provided a high degree of control at the two levels of concentration employed (Plates I-II). In experiments 1 and U the Omadine treatments and Panogen 15 gave significantly better control in relation to Actidione. Hexachlorobenzene gave significantly better control than Acti- dione in experiments 1, 2 and 3« Panogen 15. and copper and sodium Omadine gave better control than hexachlorobenzene on Chinese cabbage in experiments 3 and 5 . Highly significant differences in control were obtained in experi­ ments S y 6 and 7 between treated and untreated beans inoculated with C. lindemuthianum, except with the Acti-dione treatment in experiment 7 (Table III). Panogen 15 and sodium, copper and zinc Omadine treatments provided highly significant control in comparison to Acti-dione in 29 30 TABLE II. Influence of various antifungal materials on disease incidence of Phoma lingam. Experiment No Concentration of active ingredients Chinese Cabbage H 10 8.00 8 .00 7-36 7 .6 3 ppm U .oo U .86 5.5o U.38 --- 200 PPm 3.00 3.1U 3.88 3.75 3.a 100 ppm ---- ---- ---- ---- 3.9 h.h ppm 2.71 2.86 3.13 2.75 3.1 2 .2 ppm ---- ---- ---- ---- a .5 200 ppm 2 .? ! 2.143 2 .50 3.38 3.1 100 ppm ---- ---- ---- a .6 200 ppm 2.57 2 .29 2.75 3 .3 100 ppm ---- ---- ---- ---- a .7 200 ppm 2.U3 2.57 3.25 2 .63 2.7 100 ppm ---- ---- ---- ---- 3*5 200 ppm 2.29 2.U3 2.63 2.6 3 2.9 100 PPm ---- ---- ---- ---- 3.5 .05 0.7U 0.72 0.39 0.7U 0.55 .01 0.99 0.96 0.53 0.98 0.73 Hexachlor ob enz ene Panogen 1 5 ' Zinc Omadine Manganese Omadine Copper Omadine Sodium Omadine rc 5.86 Acti-dione L.S.D. Cabbage 1 o o Check Treatment Means "Methyl mercury dicyandiamide at U.U ppm is roughly equal to 3 ppm °T mercury. PLATE I. Spray trials for control of Phoma lingam on Chinese cabbage (experiment 10). Left: untreated check. Center: Right: Left: Center: Right: Left: Center: Right: treated with hexachlorobenzene at 200 ppm. treated with hexachlorobenzene at 100 ppm. untreated check. treated with Panogen 15 at h ppm. treated with Panogen 15 at 2.2 ppm. untreated check. treated with zinc Omadine at 200 ppm. treated with zinc Omadine at 100 ppm. PLATE I 31 PLATE II. Spray trials for control of Phoma lingam on Chinese cabbage (experiment 10). Left: Center: Right: Left: Center: Right: Left: Center: Right: untreated check. treated with manganese Omadine at 200 ppm. treated with manganese Omadine at 100 ppm. untreated check. treated with copper Omadine at 200 ppm. treated with copper Omadine at 100 ppm. untreated check. treated with sodium Omadine at 200 ppm. treated with sodium Omadine at 100 ppm. PLATE II 32 33 TABLE III. Influence of various antifungal materials on disease Incidence of Colletotrichuin 1indemuthianum on beans . Concentration of active ingredients Experiment N o . Check Acti -dione Hexachlorobenzene Panogen 13 11 8.25 7 .00 9.00 6.5 3 ppm 2.88 5.86 8.00 --- 200 ppm 3.25 5.15 3.29 3.1 100 ppm ---- --- ---- 3.3 3.3 PPm 2 .50 3.15 1.57 1.3 --- 2 .2 ppm 200 ppm 2.25 1.86 100 ppm --- --- 200 ppm 3.13 3.13 3.29 5.5 100 ppm ---- ---- ---- 5.9 200 ppm 2 .00 1.57 3.29 5.3 100 ppm ---- ---- ---- 5.6 200 ppm 2 .00 1.86 3.71 3.0 100 ppm ---- ---- ---- 3.9 .05 1.36 1.00 2 .08 0.51 .01 1.93 1.33 2 .76 0.67 Zinc Omadine Manganese Omadine Copper Omadine Sodium Omadine L.S.D. £ Treatment Means 6 7 1.3 3.13 3.5 5.5 3b experiments 6 and 7 (Plates III—VI) . Copper, sodium and zinc Omadine gave better control than hexachlorobenzene In experiment 5. Experiments 5 and 6 (Table III) were completed during the months of* January and Februa.ry during which time the greenhouse maintained an even temperature of about 22 C. Experiment 7 was completed during late March: and 11, in April when the greenhouse temperatures rose appreciably during the daylight hours (Table III). Control of C_. lindemuthianum was reduced at the higher temperatures during experiments 7 and 11 (Table III) for all treatments except Panogen 15- Panogen 15 gave significantly better control than other treatments in experiment 7 and highly significant control In relation to other treatments in experiment 11. Results in the first P. sorghi trial (Table IV, exp. 8) were erratic with hexachlorobenzene and zinc Omadine giving no control in this experi­ ment, but all other treatments gave good control. In the second trial (Table IV, exp. 9)* the treatment means for Acti-dione, hexachlorobenzene, Panogen 15 and zinc, manganese, copper and sodium Omadine showed a high degree of significance over the inoculated untreated checks. All of the other compounds in this experiment were better than Acti-dione. 2. Phytotoxicity Thiolutin at 200 ppm when applied as foliar sprays produced epinasty and bronzing of the leaves of tomato plants, but only a slight mottling on bean plants (55)- Foliar sprays of fungistatin XG caused epinasty of tomato, leaf crinkle on cabbage, slight leaf puckering on beans, and chlorotic spots on cucumber leaves (55). Foliar sprays of diaphine HC1 PLATE III. Spray trials for control of Colletotrichum lindemuthianum on bean (experiment 7)* Left: Right: Left: Right: untreated check. treated with Acti-dione at 3 ppm. untreated check. treated with hexachlorobenzene at 200 ppm. PLATE III PLATE IV. Spray trials for control of Colletotrichum lindemuthianuin on bean (experiment 7)■ Left: Right: Left: Right: untreated check. treated with Panogen 15> at U-U ppm. untreated check. treated with zinc Omadine at 200 ppm. PLATE IV PLATE V Spray trials for control of Colletotrichum lindemuthianum on bean (experiment 7)* Left: Right: Left: Right: untreated check. treated with manganese Omadine at 200 ppm. untreated check. treated with copper Omadine at 200 ppm. PLATE V PLATE VI. Spray trials for control of Colletotrichum lindemuthianum on bean (experiment 7). Left: Right: untreated check. treated with sodium Omadine at 200 ppm. Reading from left to right: untreated check, treated with Acti-dione at 3 ppm, Panogen 15 at U.U ppm and hexachloro­ benzene at 200 ppm. PLATE VI 39 TABLE IV„ Influence of various antifungal materials on disease incidence of Puccinia sorghi on corn. Concentration of active ingredients Experiment N o . Check Treatment Means 8 9 5.00 3.50 3 ppm 2 .71 2 .70 Hexachlor ob enz ene 200 ppm U.U3 1.90 Panogen 15 h-h ppm 2.57 1.80 Zinc Omadine 200 ppm U .29 2 .00 Manganese Omadine 200 ppm 2.86 1.80 Copper Omadine 200 ppm 2.U3 1.60 Sodium Omadine 200 ppm 2 .00 1.90 Acti-dione L.S.D. .05 .01 ho produced a toxic reaction on bean, cucumber (Plate VII), tomato and cabbage, but not on corn. This toxic reaction generally consisted of chlorotic spots or chlorotic leaf margins on leaves which were immature at time of application, Diaphine HC1 produced chlorotic rings on cabbage which were similar in appearance to certain virus symptoms. In most cases, however, the host plant recovered, and any new growth that appeared was either free of injury or only slightly injured. At lower concentrations of 100, 50 and 25 ppm, diaphine HC1 still produced marked chlorosis on cucumber and beans, but a marked reduction in phytotoxicity was exhibited at lower concentrations on cabbage and tomato (55)• Rimocidin sulfate applied as foliar sprays at 200 ppm caused slight puckering on bean and cucumber leaves, but the damage was not severe (55) • Acti-dione when applied as a foliar spray at 3 and 5 ppm caused necrotic spots on the leaves of tomato, cucumber, bean and cabbage plants; however, phytotoxicity has been reported with this material (18). Foliar sprays of Panogen 15 and Nystatin were not phytotoxic at U-U and 200 ppm respectively, but hexachlorobenzene at 200 ppm caused necrotic spots on foliage. At 200 and 100 ppm, copper Omadine foliar sprays were more phytotoxic than zinc, manganese and sodium Omadine. Single foliage applications of copper Omadine at 200 and 100 ppm on Chinese cabbage caused appreciable plant stunting, while zinc, manganese and sodium Omadine at 200 ppm produced only slight stunting and leaf puckering (Plate VII). Single foliage applications of the four Omadine salts at 200 ppm were not phytotoxic to corn and beans. Wherever phytotoxic reactions were caused by foliar sprays of Omadine salts, the plants recovered quickly with new growth appearing normal. PLATE VII. Phytotoxicity caused by one foliar spray of diaphine HC1 at 200 ppm on cucumber. Left: untreated check. Right: treated plant showing chlorosis. Phytotoxicity caused by one foliar spray of diaphine HC1 and sodium Omadine at 200 ppm on bean. Left: Center: Right: untreated check. sodium Omadine treated plant showing puckering. diaphine HC1 treated plant showing chlorosis. PLATE VII U2 Seedlings grown in hydroponic solutions containing Omadine salts at 5 ppm exhibited root stunting and little secondary root formation. Root stunting was severe in 0.11 ppm Panogen 15 > but less severe in 5 ppm hexachlorobenzene, while the plants in 3 ppm Acti-dione solution died within three days. After 8 days, the young leaves of plants in the hexachlorobenzene solutions showed necrotic brown spots. Tomato and cucumber seedlings grown in sand were harvested several days after application of 50 ppm sodium Omadine as a root drench. The roots were stunted, discolored and generally severely injured (Plate X). Roots severely injured were not recovering from this injury, but new roots were being regenerated up the stem at or abov^ the soil line. 3- Experiments on the systemic activity of several antifungal compounds in the control of C_. lindemuthianum on bean In the detached leaf culture experiment, C_. lindemuthianum inocu­ lated bean leaflets were placed in jars with the petioles extending into a nutrient solution containing one of the antifungal compounds. Hexa­ chlorobenzene, Panogen 15 and zinc, copper and sodium Omadine showed a high degree of significance over inoculated checks, while manganese Omadine was significantly better (Table V). Fungal and bacterial contaminates were a problem in this experiment, and the Acti-dione treatment was discarded because of excessive contamination. Leaflets in control jars containing eosine showed that the dye was translocated throughout the xylem system of the blade inside of 2b hours. It was therefore assumed that the antifungal compounds were also translocated throughout the xylem system in this period of time. U3 TABLE V. Influence of various antifungal materials via systemic action on disease incidence of Colletotrichum lindemuthianum on bean leaflets in detached leaf culture. Concentration of active ingredients Check Treatment Means 8.63 Hexachlor ob enz ene 5 ppm 5.13 0.11 ppm 3.88 Zinc Omadine 5 ppm U-50 Manganese Omadine 5 ppm 6.63 Copper Omadine 5 ppm 5.75 Sodium Omadine 5 ppm 5.75 Panogen 15 L.S.D. .05 1.63 .01 2.18 In the hydroponic experiment in which beans were grown in solutions containing test compounds and then inoculated, the disease incidence was erratic. However, sufficient infection occurred to determine that foliage infection by C_. lindemuthianum was not controlled by an;y systemic activity of Panogen 15* Acti-dione, hexachlorobenzene, or the Omadine salts. Studies of Sodium Omadine 1. Spore germination tests Dosage response (DR) curves for C. lagenarium, C. phomoides and H. carbonum under the influence of sodium Omadine are presented in Graph I. Uh GRAPH I Dosage response curves of the spores of three fungal organisms under influence of sodium Omadine at several concentrations. 100 ED ED - 50 Colletotrichum Percent Inhibition' olletotrichum phomoides 20 Helminthospori carbonum .001 .005 .01 Sodium Omadine ppm Logarithmic' three by three inch cycles as The three DR curves are linear in nature, with those for the Colletotri ­ chum species closely parallel, being similar in slope and position. i The ED SO values for C_. lagenarium, C_. phomoides and H. carbonum were 0,007* 0.011 and 0.08 ppm sodium Omadine respectively; and total inhibi­ tion occurred at 0.05, 0.0S and 0.10 ppm respectively. Deformities of germ tubes below ED SO values were noted in several cases. The most noticeable of these was a swelling or vesicle occasionally produced at the tip of germ tubes of H. carbonum (Plate VIII) at 0.0S ppm of the compound. 2 . Stability of sodium Omadine on bean foliage Leaf discs, punched from bean leaves 2 hours after being sprayed with sodium Omadine at 200 ppm, developed an inhibition zone 2.SS nim wide when bioassayed. Leaf discs removed U8 hours after spraying developed inhibition zones 1.1} mm wide and those removed 96 hours after spraying showed an inhibition zone 0.275 run wide. 3. Growth response of L_. minor to sodium Omadine Data from the two experiments using sodium Omadine at concentrations of 5 to 0.1 ppm and 0.1 to 0.005 ppm sodium Omadine are presented in Graph II. These data represent a growth period of four weeks and are presented as percent change from control on a wet weightbasis. The -9h percent as given in Graph II for the concentration0.1 ppmis an 2 average of two experiments at this concentration. The lethal threshold 1 The effective dose for 50 percent spore inhibition is hereinafter re­ ferred to as ED 50, often termed the lethal dose (LD) by some authors (lU). 2 The values for the sodium Omadine concentration 0.1 ppm in the two experiments were -93-5 percent and -9U-2 percent respectively. PLATE VIII. Spore germination test. The effect of sodium Omadine on spores of Helminthosporium carbonum. Twenty-four hour incubation at 25° C. 1. Control (magnified 525 diameters). 2. 0.05 Ppm sodium Omadine (magnified 1,000 diameters). Note the swelling or vesicle at the end of the germ tube. 3. 0.1 ppm sodium Omadine (magnified 1,000 diameters). U. 1.0 ppm sodium Omadine (magnified 525 diameters). PLATE VIII h7 GRAPH II Influence of sodium Omadine at seven concentrations on the growth of Lemna minor given in percent change from control on wet weight basis (after 1+ weeks) ppm sodium Omadine (Control) o$ 5 1 0.5 0.1 0.05 -25$ 0.01 0.005 l I I -50$ I i i -75$ I -75% 1 (Death)-100$ I i -91% for growth of L. minor under these conditions was between 0.5 and 0.1 ppm sodium Omadine. Of significance is the fact that 0.005 ppm of the compound caused a marked retardation in growth. At all sub-lethal concentrations there was a considerable decrease in frond size as com­ pared to the controls. A comparison between untreated checks and 0.05 ppm sodium Omadine at the end of four weeks growth is seen in Plate IX. The influence of 0.05 ppm sodium Omadine upon L_. minor over a six week growth period is very striking (Graph III). The lag period of growth in the treatments has been considerably extended. The plants appeared to have recovered from the influence of the treatments at the end of the fifth week. Frond color and size appeared more normal at the end of the sixth week. h* Translocation studies of sodium Omadine Bean plants placed in Hoagland’s solution containing 5 PPm and 25 ppm sodium Omadine were removed after four days and stem and root sections cut out and bioassayed. A measurable amount of activity was found in stem sections two inches from the root tip in those plants grown in solutions containing 25 ppm, but only 0.5 inches from the root tip in solutions 5 ppm. solutions. The roots extended approximately 1.5 inches into the However, it should be noted that these roots were slightly discolored and less turgid than roots of the control plants, indicating injury and perhaps death of the roots. The results of bioassay experiments on guttation water from corn, cucumber and tomato seedlings grown in treated sand, indicate that the compound was not present in the active state in the guttation water of H ctH P h EH a PH CD 50 GRAPH III Influence of 0.05 ppm sodium Omadine on the growth of Lemna minor. 110 100 Average Wet Weight per Culture in mgin 90 80 70 60 Control 5o hO 30 Treatment 20 10 0 6 Time in Weeks these plants at concentrations above 1 ppm. Due to the lack of bio — logical activity, results of these experiments are not presented in tabular form. A bioassay of the sap from the cut ends of cucumber seedlings was made simultaneously. No activity was demonstrated in the sap from treated plants. However, it was noted that considerable bacterial growth was present in the bioassay discs containing exudate from untreated plants, while the discs containing exudate from treated plants were free from bacterial growth. No activity was demonstrated in the split-stem bioassay of tomato seedlings grown in treated sand. Data from bioassay experiments on expressed sap from cucumber, tomato and bean seedlings grown in treated and non-treated sand indicated that the compound was not present in the stems or leaves in the active state at concentrations above 1 ppm. Table VI presents the results of the bioassay of sand moisture 16 days after application of seedlings were grown. ^0 ppm sodium Omadine to sand in which tomato "When compared to the bioassay of aqueous solutions of sodium Omadine (Table VII), the sand moisture demonstrated a bio­ logical activity equal to 2f> ppm sodium Omadine. The juice expressed from tomato seedlings which had been sprayed 12 hours previously with 100 and 200 ppm sodium Omadine respectively was bioassayed against G. cingulata (Table VIII). The results indicate the presence of 1—2 ppm of the active compound in the leaves and stems, but not in the roots of tomato seedlings. Table IX summarizes results of The limitations of the bioassay technique were such that quantities of the inhibitor were undetectable below 1 ppm. 52 TABLE VI. Bioassay of moisture from sodium Omadine treated and non­ treated sand in which tomato seedlings were grown? sixteen days after drench treatment. Inhibition zone in mm from edge of discs Average of 5 bioassay discs Moisture from sodium Omadine treated sand Moisture from non-treated sand Plate 1 11.25 0 Plate 2 10.75 0 Plate 3 10.50 0 Over-all average 10.83 0 Table VII. Bioassay of distilled water dilutions of sodium Omadine. Inhibition zone in mm from edge of discs Average of 8 bioassay discs Concentrations: 5 PP^ 10 ppm 25 ppm 50 PPm 75 PPjf 100 ppm 6.33 8.12 10.75 13.75 16.75 18.67 TABLE VIII. Bioassay experiments on juice expressed from roots, stems and leaves of tomato seedlings sprayed with sodium Omadine. Inhibition zone in mm from edge of discs Average of 2h bioassay discs Roots Stems Leaves Foliage spray 200 ppm sodium Omadine 0 h .2 1 3.33 Foliage spray 100 ppm sodium Omadine 0 3.21 2.56 Untreated Control 0 0 2.52 TABLE IX. Bioassay experiments on juice expressed from roots, hypocotyls and leaves of radish seedlings sprayed with sodium Omadine. Inhibition zone in mm from edge of discs Average of 36 bioassay discs Roots Hypocotyls Leaves Foliage spray 200 ppm sodium Omadine l.?8 2.86 1.53 Foliage spray 100 ppm sodium Omadine t* 1.85 0.90 0.U2 1.75 1-50 Untreated Control t - slight clearing, but not measurable. 5/4 bioassays of juice expressed from the roofs, hypocotyls and leaves of radish seedlings sprayed with sodium Omadine 12 hours previous to bioassaying. The results indicate the presence of approximately 1 ppm of the active compound in the roots and hypocotyls when the foliage had been sprayed with 200 ppm of the compound . No activity was observed from the 100 ppm treated plants. No activity was found in the leaves from the 200 ppm treated plants. 5. Physiological studies on the influence of sodium Omadine on host plants a. Ultraviolet spectrophotometric determination The ultraviolet absorption spectra of water of guttation from cucumber seedlings grown in treated and untreated sand are presented in Graphs IV and V , The absorption spectra for the two samples from treated seedlings are more variable than the checks, and their curves are considerably below those of the checks (Graph IV). Three later experiments were completed in which water of guttation from treated and untreated cucumber seedlings was collected at 12-hour Intervals. The absorption spectra curves for the samples from treated plants 12 to U8 hours after treatment were considerably below those of the untreated control plants. However, 60 to 8U hours after treatment the absorption spectra curves approached those from the untreated control plants (Graph V). Ultraviolet spectro­ photometric determinations of various aqueous dilutions of guttation water from treated plants and control plants proved that the major differences between guttation samples from treated plants versus .0 GRAPH IV. Ultraviolet absorption spectra for guttation water from 50 ppm sodium Omadine drench and non­ treated cucumber seedlings grown in greenhouse temperatures of 68-72° F. .8 .6 .a .2 First check Optical Density Second check 1.0 .8 - 12 hour treated .6 2h hour treated .36 hour treated .a a8 hour treated .2 0 205 225 265 um ( A) 285 305 325 ■g-a -P 0 CM A1ISN3Q IV O Ild O O 0 0 It 3o G o ■H & g — -P o c a lb ro CO or a: £ coo O hDO CM ro CO -pid -P 0 vO P 0)fH o w o CO z CL cr CO ID cd e> 01 (T 01 -o o LlJ h“ CO ro CM or i- o Ld CO z LlI I— _l 01 Ll O o CM LU < CM CM LO -O CD CM CO CM A1ISN3Q IVOIldO CM CM 57 untreated control plants were differences in concentration. Substances in the guttation water from treated plants were not present in as great a concentration as in the untreated control plants. Guttation samples from treated and control plants were analyzed for amino acids and reducing sugars. Ninhydrin failed to give a color reaction to a spot on filter paper to which a one ml sample of guttation water had been concentrated. Further tests showed that no amino acids were found in guttation water from both treated and control plants. Somogyi *s test for reducing sugars in guttation water proved negative. An interesting temperature phenomenon was observed in these studies. The plants in the first experiments were grown in the greenhouse in temperatures of 68° to 70° F. Several later experi­ ments were run when greenhouse temperatures ranged from 76° F at night to 90° F in the day. When the ultraviolet absorption spectra was determined for samples of guttation from plants grown in higher temperatures, it was found that the absorption spectra curves for the treated and control plants were identical. Furthermore^ the guttation samples were examined undiluted3 and the character and slope of these latter curves (Graph VI) were entirely different from the absorption spectra curves of guttation water from plants grown in lower temperatures. The nature of this difference due to temperature was not examined. The quantity of guttation water produced by the seedlings at these higher temperatures was many 56 GRAPH VI Ultraviolet absorption spectra for undiluted guttation water from 50 ppm sodium Omadine, and water drench treated cucumber seedlings grown in greenhouse temperatures of 70-90 C. 1.6 • h Optical Density 1.2 1.0 .8 .6 .2 0 285 225 urn ( A ) 59 times that produced at lower temperatures. It was also found that addition of fertilizer to the sand slightly altered the slope and character of the curve, but this alteration, due to a different level of fertility, was negligible. It was noticed that less water of guttation was collected from treated seedlings than the untreated controls. Therefore, pot saucers containing an equal number of cucumber seedlings were treated with a water drench and a drench application of sodium Omadine at concentrations of 50, 100 and 200 ppm respectively. The following volumes of water of guttation were collected 2h hours later: 0.8 ml from the water check, and 0.5 ml, 0.25 ml and 0.15 ml respectively from the treatments. The volumes of guttation water collected were inversely proportional to dosage of sodium Omadine. Pot saucers each containing eleven cucumber seedlings were treated I4 days apart with two drench applications of distilled water and sodium Omadine at 50, 100 and 200 ppm respectively. The pot saucers were placed in the greenhouse at temperatures of 55 to 65° F. Four days after the second treatment, the sand was flushed from the roots of the seedlings. The roots of the plants of all treatments (Plate X) were yellowed and stunted, with the 200 ppm treatment showing the greatest damage. Twisting and curl­ ing of cotyledons of seedlings from the 200 ppm treatment were also noted. PLATE X . Cucumber seedlings grown in pot saucers in sand to which two drench treatments h days apart of sodium Omadine were applied. Photographed 8 days after the first treatment. Upper left: water-drench control. Upper right: 50 ppm sodium Omadine. Lower left: 100 ppm sodium Omadine. Lower right: 200 ppm sodium Omadine. 61 b. Free and combined amino acid studies of treated plants Semi—quantitative determinations of free and combined amino acids of treated plants were made via paper chromatography. No differences in amino acids were observed between plants treated with foliar applications of sodium Omadine and untreated control plants. Also, no differences in free amino acids were observed between plants treated with root drench applications of sodium Omadine and water drench control plants. c. Reducing sugar studies of treated plants Data from various concentrations of D-glucose were determined via the Somogyi method and read in a Klett-Summerson photoelectric colorimeter. These data, plotted on Graph VII, served as a standard by which unknown quantities of reducing sugars could be estimated in equivalent quantities of D-glucose. The colorimetric readings of extracts of control and treated plants as determined via the Somogyi method could not be directly evaluated, since the color change was a logarithmic function of the quantity of reducing sugars present in the extracts. By converting the colorimetric readings to equivalent amounts of D-glucose, quantitative differences between the reducing sugars of the treated plant extracts and control plant extracts were expressed in percent change from controls (Table X). The amounts of D-glucose equivalent to reducing sugars in the extracts from the foliar* treated cucumber, bean, Chinese cabbage and tomato plants were all below those of the untreated control 62 GRAPH VII Concentrations of 0.05 - 1% D-glucose as determined via the Somogyi method and read in a Klett-Summerson Photoelectric Colorimeter. 220 210 200 190 Colorimetric Scale 180 170 160 150 lUO 130 120 110 100 .02 i .5 Percent D-Glucose Solutions Semi-logarithmic: 3 cycles x 10 divisions 1 63 Reducing sugar determinations of extracts from control and sodium Omadine treated plants as ascertained via the Somogyi method. Foliar spray treated plants harvested 2I4 hours after 200 ppm sodium Omadine treatment. Root drench treated plants harvested U days after application of 50 ppm sodium Omadine, Determinations are based on an average of eight plants per treatment. TABLE X, fOt CO 1 CO •H p P i rH P oj O oi cir pi CCDO jp f t ! P i 1—1w 0 0 O P ft £ 0 i p!oj P P 0 ft 0 CD P i 0 bO P CD P x> P i1 0 CD CD f o i £ p 0 CD CO 1—I t CD P i Pi cxJ Oj ft ft ft ft ft > O CD bO sd p; o •H -oPj o O C \J Pi O ■H P cd •oH rH ft ft f t (ft c— <— 1 CD CO P p ct3 Pi CD 1— 1 O — oj uj O P ft f t ft ft P CD Pi f t CD O CO — 0 0 0 P Pi p i > •H — bO 0 1 ft4 E f t < *i f t 1} 1 f t O *H f t O P ft CD CO CD b O P rH £ Oj O •H *H CD O P 1 1 P O CD O O ft ft O On O 1— 1 << XO! Pi P o p o o CXJ CXJ 1— 1 ft f t ft is O ft CD Pi CO -|.-3 O CO Pi *H CD CD P O pi 1— 1 & CD O 1i P 0 HD r— 1 cd Pi •H 0 CO !-1 P ft CO O CXI PA O I— i— 1 CXi 1— I PA -ft 0 ■LA 0 OX CA O O O 0 0 O 0 O O IA 1— 1 HD CO 1— 1 A 1 f t A rH r— 1— ft O CO I— 1 P A O P A r— i • • ft• HD CA O • P CO Pi PP P f t P — oj Pi Pi Pi 1— 1 A CO rH 1— 1 1— 1 ft (D bD a} O ( D CO CO -P ft iH P o* pi o s CD ft CD i •PH X ! o CD bO oj ft o p oj 6 o ft p CD ■P8 o opi Pi oj CD ft 3O CD (O CD Pi S o o p o Eh 6h plants. The reverse of this was observed for the root-drench treated plants. The leaves of cucumber, bean, Chinese cabbage and tomato plants treated with one foliar application of 200 ppm sodium Omadine from which extracts were made after 2U hours, contained 27} 50, 53 and 22 percent less equivalent amounts of D-glucose re­ spectively than the controls. The leaves of the same variety of plants treated with one drench application of 50 PPm sodium Omadine from which extracts were made after H days contained 87, 33, 13, and 78 percent more equivalent amounts of D-glucose respectively than the controls. 6. Inactivation of sodium Omadine Boiled and unboiled guttation water from corn and cucumber seedlings when bioassayed at 0 and 12 hours showed no inactivation of sodium Omadine. The activity of sodium Omadine was reduced when combined with juice extracted from untreated radish and tomato seedlings. Concentrations of 5 to 100 ppm sodium Omadine in sap expressed from radish leaves and hypocotyls were bioassayed immediately and after incubating for 12 hours. All concentrations in leaf sap below 10 ppm at 0 and 12 hours showed total inactivation (Table XI) . The activity of the 100 ppm solutions were greatly reduced at the end of the 13“hour period. The juice from the hypocotyl did not inactivate the compound as completely as that from the leaf. In similar experiments with sap from tomato leaf and stem tissues (Table XII) biological activity was reduced. However, 65 TABLE XI. Bioassay experiments on sodium Omadine treated extract expressed from radish seedlings. Inhibition zone in mm from edge of discs Average of 12 bioassay discs Plated Immediately Check 10 ppm 50 ppm 5 ppm Plated after 12 hours 10 ppm 25 PPm 50 ppm 100 ppm Sap from leaves 1.63 1.1+2 1.1+2 1.75 1.73 1.5U 1.63 5.00 Sap from hypocotyl 1.00 1.75 3-33 1.25 2.25 1-33 1.58 1+.08 Bioassay experiments on sodium Omadine treated extract expressed from tomato seedlings. TABLE XII. Inhibition zone in mm from edge of discs Average of 12 bioassay discs Plated Immediately Check 10 ppm 50 ppm 5^ppm Plated after 12 hours 10. ppm 25 ppm 50 ppm 100 ppm Sap from leaves 5.50 5.58 6.25 5.U2 5.83 6.58 6.17 5. 6 7 Sap from stem 3.75 U.83 5.75 3.67 3.75 14.75 U-75 5.08 the inactivation was not as complete as the inactivation by the sap from radish. Antifungal activity of sodium Omadine at 50 and 100 ppm in boiled extracts from radish leaves and hypocotyls and tomato leaves was not reduced. The data for this experiment are now shown. There were no differences of reducing sugars or free amino acids between distilled 66 water:expressed juice and 100 ppm sodium Omadine: expressed juice from radish, cucumber and tomato leaves after a two hour incubation period. An aqueous solution of sodium Omadine at 100 jig/ml and D-glucose at 225> mg/ml when bioassayed against G. cingulata after an incubation period of 10 days, gave an inhibition zone the same size as the sodium Omadine control (Plate XI). a 2 mm inhibition zone. 100 }ig/ml The D-glucose control solution gave No reduction in inhibitory properties of sodium Omadine by the D-glucose was observed. Studies Concerning Mode of Action of the Omadine Compounds 1. Purification and investigation of chemical composition a. Studies of technical grade sodium Omadine by paper chroma­ tography Chromatograms of technical grade sodium Omadine, lot number Id. JYP-8Cr-113U yielded three inhibitory areas when bioassayed (Figure I). an On fifteen replications the first area, or spot, had value of 0 .0 9 9 ? the second, 0 .6 8 7 ? and the third, 0.887. The first spot had an elongated "tail" below it which disappeared when 19 and 20 p.g samples were chromatogramed. Discs from the center of these three spots, including one from the tail of the first spot, gave the following comparative inhibition zones when bioassayed; the first spot produced the greatest zone of inhibi­ tion; the tail from the first spot produced the second largest; the second and third spots were about equal in inhibition and slightly less than the tail from the first spot (Plate XII). The autobiograph of the technical grade compound, Figure II, shows PLATE XI Bioassay plates of sodium Omadine at 100 pg/ml alone and in combination with D-glucose at 229 mg/ml. Solutions bioassayed after standing at room temperature for 10 days. Upper four bioassay discs: sodium Omadine. Lower four bioassay discs: sodium Omadine plus D-glucose. 68 FIGURE I Chromatograms of UO p.g of sodium and disulfide Omadine showing quench-spots as observed under fluorescent light Sodium Omadine Omadine Disulfide 1 Rf 0.099 Q Rf 0.68? Rf 0.887 Solvent Front O O o Rf 0.690 PLATE XII Bioassay from fluorescent "quench" spots of chromatograms of technical grade sodium Omadine., developed in water-saturated n-butanol. The discs in the two bioassay plates are from corresponding spots from two different chromatograms. Upper row left: Upper row right: Lower row left: Lower row right: third "quench” spot. second "quench" spot. tail from first "quench" spot. first "quench" spot. FIGURE II Autobiographs of I4.O pg of t-echnical grade and purified sodium Omadine. Technical Grade #Id. JYP-8Cr--113U Inhibition zones Purified inhibition zonss around these active spots and gives a clearer picture as to relative quantities of inhibitor present. The first spot was eleuted with glass distilled water and rechromatogramed. Three active spots with the same Rf values as before were obtained. The second spot when treated similarly yielded mainly the second spot and a smaller third spot, and a very small quantity of the first spot. The third spot when rechroma­ togramed yielded only the third spot. Further aqueous eleutions from all three spots were given the precipitate test for the presence of sodium Omadine, to which the first spot gave a positive reaction, and the second and third a negative reaction. Omadine disulfide when chroma to grained gave a biologically active "quench" spot which had the same R^* value as the second spot from chromatograms of sodium Omadine (Figure I) . Eleutions of the 1 second spot were co-chromatogramed with Omadine disulfide and resulted in a large active spot in the number two position, and slight spots in the first and third positions. When disulfide and sodium Omadine were co-chromatogramed, spots one and three were normal, but the second spot was very large. This is further proof that Omadine disulfide and the substance causing the second spot on chromatograms of sodium Omadine are identical, both being the Omadine disulfide derivative. The eleuted material and the solution of Omadine disulfide were applied to the same spot and chromatogramed together. 72 b. Purification The melting point (MP) of technical grade sodium Omadine, Id. JYP-8Cr-113U, was 21U-2l8° C. if led The MP for sodium Omadine fractions in tertiary—amyl, secondary—butyl and iso—propyl alcohol were 250-253° C, 25l-25U° C, and 2?0-27U° C respectively. Bioassay experiments of l.i ppm of "purified" fractions in tertiary-amyl alcohol gave an average inhibition zone of 9*7 mm, while average inhibition zones from secondary-butyl alcohol and iso-propyl alcohol were 9.85 mm and 8.88 mm respectively. Technical grade sodium Omadine at U ppm gave average inhibition zones of 8,25 mm. The "purified" fraction from iso-propyl alcohol was discarded since it showed the least amount in gain of biological, activity, and since its MP was higher than the MP of the chemically pure compound (U5)The ultraviolet absorption spectra for 10 pg/ml of sodium Omadine "purified" in tertiary-amyl and secondary-butyl alcohol and technical grade sodium Omadine respectively were determined (Graph VIII) . Absorption spectra curves from sodium Omadine "purified" from the alcohols showed it to be at higher concentrations than the technical grade sodium Omadine. The absorption spectra curves for sodium Omadine fractions "purified" from the alcohols show excellent similarities to the ultraviolet absorption spectra curve for chemically pure sodium Omadine as presented in a brochure from the Squibb Institute for Medical Research (62). The absorption The chemically pure compound was reported to have a MP of 252 25U U5). C 73 GRAPH VIII Ultraviolet absorption spectra oi* sodium Omadine samples at 10 pg/ml. 1.6 Optical Density Fraction purified in secondary-butyl alcohol Fraction purified in tertlary-amyl alcohol Technical grade Id. JYP-8Cr-lllU 1.0 20£ 22^ 2U£ 26£ (A ) 303 325 3U0 lb spectra curve for "the fraction "purified” in second ary-butyl alcohol was slightly higher than that for tertiary—amyl alcohol, and there­ fore, the former was selected for all further use whenever a "purified” sample of sodium Omadine was required. Chromatograms of the "purified” sodium Omadine gave the same three biologically active spots as the technical grade. However, autobiographs for chromatograms from the "purified” compound yielded greater activity in the first spot and less activity in spots two and three when compared to autobiographs for chromatograms from the technical compound (Figure II). c . Chemical tests Chemical tests for the presence of -SS and -SH groups in chromatograms of technical grade and "purified” sodium Omadine did not indicate the presence of either sulfur group. Methionine and cysteine gave positive reactions in each of the four tests, however, Omadine disulfide, whose chemical structure is known to contain an -SS group (1*5,62) did not give a positive reaction. 2. Location of the action of the Omadine compounds Plates XIII through XVI show the growth of spores of G. cingulata which had been washed, brought in contact with solutions of several Omadine derivatives and combinations of sodium Omadine plus water-soluble metal salts and appropriate controls, rewashed, and tested for viability on culture plates. Plates XIII and XIV show the growth of spores exposed 75 to "the following treatments: water check; sodium, copper, ferric and disulfide Omadine derivatives; sodium Omadine plus cupric chloride; and cupric chloride check. All treatments were present at 1 ppm. Results of growth of spores subjected to a 12 hour treatment is presented in Plate XIII. Good growth was present from spores from the water check and sodium Omadine treatment; spores from the cupric chloride and ferric and disulfide Omadine treatments gave moderate growth; the copper Omadine treatment resulted in less growth; and the sodium Omadine:cupric chloride treatment greatly reduced the viability of the spores. Growth of spores subjected to a 2h hour treatment (Plate XIV) corroborates and amplifies these results. Good growth is noted from spores in the water check and somewhat less in the sodium Omadine treatment; fair growth from the Omadine disulfide treatment; less growth from the cupric chloride treatment; poor growth from the copper and ferric Omadine treatments; and greatly reduced growth from the sodium Omadine:cupric chloride treatment. Growth of spores after 2U hour exposures to each of cupric chloride, zinc chloride, manganous sulfate and ferric chloride alone and in combi­ nation with sodium Omadine; and sodium Omadine and water checks is presented in Plates XV and XVI. Good growth was present from spores in the water check and sodium Omadine treatment. in all other treatments. Less growth was present The sodium Omadine:cupric chloride treatment produced the greatest reduction in spore viability with very little growth in evidence from this treatment. Cupric chloride alone reduced spore viability more than did the sodium Omadine, but not nearly as 76 .much as did the two compounds together. The combination of zinc chloride and sodium Omadine resulted in less dense growth than either the zinc chloride sodium Omadine alone. The ferric chloride: sodium Omadine treatment allowed much less growth than was allowed by either of the two compounds in separate treatments. The manganous sulfate:sodium Omadine treatment resulted in a less dense growth than either of the two compounds in separate treatments. After 2k hours in 2 percent sucrose, the percent inhibition of spores from the preceding experiment was determined (Table XIII). These data show that the sodium Omadine treatment reduced spore TABLE XIII. Percent inhibition" of spores of Glomerella cingulata after 2k hours in 2 percent sucrose, after prior 2k hour exposure to treatments. Compounds at 1 ppm Percent inhibition of single compound Percent inhibition of compound plus sodium Omadine at 1 ppm Sodium Omadine 18.h ---- Cupric chloride 52.0 89.0 Zinc chlorid e 29.5 UU.5 Ferric chloride 25.5 68.5 Manganous sulfate 15.0 U2.5 These data have been corrected to percentages of inhibition of the viable spores as determined from a distilled-water check. 77 germination by 18.5 percent. When cupric chloride, zinc chloride, ferric chloride or manganous sulfate was added to the sodium Omadine, a greater percentage of the spores were inhibited, especially noticeable was the reduction caused by cupric and ferric chloride. However, since each of the four metal salts caused a reduction in spore germination, in evalu­ ating the reduction of spore germination by a combination of sodium Omadine and one of the metal salts, the inhibition caused by sodium Omadine and that by the metal salt alone must be taken into consider­ ation. The 89 percent inhibition resulting from the sodium Omadine: cupric chloride treatment is greater than the additive effect of the two compounds alone, which would be ?0.5 percent. The UU.5 percent inhibition of the sodium Omadine:zinc chloride treatment is in all respects equal to the additive effect, i_i_8 percent, of the two compounds alone. The 68.5 percent inhibition of the sodium Omadine:ferric chloride treatment is greater than UI; percent, which would be the additive effect of the compounds alone. The U2 percent inhibition of the sodium Omadine: manganous sulfate treatment is also greater than 33*5 percent, the additive effect of the two compounds alone. To summarize, sodium Omadine alone did not cause a great amount of spore inhibition, but in combination with cupric chloride resulted in a multiple effect causing almost 90 percent inhibition. A striking multiple effect is also noted by ferric chloride: sodium Omadine, and to a lesser degree by manganous sulfate:sodium Omadine. •\X) CD X XI X -P CD CO XI cd p P to p o *P cd CD Is p X X CD P o P a CD CO cd O CD p r P Pi X p Pi E o o X X -P o cd P XI CM CD X 1 —) E o -P p XI o p P w •H o CD X P P Is CO -P CO cd X X !>> o P -P p cd p p x r—1 o CD P a §• E P o O o £ •P o P P » O P to E p cd p P o Pi I — 1 o o P Pi fd •H •H CO CO i CD p —1 P cd p CD CD -p -P Oh p P e o CO I— 1 1— I -p p Pi -p CO CD P CD p •S' CO CD p o p> P bO CD o O P P CO cd Pi to CD Pi CD - p CO CO P p o o P O rd o o P Pi CO -P p Pi X CO P i •H o CD -P o p X P CD -p X O E X bO P -P - P £ p P P o o X -P CD p p P o P CD pi P P h p CD • 1—1 1—1 1— 1 X w 1-3 P o E P •P X O to ■p p p CD • P CD X X •P P P E O O i —1 XI E o p *1—1 o •p X o p P •P X cd E O P CD Pi ph CD •H CO o rP O P -P P o o p CD -P P Is X CD i —1 i— 1 *P -P CO •ft X) •• -p p CD X pH p •• -p X CD bO X • p •P P p O o r— 1 p x o +=> cm o cd • p i —l p Pi E p o o P ph X P Is P o P CD P p •P CD X Pi cd ft, g E=> O CD P O o .. -p x • bO CD •P X P •P o -p P CO +=> •p P X CD i— i CD P E •P o XI P cd cp E O |3 o •-> P CD P P *P cd X Is cd o E X O PLATE XIII •H P r -» •S 03 P 6 O P {-H 5 03 P 6 o E5 p •rH 03 O CD 03 *\ CD dd dd p CO dd Cd sd P CO Is o • p cd CD !3 P 0 3 0 3 CD P a *\ O P CL) CO cd O 03 P a) dd CO St i—( & P h eT o o 0 3 si -P cd PJ s t C D dd dd CNJ s o P si -p o P cd H o CD •H dd |S p o cm CO P -p p p P sd •rH 03 cd 03 dd sd o CCS P cd p i cd 0 3 i— 1 o CD 5 tx! 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TO CD o Pi ft ft CO ft ft Pi o pi aj CD O ( —1 * ft CO X! co PI ft ft cd o o P p o ft r—1 1ST O P ft -ft P Pi TO O CD o Pi p •rH CD f t -ft aj aj £ Is O o o o aj (ft CO ft CD I— 1 —I •H aj ft Pi £ pj o o Pi f t ft Pi P! o o ft •H CO CO Pi CD CD - f t f t aj CO f t pi Pin CO Pi CD a j P i bO o aj ft CO CD CO ft o o P -ft f t K o CD Pi f t ft ft si ft bO t pi Pi pj O g , 1 ft * CO •H ft Ph h ft -ft aj -ft PI CD CO CD P Ph CO aj Is CO i Pi CD o £ •n CD *iH P O i— I ft O o •rH P ft P o *N CD P •rH f t g o £ pj •rH ft O CO ft £ P £ pi •rH ft O CO «< CD ft a j -ft -ft aj ai ft - f t CD Pi o P as P -, ft •rH P O i— 1 bD f t ! o *H P a O ■H ft P PF ft pj o ft CD i— 1 f t P is TO o CD p P p •rH CD f t f t TO Ph £ »• ft ft ft O »• ft ft bD •rH P O ft • ft CD ft CD f t i—1 ■H P Is O o p ft o p CD o Is p O •rH ft tH [ —1 PLATE XV !=* X! C£J E-h CM •N p X p p p X p P co P P p p CO Is o *p p P X X § p p p •> o p p CO p o p p rP P h X p PhE o o X p P o P cd P -0 P p CNJ E o P p P o p P w o P •p p p P Is P in CO cd X P !>* o P p p cd •3 P X p i— 1 o P 5: E 0 P o o o o p P •p • o P CO P E cd p P o P h i— i o o P P h i — l•p •p p p in m 1— 1 P p P p p E > o p i— 1 -p o p o p o CO p p p p p o o Ph o CO p o p p •p p p p p P PH P P CO rH p P hP CO P P P p CO P p bD p o P p Ph P h CO P m CO p o p o P Is p P O P X bD p s 0 o o p p o p X p p h W p X o p p p o pH CO p p p E P P p P P e P p X O P CO bD P cd i —I E o p TS -P P P cd o X *rH O •rH p o i —i ,oP o •rH p p p CD p £ P *H CD X P cd cd E .3 O p p •p X •CDo 06 OE 1—1 X ♦rH O P CO CO •P cd 1— I *1 I •T I X E P Xo 'v co CD X •rH *♦ P P o Jp I— I -P ,P bD bO Pl •H O o O •rH -P P •P P O P ^ P p P P P rH P p CD U7,62). Therefore, the results shown in Plates XIII and XIV for the commercially prepared salts were at concentrations lower than 1 ppm. Sodium Omadine reduced spore germination slightly. However, this is expected, since it is highly improbably that all excess metals were removed from the exterior of the spores. sodium and permeation could occur. These metal ions replaced the Also, it is possible that sodium Omadine molecules adhered to the surface of the spores, due to the 100 attraction from electric charges in the polar groups in the semipermeable membrane (6,69). If the spores come in contact with free metals, replacement of the sodium ion takes place, allowing permeation. When sodium Omadine was used in these studies, it was employed in tests with other water-soluble salts present, and substitution of metals undoubtedly occurred. When distilled water was employed, as differen­ tiated from glass-distilled water, sufficient metals were also present to allow substitution on the Omadine molecule. Horsfall (2l|) stated that most nitrogen compounds are inherently toxic, but that many do not display activity as fungicides unless they contain a "shaped charge" to make them permeable. He further stated (2U) that pyridine could be made into a fungicide by the addition of a styryl group in the number two position on the pyridine ring, or by a carboxy ester in the number three position. A fungicidal pyridine compound closely related to the Omadines is 3-pyridine thiol, the metal salts of which possess considerable fungitoxicity (6l). This study has shown that trace amounts of the Omadine derivatives produced remarkable growth retardation of L_. minor and inhibition of fungal spores. The inhibitory reaction produced in all cases far exceeded the inhibitory reaction of the water-soluble metal salts. Therefore, the hypothesis was made that once the Omadine molecule enters the cell, it undergoes reduction, leaving the pyridine ring, which is the toxic property. The zinc chloride:sodium Omadine treatment as previously discussed in the culture plate and spore germination tests, did not give a 101 reduction of viability below the additive effects of the two compounds alone. The fact that the zinc chloride caused some reduction in spore viability is evidence that zinc alone is toxic to these spores. It may be that spores of this fungus do not possess the ability to reduce the zinc chelate as quickly as other metal chelates, and therefore the toxic moiety of the compound is not released in sufficient quantities to cause the inhibition produced by other metal chelates. CHAPTER V SUMMARY 1. Acti-dione, Panogen 15, hexachlorobenzene and the Omadine salts of copper, zinc, manganese and sodium when applied as foliar sprays controlled P_. lingam on cabbage, C>. lindemuthianum on bean and P. sorghi on corn. The best overall control was obtained with sodium Omadine, copper Omadine and Panogen 15. 2 . Diaphine HC1 at a wide range of concentrations produced a marked chlorosis which on cabbage was similar in appearance to certain virus symptoms. Copper Omadine produced noticeable stunting on Chinese cabbage, while zinc, manganese and sodium Omadine produced only a slight stunting. Plants recovered quickly from any phytotoxic reactions produced by foliar applications of the Omadine compounds. The roots of cucumber seedlings grown in sand treated with sodium Omadine were stunted and discolored. They did not regenerate from their growing tips if injured severely, but new roots were formed near or above the soil line. 3. Concentrations of sodium Omadine necessary to produce total inhibition of the spores of lagenarium, C_. phomoides and H. carbonum in distilled water were 0.05, 0.05 and 0.10 ppm respectively. Vesicles or swellings were occasionally produced at the tips of the germ tubes of H. carbonum at concentrations of 0.05 ppm* 102 103 U . The lethal threshold for growth of L^. minor under the influence of sodium Omadine was between 0,5 and 0.1 ppm. Concentrations as low as 0.005 ppm produced a marked decrease in growth. 5. Sodium Omadine does not act as a systemic fungicide, and up to 50 ppm of the compound is inactivated immediately upon contact with extracts from radish and tomato. 6 . Leaves of plants treated with foliar sprays of sodium Omadine were found to contain less reducing sugars than untreated controls, while plants treated with root drenches of the compound had higher concentrations of reducing sugars than the controls. It was thought that the lower amount of reducing sugars in treated leaves was due to some change in ability of the treated leaves to produce a normal amount of sugars. The greater amount of reducing sugars in plants treated with root drench applications was explained on the basis of root damage, disruption of the translocation, and perhaps an altering of the processes by which soluble sugars are converted into starch. 7. The ultraviolet absorption spectra of guttation water of cucumber seedlings grown at temperatures of 68° to 70° F in sand treated with sodium Omadine indicated a lower amount of inorganic substances were probably present than from control plants. At higher temperatures these differences were not present, and the absorption spectra curves for control plants from both high and low ranges of temperatures were entirely different. 8 . The hypothesis was made that limited amounts of -SH groups were present in aqueous solutions of sodium Omadine, and that therefore lOli several chemical reactions characteristic of the mercaptans were possible. Circumstantial evidence was presented as the basis for the hypothesis, and evidence is presented to show that some of the reactions characteristic of mercaptans do take place. 9. Evidence was presented to the effect that in a solution contain­ ing water-soluble metal salts there is a replacement of the sodium ion attached to the Omadine molecule by the metal element in solution. 10. A second hypothesis was made that the Omadine salts act as chelators in which the preformed metal chelates, possessing a fat-water solubility balance, permeate the cellular membranes and preform a toxic reaction within the cell. 11. Evidence is given in support of this hypothesis. A third hypothesis was made that once the Omadine molecule enters the cell it undergoes reduction, leaving the pyridine ring which is the toxic property. of this hypothesis. Circumstantial evidence is presented in support LITERATURE CITED 1* Allison, Patricia and G. L. Barnes. 1956. Plant disease control by a new class of chemicals, 2—pyridinethiol—1—oxide and derivatives. (Abs.) Phytopath. 1+6; 1. 2. American Phytopathological Society. Committee on Standardization of Fungicidal Tests. 191+3* The slide germination method of evaluating protectant fungicides. Phytopath. 627-632. 3. American Phytopathological Society. Committee on Standardization of Fungicidal Tests. 191+7. Test tube dilution technique for use with slide germination method of evaluating protectant fungicides. Phytopath, 37.: 3514-356. 1+. Awapara, J. 191+8* Application of paper chromatography to the esti­ mation of free amino acids in tissues. Arch. Biochem. !£_; 172-173* 5. Block, R. J., E. L. Durrum and G. Zweig. 1955* A manual of paper chromatography and paper electrophoresis. Academic Press Inc., New York. 1+81+ p, illus . 6. Bonner, J. and Arthur ¥. Galston. 1952. Principlesof plant physiology. ¥. H. Freeman and Company, San Francisco. 1+99 V , illus. 7. Cation, D. 1953- Experiments with Actidione for control of cherry leaf spot (Coccomyces hiemalis). Phytopath. 1+3: 1+68, 8. Cohen, R. 1951* Four new fungicides for Coccidiodes immitis. 1. Sodium caprylate. 2. Ethyl vanillate. 3* Fradicidin. 1+. Thiolutin. Arch. Pediat. 68^ 259-261+* 9. Couch, Houston B. and Herbert Cole, Jr. 1956. Fivecompoundstested for control of Merion bluegrass rust. PI. Dis. Rept. 1+0: 103-105* 10. Couch, H. B. and H. Cole, Jr. 1957* Chemical control of meltingout of Kentucky bluegrass. PI. Dis. Rept. Uls 205-208. 11. Davisson, J. ¥., F. ¥. Tanner, Jr., A. C. Finlay and I. A. Solomons. 1951 * Rimocidin, a new antiobiotic. Antibiotics and Chemotheraphy 1: 289-290. 12. Degering, E. F. 1951* An outline of organic chemistry. Sixth Ed. Barnes and Noble, Inc., New York. 1+20 p. 105 106 13* Dent, C. E., W. Stepka and F. C, Steward. 1997* Detection of the l60e'68f683aCldS ce-^-^-s by partition chromatography. Nature 1)4. Dimond, A. E., J. G. Horsfall, J. W. Heuberger and E. M. Stoddard. 19U1. Role of the dosage-response curve in the evaluation of fungicides. Conn. Agr. Expt. Sta. Bui. 991. 15. Felber, Irma J. and C. L. Hamner. I9I48. Control of mildew on bean plants by means of an antibiotic. Bot. Gaz . 110: 329-329. 16. Gallegly, M. E. and J. C. Walker. 1999. Plant nutrition in relation to disease development. V. Am. Jour. Bot. 369 613-623. 17 • Gopalkrishnan, K. S. and J. A. Jump, 1992. The antibiotic activity of thiolutin in the chemotherapy of the Fusarium wilt of tomato . Phytopath. i|2: 338-339. 18. Gottlieb , D ., H. H. Hassan and M. B. Linn. plant protectant. Phytopath. 9<3: 218. 1990. Actidione as a 19. Gottlieb, D., Marvin Legator and Betty Bevan. 1991• A plate method of antibiotic assay with Mycobacterium tuberculosis 607 and Brucella abortus. Antibiotics and Chemotherapy T: 97-98. 20. Hamilton, J. M. and Michael Szkolnik. 1993- Factors involved in the performance of Cycloheximide (actidione) against Coccom.yces hi emails . (Abs.) Phytopath . 1+3: 109 . 21. Hazen, E. L. and R. Brown. 1991- Fungicidin, an antibiotic pro­ duced by a soil actinomycete. Proc. Soc. Exptl. Biol, and Med. 76: 93. 22. Heuberger, J. W. and P. L. Poulos. 1993. Control of fire blight and frog -eye leaf spot (black rot) diseases of apples in Delaware in 1992. PI. Dis. Rept. 3 J i 81-83. 23. Hobby, G. L., P. P. Regna, N. Dougherty and W. E. Stieg. 1999. The antifungal activity of antibiotic XG. Jour. Clin. Invest. 28: 927-933. 2I4. Horsfall, J. G. 1996. Principles of fungicidal action. Botanica Co., Waltham, Mass. 279 P* Chronica 29. Howard, F. L. 1939. The value of testing fungicides in the labora­ tory before use in the field. Proc. Am. Hort. Soc. 379 909-ul926. Hrushovetz, S. B. 1999 • The effect of infection by Helminthosporium sativum on the amino acid content of wheat roots. Can. Jour. Bot. 32: 971-979. 10? 27. Hutner, S. H. 1953. Discussion following: L. G. Nickell 1953. Antibiotics in the growth of plants. Antibiotics and Chemotherapy 23 U58 -U59 • 28. Kenaga, C. B. 1957* Multiple spotting apparatus for paper chroma­ tography. (Abs.) Phytopath. IjJ: 19. 29. Kenaga, C. B. and R. L. Kiesling. 1956. Studies on the effects of several salts of l-hydroxy-2(lH)-pynidinethione on several economic plants. Phytopath. \ \ J j 19. 30. Kenaga, C. B., R. L. Kiesling, S. M. Ringel and E. S. Beneke. 1955. Physiological studies and trial applications of antifungal materials for the control of plant diseases. Michigan State University (unpublished manuscript). 31. Kunkel, L. 0. 1918. A method of obtaining abundant sporulation in cultures of Macrosporium solani E. and M. Brooklyn Bot. Gard. Mem. I : 306-312. 32. Leben, C. 19U9 • The determination of antimycin on plant leaves. (Abs.) Phytopath. 39/ 1333- Leben, C. and G. W. Keitt. 19U9 • Laboratory and greenhouse studies of antimycin preparations as protectant fungicides. Phytopath. 39: 529-5UO/ 3U. Leukel, R. W. 1951- Cooperative seed-treatment tests on small grains in 1951. PI* Dis. Rept. 35: UU5-U51 • 35. Loo, Y. H., P. S. Skell, H. H. Thornberry, J. Ehrlich, J. M. McGuire, G. M. Savage and J. C. Sylvester. 19U5- Assay of streptomycin by the paper-disc plate method. Jour. Bact. 50: 701-709. 36. Malcolmson, Jean F. and R. Bonde. 1956. Studies in the control of bacterial and fungous decay of potato seed pieces. PI. Dis. Rept. U0 : 708-713. 37. Marsh, R. ¥. 1936. Notes on a technic for the laboratory evalu­ ation of protective fungicides. Trans. Brit. Myc. Soc. 20_: 305-309. 38. Martin, Hubert. 1953. Guide to the chemicals used in crop pro­ tection. Can. Dept. Agri., second Ed. 39. Mason, C. L. 19U8. A study of the fungicidal action of 8-quinoli.no1 and some of its derivatives. Phytopath. 38: 7UO-751* U0. McCallan, S. E. A. and R. H. Wellman. 19h3. A^ greenhouse method of evaluating fungicides by means of tomato foliage diseases. Contrib. Boyce Thompson Inst. 13/ 93~13U. 108 ill. Murneek, A. E. 1952. Thiolutin as a possible inhibitor of fire blight. Phytopath. 1+2: 57. 52. Nelson, N. 1955* A photometric adaptation of the Somogyi method for the determination of glucose. Jour. Biol. Chem. 153: 375-380. 53 * Nickell, L. G. and A. C. Finlay. 1955. Growth modifiers. Anti­ biotics and their effects on plant growth. Jour. Agri. and Food Chem. 2 : 178-182. 55* Norman, A. G. 1955• Some effects of pyridinethione on the growth of higher plants. Plant Physiology 30:supplement. Proceedings of the Plant Physiology Meetings, Sept. 5-8, 1955* 55* Olin Mathieson Chemical Corporation, Industrial Chemicals Division. 1956. Mathieson pyridinethione and derivatives. A brochure (personal communication). 56. Olin Mathieson Chemical Corporation, Baltimore, Md. 1956. Agricultural chemicals, pyridinethione, disulfide derivative. A technical data sheet (personal communication). 57- OlinMathieson Chemical Corporation, Baltimore, Md. 1956. Agri­ cultural chemicals, pyridinethione, ferric salt. Technical data sheet (personal communication). 58. Olin MathiesonChemical Corporation, Baltimore, Md . 1956. Agri­ cultural chemicals, pyridinethione, manganese salt. A technical data sheet (personal communication). 59. OlinMathieson Chemical Corporation, Baltimore, Md. 1956. Agricultural Chemicals, pyridinethione zinc salt. A technical data sheet (personal communication). ,50. Pansy, F. E., H. Stander, ¥. L. Koerber and R. Donovick. 1953* In vitro studies with 1-hydroxy-2(1H) pyridinethione. Proc. Soc. Exp. Biol. Med. 82_: 122-125. 51. Purdy, Laurence H. 1956. Organic chemicals containing chlorine as seed treatments for wheat smut control. (Abs.) Phytopath. 56 ' 23* 52. Rands, R. D. 1917. The production of spores of Alternaria solani in pure culture. Phytopath. J_' 316-317* 53. Ringel, Samuel Morris. 1956. In vitro studies of^Colletotrichum phomoides under the influence of sodium pyridinethione and other antifungal materials. A thesis for the degree of Ph. D. Michigan State University. 109 55* Rmgel, S. M. and^E. S. Beneke, 1956. The influence of certain sugars on the antifungal activity of sodium pyridinethione. Mycologia 58: 329-336. 55* Ringel, S. M., E. S. Beneke, C. Kenaga and R. L. Kiesling. 1955. Physiological studies and trial application of antifungal anti— biotics for the control of plant diseases. Michigan State University (unpublished manuscript). 56. Sander, Evamarie and Patricia Allison. 1956. Bioassay of the translocated fungicide, 2—pyridinethiol-1-oxide, in cucumber seedlings. (Abs.) Phytopath. 56: 25* 57* Shaw, E., J. Bernstein, K. Losee and ¥. A. Lott. 1950. Analogs of aspergillic acid. IV. Substituted 2-bromopyridine-N-oxides and their conversion to cyclic thiohydroxamic acids. Jour. Am. Chem. Soc. 72: 5362-5365. 58* Siang, W. N., and C. S. Holton. 1953. Mode of action of hexachlorobenzene on wheat bunt fungi in vitro. PI. Dis. Rept. 37 - 63-6559- Skiver, R. E. 1956. The use of antibiotics and fungicides in mist propagation. PI. Dis. Rept. 50: 1075-1080. 60. Somogyi, Michael. Chem. 195- 19-23. 1952. Notes on sugar determination. Jour. Biol. 61. Soo-Hoo, G. and E. Grunberg. 1950. The antifungal activity of metal derivatives of 3 -pyridine thi ol. Jour. Invest. Derma. l5_: 169-172 . 62. The Squibb Institute for Medical Research. New Brunswick, New jersey. 1955. 2-pyridinethiol, 1-oxide. A brochure (personal comrnunication) . 63. Steward, F. C., R. H. Wetmore, J. F. Thompson and J. P. Nitsch. 1955. A quantitative chromatographic study of nitrogenous com­ ponents of shoot apices. Am. Jour. Bot. 5l: 123-125* 65* Matsukawa, Tiazo and Shojiro Yurugi . 1953* On a new derivative of thiamine with cysteine. Science 118: 109-111- 65. Thornberrv. H. H. 1950. A paper-disk method for the quantitative evaluation of fungicides and bactericides. Phytopath. 51: 519-529* 66. Toennies, G. and J. J. Kolb. 1951- Techniques and reagents for paper chromatography. Anal. Chem. 23* 823-826. 67. Vaartaja, Olli. 1956. off of tree seedlings. Screening fungicides for controlling dampingPhytopath. 56_: 387-390. 110 68. Vaughn/ John R. 1951. against turf diseases. 69. Cycloheximide, an antibiotic effective (Abs.) Phytopath. 1+1: 3 6 . Veldstra, H. 19U1+. Researches on plant growth substances V. Relation between chemical structure and physiological activity II. Contemplations on place and mechanism of theaction of the growth substances. Enzymolgia 11: 97-136. 70. Vincent, J. G. and Helen W. Vincent. I9 I+U* Filter paper disc modification of the Oxford cup penicillin determination. Proc. Soc. Exp. Biol, and Med. 162-161+. 71. Wallen, V. R. and A. J. Skolko. 1950. Antibiotic XG (from Bacillus subtilus) as a seed treatment for the control of leaf and pod spot of peas caused by Ascochyta pisi. Can. Jour. Res. Sec. C. Bot. Sci. 28: 623-636. 72. Wallen, V. R., M. D, Sutton and A. J. Skolko. 1950. The effect of Actidione on the growth of certain pathogenic fungi and on the germination of seed. Phytopath. 1+0: 156-160. 73. Whiffen, Alma J. 191+8. The production, assay and antibiotic activity of Actidione, and antibiotic from Streptomyces griseus. Jour. Bact. 56: 283-291. 7l+. Whiffen, Alma J., N. Bohonos and R. L. Emerson. 19U6. The pro­ duction of an antifungal antibiotic by Streptomyces griseus. Jour. Bact. 52: 610-611. 75. Winegard, H. M. and G. Toennies. 191+8. Detection of sulfurcontaining amino acids on paper chromatograms. Sci. 108: 506-507. 76. Yarwood, C. E. 1937. Sulphur and rosin as downy mildew fungicides. Phytopath. 27 : 931-91+1. A P P E N D I X Ill Spotting Apparatus for Paner Chromatography1 Considerable variability in paper chromatography may result from improper technique in application of samples to the paper. This vari­ ability is often multiplied several-fold when sample volumes of l/lO ml or greater must be applied, for it necessitates repeated application of the sample in small and equal amounts to a particular point on the paper to build up the required volume.When multiple spotting must be done, in addition to being inaccurate, it becomes a time-consuming, and tedious chore. Sample factory for applicator-apparatus known to the author, appear very satis­ application of small volumes, but leave something to be desired when large volumes must be employed. Therefore, to circumvent this tedious task of multiple spotting of large volumes, a multiple spotting apparatus for paper chromatography was constructed (Plate A) which allows the simultaneous application of ten spots to various arrangements of filter papers held vertically on a separate adjustable drying rack. Up to 1 rah of material may be applied per spot from matched tuberculin syringes fitted with No. 26 needles filed flush with the shank. The syringes are filled with the sample, and clamped in a parallel series one and one-half inches apart across a narrow bar, which is fastened at right angles to a lower framework. An electric motor geared down to a few rpm and mounted within the framework rotates a long threaded driving screw, the forward end of which is mounted in 1The text of a paper presented at the forty-eight annual meeting of the American Phytopathological Society in conjunction with the Potato Association of America at Cincinnati, Ohio, in December, 1956. 112 ball-bearings below the cross-piece holding the syringes. A slide, mounted on aluminum right—angle pieces rides snuggly in a channel of* similar right—angle pieces fastened to the framework on either side of the driving screw. The slide rides above the driving screw and moves down the channel by means of a "split—nutTT held against the driving screw by a coil spring attached to the slide. At the forward end of the slide a metal crosspiece is mounted which contacts the syringe plungers. The motor turns the driving screw causing the split-nut- mounted slide to move forward, the slide cross-piece presses the syringe plungers resulting in the substance within each syringe to be exuded. The syringes are matched as to volume per given barrel length and there­ fore equal volumes are exuded from each syringe. The electricity supplied to the motor is first connected to a 60second-cycle percentage timer, which may be set to regulate the current supplied to the motor in various intervals of a minute, or in continuous operation. A heat gun is mounted on the basal piece of the adjustable spotting rack (Plate B) . A copper manifold attached to the drier runs just beneath and behind the paper or papers to be spotted. The filter paper sheets are fastened vertically at their upper and lower edges to adjust­ able glass—strips . For two-dimensional chromatograms, each filter—paper sheet is clipped vertically along one edge of the rack, folded back and fastened at its upper-corner to a heavy wire support which is attached to the verticle posts of the rack. The multiple -spotter is moved up to the drying rack to which the papers have been attached (Plate C). 113 The blunt noses of the syringe needles are placed against the papers 1/2 inch above the lower glass strip. Drying of the samples is facili­ tated by hot or cold air passing from the manifold and directed on the spots. With the heating element of the heat gun turned on, the temperature of the air stream at the point of sample application is between 36-38° C over the entire length of the manifold. Both the heat gun and the multiple-spotter are connected to an electric interval timer clock, which shuts off the apparatus after a predetermined length of time. By adjusting the percentage timer, the area of the applied spots may be accurately regulated (Plate D). A dilute solution of ink when applied to Whatman filter paper number 1 at a percentage timer setting of 5 percent produced sample spots of less than one-quarter inch in diameter. The sample spot diameters ranged from one-quarter inch up to three-quarters of an inch when a percentage timer setting of 100 percent (or continuous operation) was employed. Percentage timer settings of I4O percent through 100 percent resulted in sample spots which were slightly elongated due to the force of the heated air from the manifold. The size of the sample spots remained consistently even at all percentage timer settings. Calculations as to unit-time per unit volume for any percentage timer setting is easily computed. By means of a photoelectric densitometer, one—dimensional chromato­ grams of amino acid mixtures applied in equal volumes were read as to maximum density for each ninhydrin reacting spot. On 36 replications the densitometer showed little variation among spots of the same amino acids. Ilk With this apparatus large sample volumes may be employed; as many as ten sample spots may be applied to any size filter paper or papers, including one spot per sheet for each of 10 sheets when two-dimensional chromatograms are to be used; the spots are uniform within each run and among replications; the area of the spot is easily controlled; and the sample may be dried in either hot or cold air streams. Therefore, when larger volumes are to be employed for paper chromatography, replications may be made with greater ease, accuracy, and at a considerable saving of time. PLATE A. Multiple sample spotter for paper chromatography PLATE B. Adjustable spotting rack (upper) set up for one-dimensional chromatograms j (lower) set up for two-dimensional chromatograms. PLATE C. 117 Multiple sample spotte:* in position in front of the adjustable spotting rack which is set up for two-dimensional chromatograms. PLATE D. 118 SAMPLE SPOTS AT VARIOUS PERCENTAGE TWER SETTINGS o o o 0 0 o 5% 10% o O O o O O O o o O 15% 0 © O o o o o o Q o 20% 0 © o o o 0 Q o O o 25% • • • • e • • O • • 30% ©i 0 • • © • o © © © 40% a • • • 50% 0 • 0 0 0 0 0 0 # 0 0 0 0 70% 0 0 0 0 100% |i]M4U4mprimquj|iniimiiL|iinnuiii|ni|rii!iTgiii|ippniimiininrqi4m] 119 TABLE A. Composition of basal medium used in culture of Lemna minor kno3 0.002M Thiamine 100 >ig/l Ca(N03)2 0.003M Pyridoxin 800 KH2P04 0.001M Nicotinamide 800 pg/l MgS04 0.001M B 0.1 ppm CaCl2- 0.003M Mn 0.1 ppm KG1 0.002M Zn 0.3 ppm MgCl2 o.oouyr Cu 0.1 ppm Sucrose 2% Mo 0.1 ppm Fe 0.5 ppm TABLE B. )ig/l Hoagland's complete nutrient solution. Stock solutions: H 0 No. 2 227.7 kno3 109.70 gm KH sP04 29 •5>2 gm 106.70 gm MgS04 -7H20 No. 3 gm/liter of solution Ca(N03)2*H20 /uter of solution NaCl 7.05 gm h 3b o 3 0.282 gm Cu C12 ’H 20 0.001; gm ZnCl2 MnCl2-UH20 O.OO3 gm /lOO ml solution 0.039 gm FeCl3 *6H20 0.500 Solution prepared: gm UO ml of No. 1$ 50 Ml of No. 2} 1 ml of No. 3 and brought up to 10 liters with distilled water.