PATHOLOGICAL RESPIRATION AITD SYSTEMIC FACTORS IN FUSARIUM WILT OF TOMATO By RALPH PORTER COLLINS 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 1957 Approved ABSTRACT RALPH PORTER COLLINS Leaves from infected plants had higher respiration than disease-free controls as early as 1 day after inoculation with Eusarium oxysponmi f. lycopersici. Higher respiration rates continued until plants showed advanced symptoms* These comparative results were obtained using leaf discs with Warburg technique* The relationship held whether res­ piration was determined on a dry weight or on a nitrogen basis. Total nitrogen was unchanged 2 and 8 days after in­ oculation but on the lkth and lf?th days leaf tissue from diseased plants showed a 1 0 to 18 per cent increase in total nitrogen. Since leaves were not Invaded by the fungus the findings indicate systemic toxemia. The respiration of stem slices followed a similar pat­ tern. The pathogen was present in stems in the area used as early as 2 days after inoculation, but all stem sections did not contain the pathogen until 7 days after inoculation. Several substances known to be produced by F. oxysporum f. lycopersici were tested for their effect on host respira­ tion. Fusarinic acid at concentrations of 10“^ M, 10“^ M, and 10“^ M had an inhibitory effect on leaf respiration. lower concentration fusarinic acid had no effect. In Pectinase, a commercial enzyme preparation consisting of several pectolytic enzymes, had no effect on respiration of leaf discs taken from cuttings regardless of the concentration used. Cuttings were allowed to take up ethylene solutions by tran- spirational pull and respiration of* leaves was determined. Leaf discs were infiltrated with ethylene solutions, leaves were also gassed with ethylene. and Respiration was un­ affected regardless of the treatment or the concentration of ethylene used. Infection caused increased permeability to electrolytes as early as L|. days after inoculation as determined by conduc­ tivity of leaf leachings. Permeability thus determined was approximately double that of the controls 1 2 days after in­ oculation. An attempt was made to identify ethylene as a volatile from diseased plants using gas chromatography and Infrared spectroscopy. Results were Inconclusive. A bacterial con­ taminant growing on tomato leaf tissue produced a volatile identified as nitrous oxide. PATHOLOGICAL RESPIRATION AND SYSTEMIC FACTORS IN FUSARIUM WILT OF TOMATO By RALPH PORTER COLLINS A THESIS Submitted to the School for Advanced Graduate Studies 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: 10008502 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008502 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 48106 - 1346 TABLE OP CONTENTS Page INTRODUCTION.......................................... 1 LITERATURE REVIEW ................................... 2 Respiratory changes in diseased plants . . . . 2 Fusarinic a c i d ................................. 6 Physiological effects of ethylene. 6 • ......... Permeability changes in infected plants. ... 9 MATERIALS AND M E T H O D S ............................... 10 Host material, fungus material, and inoculation methods♦ . ...................... . . . . . . 10 Methods of determining respiration . . . • • • 12 Nitrogen determinations........................ 13 Method of determining permeability changes . • 111 En zyme a.ss ays................................... ll|. Methods of ethylene treatment.......... 1$ Detection of ethylene............. 16 EXPERIMENTAL RESULTS................................. 19 Respiration of leaves from diseased and healthy p l a n t s ........................................ 19 Respiration in stems of inoculated and uninocu­ lated p l a n t s ................................. 26 Determination of respiratory quotients 30 . . . » The effects of fusarinic acid on respiration • 30 Respiration of pectinase treated cuttings. 32 . * Effects of ethylene on respiration of leaves and stems...................................... 32 Effect of ethylene on disease development. . • Other possible effects of ethylene ........... Detection of ethylene.......................... Conductivity studies .......................... DISCUSSION............................................ S U M M A R Y .............................................. LITERATURE CITED. ACKNOWLEDGEMENTS The writer wishes to thank Dr. Robert P. Scheffer, under whose supervision this investigation was undertaken. The other members of the guidance committee, Dr. Everett S. Beneke, Dr. William B. Drew, and Dr. Lloyd C. Ferguson are tendered sincere thanks for their helpful suggestions and criticisms» The author is also grfcatful to Mr. Philip G-. Coleman for preparing the illustrations, to Dr. Aleksander Kivilaan for his aid in translating the German articles, and to Dr. James C. Sternberg for his interest and assistance in making the infrared and gas chromatographic analyses. Finally the author is indebted to his wife Helen for her assistance and support during the course of studies in attaining this degree and especially for her help in prepar­ ing this manuscript. INTRODUCTION It has been thought that wilt inducing pathogens pro­ duce toxins which are liberated Into the host and cause symp­ toms in advance of the fungus (20). Most experimental work on toxins is based on the assumption that S7/mptoms of dis­ ease are specific and that metabolites of the fungus produced in vitro are formed in diseased plants and have a causal re­ lationship with disease. assumptions. There are no data to support these In the present work the role of toxins In to­ mato plants systemically invaded by Pusarium oxysporum f. lycopersici (Sacc.) Snyder and Hansen, has been evaluated using host respiration and permeability as criteria. The role of fusarinic acid and ethylene, products of the fungus on the host, was studied in these respects. study, During the still other effects of ethylene became apparent and were evaluated. Metabolic changes in plants infected by obligate para­ sites (2 ) and in fruit and storage organs invaded by facul­ tative saprophytes (ff.9) have been studied. diseases have had little attention. Other types of Possible Important sim­ ilarities and differences between infection by obligate par­ asites and vascular fusaria were studied. LITERATURE REVIEW Respiratory changes in diseased plants. The respiration of plants Infected with obligate para­ sites has been studied extensively and several reviews are available (I4., 5* U 2 ). In general, respiratory increases have been noted In plants infected by strict parasites. However, in Prasium majus (L.) Infected with Erysiphe lam- procarpa (Kickx.), in Torilis nodosa (Gaertner) infected with Erysiphe communis (Wallr.)Fr.) lets infected with Erysiphe polygon! (3 ^ ) 5 (DC) and in bean leaf­ (35) decreased respiration was reported. Respiratory increases in wheat leaves infected with powdery mildew were shoim by Allen (2) and by Yarwood (55) to occur in uninvaded cells of the host. From these experi­ ments Allen et al. concluded that toxic substances produced by the mildew diffuse Into underlying host cells and cause changes in both anaerobic and aerobic respiration. Mildewed leaves of clover dusted with sulphur to kill the mildew main­ tained an increased respiration. This indicated that in­ creased respiration of mildewed leaflets was largely due to the stimulation of host respiration by the mildew rather than by respiration of the mildew fungus (55)* Areas of diseased leaves removed from the affected zone failed to show an in­ crease in respiratory rate indicating that the spread of the mildew toxins was restricted (2 , 3)* Experiments utilizing tagged substrates indicated that a diffusible substance (or substances) produced at the cite of incipient infection by 3 the host cells, by the fungus, or by both enhanced the m e ta­ bolic activity of the host tissue in the area of infection. The accumulation of various substances by leaves of wheat and barley infected with Puccinia and Erysiphe was dependent upon aerobic respiration (h-3 ). The nature of the increased respiration in plants in­ fected xtfith obligate parasites is not clearly understood but several theories have been proposed. It is said that in­ creased respiration of Infected wheat leaves is due to an Inhibition of the Pasteur effect. The Pasteur effect Is the suppression of carbohydrate breakdown aerobically as compared with the rate of breakdown anaerobically. In wheat infected with powdery mildew an increase In inorganic phosphate and an inhibition of the Pasteur effect was found (38). Allen held that mildew toxins acted as uncopulers and accelerated the release of inorganic phosphate and regeneration of ADP. He also Interpreted Sempiofs data as indicating an in­ hibition of the Pasteur effect. Other workers have followed Allen*s ideas on inhibition of the Pasteur effect. Parkas and Kiraly (17) found that wheat infected with stem rust and powdery mildew became pro­ gressively less sensitive to malonate. At the same time glycolysis inhibitors suppressed oxygen uptake equally in healthy and diseased plants. This is interpreted to be a completion of glycolysis and oxidation of glycolytic products without participation of the tricarboxylic acid cycle (1 7 ). These ideas appear consistent with the idea of Pasteur effect k inhibition since Pasteur effect means inhibition of fermen­ tation under aerobic conditions. The increase in respiration in rust-infected plants is thought by Daly et al* (11) and by Shaw and Samborski to occur via the r,hexose monophosphate shunt". (li-3) It was ob­ served that per unit of oxygen consumed the number 1 carbon atom of glucose supplied exogenously is oxidized to carbon dioxide faster by rusted and mildewed leaves than by healthy leaves (H3)* It was also shown that the relative contribu­ tion of the number 1 carbon atom of glucose to the amount of carbon dioxide evolved Increased with the incidence of dis­ ease. However, the hexose monouhosphate shunt may be oper­ ative in the pathogen rather than in the invaded tissue, since Shu et al. have shown that this pathway Is followed by germinating rust spores (Mi.). In uninfected tissue the Embden-Meyerhof pathway x^as predominant. The respiratory pattern of plants Infected with facul­ tative saprophytes is similar to that of obligate parasites. Carbon dioxide production by half roots of sweet potatoes infected with Rhizopus tritici (Salto) was higher than the controls 1 day after inoculation and by the second day was 9 times that of the uninfected half ($0). Potato tissues infected w5th Bacillus phytophthorus (Appel.) also showed large Increases in respiration (15) • Comparison of respira­ tory rates of tobacco tissue Infected with Fhytophthora parasitica var, nicotianae (Tucker) and healthy tissue showed increased respiratory nates of Invaded tissues (£3 )* Since 5 it was impossible to separate host respiration from the p a t h ­ ogen respiration some of the increase was undoubtedly due to the fungus. Sweet potatoes infected with Ceratostome11a fimbriata (Elliott) had a greatly augmented respiration (6 , 7, U8). The role of toxin production in a disease caused by a facultative saprophyte was studied by Uritani and colleagues (6 , 7f 8 , !±8 )• Several abnormal metabolites were found in sweet potatoes infected x-nlth Ceratostome 11a fimbriata* of them, ipomeamarone, a sesquiterpene, One accelerated the res­ piration of sweet potato slices by 3 0 -Li-O per cent in a solu­ tion of 1:1l, 000 dilution (l>-8). Ipomeamarone stimulated not only the respiration of sweet potato slices but acted as an uncoupler of oxidative phosphorlyation carried out by cyto­ plasmic particles of mung bean and sweet potato. It was suggested that respiratory changes in Ceratostome11a infected sweet potatoes is due to inhibition of the Pasteur effect thus following the pattern found in rust and powdery mildew infected plants. In later work, the Japanese workers found that ipomeamarone action probably accounted for only a small percentage of the respiratory increase. Tissue adjacent to the area of infection had a significant increase in functional protein and an increased activity per unit of protein (7 » 8 ). The respiratory changes in Fusarlum wilt of tomato, systemic invader, have received little attention. a Bloom (10) measured the rate of respiration of leaves and cuttings from inoculated infected plants using gas train methods and gravi- 6 metric determination of carbon dioxide, Pie found respira­ tion of shoots and leaves from diseased plants increased until the first symptoms of wilt appeared, Fusarinic acid* Fusarinic acid, a product of the tomato wilt Pusarium (2 0 ), has been identified as 5>-n-butyl- 2 -pyridin carboxylic acid Fusarinic acid acts on plants to produce necrotic areas between the leaf veins and streaks on stems of tomato plants. Synthetic fusarinic acid at a concentration of 2 x 1 0 ”^' M inhibited respiration of tomato leaf mately 16 per cent (33)* 1 0 “k- M gave more tissue approxi­ Concentrations greater than 2 x inhibition* The respiration of both healthy and nitrogen deficient tomato plants was decreased after treatment with fusarinic acid but the inhibition was more pronounced in nitrogen deficient plants (5 7 )* A similar re­ sult has been observed in tobacco leaves treated with fusar­ inic acid (1 6 ). Physiological effeets of ethylene. The influence of ethylene on fruit respiration has long been known. Lemons exposed to concentrations of ethylene varying from 1 to 1 , 0 0 0 p.p.m. in air had 1 0 0 to 2 0 0 per cent increase in carbon dioxide evolution (13)* respiration in bananas plums (Ll7). in 1 9 111 (19), persimmons Ethylene increased (1 2 ), pears (2 l+), and it was shown that citrus fruit infected with Penicillium digitaturn (Sacc.) produced a volatile which increased the respiration of uninfected citrus fruits 1 0 0 per cent at 15°C. (9). The volatile material produced by P* 7 digitatum x-ras identified as ethylene (5 6 )♦ The effects of ethylene on several fruit pectins are known. "Ethylene treated pears in early stages of maturity had a more rapid transformation of insoluble protopectin to soluble pectin than had untreated pears (2b.)m This reaction in the presence of ethylene was similar to changes which would normally occur in the more mature fruit. ethylene was slow and lasted a long time. The response to It was noted that respiration and pectins became insensitive to ethylene at approximately the same time. Decreased insoluble protopectin and definite increases in soluble pectin have been reported in the follox^ing fruits: gooseberry, peel of ponderosa lem­ on, Italian prune, Elberta peach, hull of English walnut, and Bartlett and Anjou pears (25)* In normally ripening melons insoluble pectin gradually decreased and soluble pec­ tin increased, while In ethylene treated melons increase of soluble pectin x-ras much greater in the early stage of ripen­ ing. Since the conversion of protopectin to pectin is caused by ,rprotopectinaseff it x^ras thought that ethylene activated this enzyme (3 7 ). The causal role of ethylene In Infected plants has been studied In some detail. carpon rosae hi emails Rose leaves infected with Diplo- (Wolf), cherry leaves infected with Coccomyces (Higgins), and chrysanthemum flowers infected x-rith Asochyta chrysanthemi (Stevens) produced more ethylene than did respective healthy tissues as meastxred by the triple r e ­ sponse of etiolated Alaska peas. Injury also caused Increased production of ethylene, since shredded, healthy, rose and cherry leaves produced more ethylene than did uninjured healthy leaves (52). Oranges and grapefruit Inoculated with Penicillium digitatum and oranges with Diplodia natalensis (Evans) and Diaporthe citri (Paw.)W o l f ) produced epinasty in test pea plants sooner than in normal plants under the same conditions. Diplodia rot In oranges Increased after gassing with ethylene but Diaporthe rot was not affected (29) Pure cultures of J?* digitatum produced a volatile substance which caused epinasty in treated test plants pathogens failed to produce ethylene The (3 2 ); other (5 2 ). production of ethylene in plants infected with Pusar ium wilt of tomato was examined by Dimond and Waggoner (111-). Ethylene production by infected plants was demonstrated by the amount of epinasty produced In healthy indicator plants confined with the diseased plants. Volatile substances from diseased plants also caused the triple response in peas. It was concluded that ethylene causes the epinastic symptoms associated with Pusarium Infected tomato plants. Cultures of Pusarium produced ethylene as measured by the triple re­ sponse in peas, by epinasty in tomatoes, by bromine addition, and by reversible trapping xtfith mercuric perchlorate. Pro­ duction of ethylene by the fungus varied with the substrate and appeared to be linked with a heat labile enzyme system, since none was produced by autoclaved cells. Cell free ex­ tracts of Pusarium on suitable substrates also liberated ethylene (lip). 9 Permeability changes in infected plants, Pew studies have been conducted on permeability changes in systemically invaded plants. Culture filtrates of P. oxy- sporum f. lycopersici were reported to cause increased perm­ eability in leaf cells of treated tomato cuttings Since culture filtrates contain man^r phytotoxic components, evaluation of such measurements is difficult, A better ap­ proach is that of Gottlieb (23), who found that tracheal ex­ tracts from plants infected with P. 0 x 7^3 porum f, lycopersici affect cell permeability as measured by deplasmolysis time. This reaction was reversible. Sap collected from physiolog­ ically wilted plants had little effect on deplasmolysis time. Direct aeasureaents of permeability changes after systemic invasion have not been reported. MATERIALS AND METHODS Host material, fungus material, and inoculation m e t h o d s » The tomato variety Bonny Best was used in all experi­ ments except those requiring Pusarium resistant plants, In which case the variety Jefferson was used. Plants were grown in sand In the greenhouse and were watered with a balanced nutrient solution (1 8 ) or with a suitable concen­ tration of a soluble commercial fertilizer (H Plant Marvel"). Plants from 6 to 8 weeks old were used when cuttings were needed. Stems were cut under water with a razor blade and the cuttings placed In the treating solutions. Cuttings from 8 to 1 0 inches in length were used, Pusarium oxysporum f, lycopersici Hansen, (Sacc.) Snyder and strain R^- 6 , was used throughout (5l)* The fungus was grown in 5>00 rnl. Erlenmyer flasks in shake culture at approximately 25°C. Under these conditions fungus growth was largely bud cells. used. The medium of Dimond et al. (lip) was This medium T'Tas formulated as follows: casamino acids 1 * 5 gm. yeast extract 1 , 0 gm, KHgPOlj. 1.5 gm. MgSO^ + 7H2 0 1.0 gm. glucose trace elements distilled x^ater 15>.0 gm. 0 . 2 ml. to 1000 ml. (pH 5>.5 to 5*6). Plants were inoculated by removing them from the sand, 11 washing the roots in tap water, and then dipping the roots directly into the hud cell suspension. Plants were then reset in sand and kept under greenhouse conditions favoring disease development. were uprooted, TJnino ciliated controls in all cases dipned in water, and reset in the same manner as the inoculated plants. Inoculum for cuttings was prepared by filtering culture fluids through cheesecloth to remove larger particles. The bud cell suspension was centrifuged for 30 minutes at blj.10 R.C.F. and the filtrate decanted. The bud cells were resus­ pended in distilled water, recentrifuged, and then filtered by suction through coarse filter paper to remove aggregates and mycelial fragments. Bud cell suspensions were adjusted to 5>0,000 spores per milliliter by use of a hemocy tome ter. Cuttings were placed In these suspensions and allowed to take up bud cells through the cut ends by transpirational pull. Cuttings were then rooted in sand. After a suitable time for symptom development, plants were rated for wilt and vascular browning. Wilt was rated on an arbitrary basis from 0 to If.; 0 Indicated no wilt and Ij. indicated severe wilt. was rated as follows: The intensity of vascular browning 0 indicated no vascular browning; 1 indicated slight browning at the base of the stem; 2 Indica­ ted browning of the lower l/3 of the stem; 3 Indicated brown­ ing of the lower 2 / 3 out the stem (I4-0 ). of the stem; Lj. indicated browning through 12 Methods of determining respiration* The Warburg apparatus was used to determine oxygen u p ­ take, following in general the method of Klinker (2?)* Twenty discs were cut from a tomato leaf with a 6 mm* cork borer and placed on moistened filter paper in the bottom of the Warburg flask* Measurements of oxygen uptake were made at 30°C* in a darkened room* Nine Warburg flasks were used in each determination. Pour flasks contained leaf discs from inoculated plants, four contained leaf discs from unin- oculated. plants, and one served as a thermobarometer. discs in each flask were taken from a single plant* The The flasks and their contents were allowed, to equilibrate for 30 minutes, after which the stopcocks were closed* Two read­ ings of oxygen uptake were taken at hourly intervals. After measurements of oxygen uptake were completed the leaf discs were removed from the Warburg flasks, dried for L|_8 hours in a 1 0 0 °C. oven, and weighed. Oxygen consumption was then ex­ pressed as microliters of oxygen uptake per milligram dry weight of tissue or as oxygen uptake per milligram of nitro­ gen (see later). In other experiments leaf discs were sus­ pended in M/30 potassium phosphate buffer solutions pH 5*9. In these cases leaf discs were vacuum infiltrated x-d.th the treating solution for 3 0 minutes. To determine respiration in stems, stem slices approxi­ mately 0 * 7 5 rom* thick were cut using a hand microtome and a straight razor. Slices were taken from the second internode above the cotyledons. The slices were washed in M/30 potas- 13 slum phosphate buffer at pH 5«9» Stein slices were then re­ moved from the buffer solution, blotted on filter paper, and rapidly transferred to a Warburg flask containing a disc of filter paper moistened with 0 * 1 ml. of distilled water* Twenty stem slices were used in each flask, and all values were the averages for 3 flasks. Respiratory quotients were found by the standard method Two flasks were used whose contents w^re respiring in the same manner. In one the carbon dioxide i^ras absorbed and in the other it was not, giving a measure of the carbon di­ oxide liberated. Hitrogen determinations. In one series of experiments respiration was determined on a nitrogen basis to check the accuracy and validity of ex­ pressing respiration in terms of dry weight. Total nitrogen was determined by the micro-Kjeldahl techniques of Ma and. Zuazaga (30) and Pepkowltz and Shive (30). Forty to 50 mg. of finely ground dried plant material were placed in a 5 0 ml. micro-Kjeldahl digestion flask and the contents digested, using concentrated sulfuric acid, until clear. Selenium plus a potassium sulphate-copper sulphate mixture x-ras used as a catalyst. Ammonia was distilled into a flask contain­ ing 5> ml. of a 2 per cent borib acid solution. A mixed In­ dicator was added to the boric acid solution and the contents titrated to a pink color with 0.02 >T hydrochloric acid. each determination 2 Lf_ flasks were used: 12 For contained mater­ ial from healthy plants and 1 2 contained material from dis­ Ill- eased plants. Method of 'determining permeability changes. Conductivity measurements of leachings from healthy and diseased plants were used as a measure of cell permeability. This method measures permeability to electrolytes; the greater the conductivity, the greater the permeability. tivity bridge used. A conduc­ (Model RC 1 6 B 1 Industrial Instruments, Inc.) was Leaves from healthy and diseased plants were carefully selected, washed in double distilled water, and placed In flasks containing 5>0 ml. of double distilled x%rater. To In­ sure that all leaves x^ere wetted they were either vacuum In­ filtrated or placed on a reciprocal shaker. After 7 hours the leaves were removed, from the water and conductivity read­ ings were made on the water using a conductivity cell with a cell constant of 0.1. All measurements were made at 25°C. Leaves were dried for L|_8 hours in a 1 0 0 °C. oven and calcula­ tions were made on the basis of conductivity in micromhos per milligram of dry weight of leaf tissue. Enzyme assays. Pectin methyl esterase activity in culture filtrates was determined by electrometric titration of free carboxyl groups produced by the action of the enzyme on pectin, by Gothoskar et al. (21). as suggested A 1.5 per cent solution of pectin (pure citrus pectin, H. F., from California Fruit Growers Exchange) was made by slowly adding 15 gm. of pectin to 600 ml. of distilled water using mechanical agitation. To dis­ solve the pectin the mixture was autoclaved at 15 lb. pressure 15 for 10 minutes. After cooling, distilled water was added to bring the solution to 1 liter. Ten ml. of a 0.5 M acetate buffer were added to 7 5 ml* portions of the pectin solution. The culture filtrate was adjusted to pH 7«0 before addition to the pectin buffer mixture. Fifteen ml. of filtrate were added to the pectin buffer mixture. Heated samples of the enzyme and filtrate solution were run as controls. The m i x ­ tures were held for 3 hours at 30°C. in a constant temperature bath. The solutions were then titrated to pH 7*0 with a 0.1 N KOH using mechanical stirring. Pectin methyl esterase activ­ ity was expressed as "mg methoxyl removed at the end of three hours by one ml of the culture filtrate or one gm of the en­ zyme sample" (2 2 ). The assay for pectin splitting enzymes (depolymerase and/or polygalacturonase) was based on viscosity measurements. A 0.5 H sodium citrate-citric acid buffer was prepared at pH it.O and heated to 50°C. Pectin was added using mechanical agitation until a 1 . 0 per cent solution was obtained and the solution was passed through several layers of cheesecloth to insure uniformity. Tx-ro ml. of culture fluid was added to 2 0 ml. of the pectin solution and incubated in a water bath at 30°C. for minutes. Five ml. portions of the mixture were placed in an Ostwald viscosimeter and the dropping time noted. Heated culture fluids were used as controls. Methods of ethylene treatment. Pure grade ethylene (99*0 mol per cent purity) was ob­ tained from Phillips Petroleum Company. Saturated ethylene 16 solutions were prepared by bubbling ethylene through distilled water for 30 minutes at l 8 °C* At l 8 ° C # 100 ml. of water con­ tains approximately 0.015 ml. of ethylene (111). Saturated solutions were diluted with distilled water as needed for the various experiments. Tomato cuttings were allox/ed to take up these solutions by transpirational pull. In other exper­ iments leaf discs were vacuum ilfiltrated with ethylene sol­ utions. In still other cases plants or cuttings were placed in desiccator jars and gassed directly with measured amounts of ethylene. Detection of ethylene. Cras chromatography and infrared spectroscopy were used to detect ethylene In emanations from healthy and diseased plants. For gas chromatography a vapor fractometer (Perkin- Elmer Corp. Model lJLj.) was used to separate and measure the volatile components collected from plants. The volatiles were passed In a stream of gas through a column made of an Inactive solid supporting material plus the partition liquid or fixed phase. bile phase Different equilibria exist between the m o ­ (carrier gas and sample) and the stationary phase (the column material), causing components to separate accord­ ing to their Individual equilibrium constants. The concentration of each component was measured by a dual thermocouple and the results were expressed as a series of symmetrical peaks on a recorder. The position of the peak along the ordinate or time axis was used as the qualitative value. The measure of the concentration of a component in 17 a mixture was the area of the peak or abscissa value ( 3 1 )• An effort was made to identify ethylene by means of infrared spectrophotometry. Analyses were made using a re­ cording infrared spectrophotometer (Perkin-Elmer Model 21) and an evacuated I], meter gas cell* This method is based on the fact that nearly all organic substances possess selective absorption at certain frequencies in the infrared portion of the electromagnetic spectrum* The spectrophotometer deter­ mines the per cent transmission or absorption of the sample at a series of narrow frequency intervals throughout a selec­ ted part of the spectrum. A plot of transmissions or ab­ sorption values versus frequency or wave length constitutes the infrared spectrum of the samples (2 6 ). Gases were collected from plant materials in 10 liter desiccators. Four to five kilograms of plant tissue were used in each analysis. Plant materials remained in the des­ iccator for 2 or 3 days and were supplied with oxygen from a large polyethylene bag connected to the desiccator. was added to the bag from an oxygen tank as needed. Oxygen Gases were removed by vacuum and passed through a series of ascarite and calcium chloride towers to remove carbon dioxide and water. The gases were first collected in a 2£ ml. trap im­ mersed in liquid nitrogen. The gases were too dilute to sample directly from the larger trap so they were further concentrated in a 10 ml. trap. This was done by removing the liquid nitrogen, in a Dewar flask, from the larger trap and placing it around the smaller trap. As the larger trap 18 warmed the gases flowed from it to the smaller trap* Gases were collected from the smaller trap by removing the liquid nitrogen bath and allowing the trap to come to room temper­ ature. Volatiles vrere then removed from the trap by inject­ ing a needle through a rubber serum bottle cap which was over the side arm. Gases were injected into the vapor fractometer by inserting the needle through a rubber dia­ phragm into the column. When the gas cell was used volatile materials were injected through a rubber serum bottle cap placed over a small opening in the evacuated cell. EXPERIMENTAL RESULTS Respiration of leaves from diseased and healthy plants. Many workers have found increased respiration in leaves directly invaded by strict parasites* Increased respiration has also been found in sound storage tissue adjacent to tis­ sue invaded by certain facultative saprophytes. of diseases, Other types such as vascular wilts, have had little study. The respiration of leaf discs from healthy and diseased plants was compared following the method of Klinker (2 7 )* Plants were inoculated by root dip and kept in the greenhouse until used. Por each Warburg determination leaves from Ij. Inoculated and k uninoculated plants were used. The leaves were carefully selected for uniformity and generally the third or fourth leaf from the base of the stem was chosen. Respiration was expressed as oxygen uptake per mg. dr;/ weight or as oxygen uptake per mg. of nitrogen. In an experiment with plants Inoculated 32 days after planting respiratory increases were noted in Infected plants 5 days after Inoculation. Respiration increased sharply on the 1 1 th day, reached a peak 1 2 days after inoculation, and dropped off rapidly thereafter (Pig. 1). In a second experi­ ment, 7 weeks old plants were used and a significant increase in respiration was found 2 days after inoculation. Leaves from Infected plants had slightly higher respiration for 8 days, but on the 10th day a sharp Increase occurred. The respiratory rate continued to climb until the llpth day, when the experiment was ended (Pig. !)• FIG. 1. Respiration In diseased plants expressed as the ratio of respiration in diseased plants to that in healthy plants under the same conditions, dry weight basis* determined on a Line A. is for plants inoculated [j. weeks after seeding and Line B. is for 7 weeks plants. 20 .8 .6 RATIO OF RESPIRATION 2.0 1.4 1.2 2 4 6 DAYS 8 A FTER 10 12 INOCULATION 14 21 The appearance of diseased and healthy plants used in experiment 2 is shown in Figures 2 , 3> and lu Two days after inoculation there were no differences in appearance of dis­ eased and healthy plants (Fig. 2). oculation (Fig. 3 ) showed epinasty, lower leaves, (Fig. and stunting. were yellowed, Plants 8 days after in­ slight yellowing of the Plants 1L|_ d?.ys after inoculation stunted, and had some wilting of lower leaves. Leaf discs from healthy and diseased leaves were made with a sterile cork borer, and the discs were placed on po­ tato dextrose agar, and incubated at 25°C. for 1 week. Fusar- ium was not isolated from leaves of diseased or healthy plants although repeated attempts were made. In experiments 1 and 2 respiration was higher in inocu­ lated plants at 2 and 7 days, respectively, when first deter­ minations were made. In a third experiment an effort was made to determine how soon respiration was affected. Signif­ icant r e s p i r a t o r increases were noted in diseased plants as early as 1 day after inoculation (Fig. £). The experiment was ended after 3 days because respiration of plants in later stages of disease had been determined in earlier experiments. This experiment was repeated using washed bud cells as inoc­ ulum. The same respiratory pattern was evident, indicating that toxic substances in cultures used as inoculum were not responsible for early respiration increases. In 2 further experiments respiration of leaf discs from diseased and healthy plants was measured and total nitrogen FIG-. 2. Inoculated plant (right) and uninoculated control (left) 2 days after inoculation. 22 PIG. 3. control Inoculated plant (right) and uninoculated (left) 8 days after inoculation. is beginning to show symptoms of disease. Inoculated plant 23 PIG. L. Inoculated plant (right) and uninoculated control (left) lk days after inoculation. showing advanced symptoms of disease. Inoculated plant FIG. Early respiratory changes in infected plants Respiration in diseased plants is expressed as the ratio with respiration In healthy plants on a dry weight basis. 1.4 1.3 1.2 RATIO OF RESPIRATION 25 1.0 9 O I DAYS AFTER 2 INOCULATION 3 26 was determined in the leaf samples* Respiration was then expressed as oxygen uptake per mg. of nitrogen. These exper­ iments offered, a check on the validit?/- of expressing oxygen ■uptake on a dry weight "basis. Lea.ves from healthy and dis­ eased plants were collected 2 , L]., and ll|. days after inocula­ tion. After oxygen uptake was measured, the leaf discs were dried and ground to a fine powder in a Wiley mill, Kjeldahl determinations were made. and micro- Oxygen uptake per unit of total nitrogen again showed that respiration in diseased plants was increased slightly after inoculation, and rose to almost double the value in healthy plants by the lLpfch day after inoculation (Fig. 6 ). No differences wnre found in total nitrogen in plant material collected 2 and I4. days after inoculation but the leaf tissue from diseased plants collected llj. days after inoculation showed a 1 0 per cent increase in total nitrogen. This difference was not enough, however, to account for the increased respiration of diseased as compared to healthy plants. The experiment was repeated with essen­ tially the same results except that total nitrogen was 1 8 per cent higher in diseased plants than in healthy plants by the 15>th day. Respiration in stems of inoculated and uninoculated plants. Respiration of leaves from diseased plants was clearly increased. But what happens to respiration in the stems, where the fungus might be present and fungus products might be in higher concentrations? Respiration in stems of diseased and healthy plants was PIG. 6 . Respi ratory changes in leaves of infected plants determined on a total nitrogen basis. Respiration infected plants is expressed in relation to respiration in healthy plants. 27 1.8 1.6 OF RESPIRATION 2.0 RATIO 1.4 1.2 2 4 6 DAYS AFTER 8 10 INOCULATION 12 28 compared by the use of stem slices, which were taken from the second node above the cotyledons. The stem slices were Trashed approximately 2 0 minutes in a M/30 potassium phosphate buffer at pH 5.9. Slic es were than removed from the buffer solu­ tion, blotted, dry, and transferred to Warburg flasks contain­ ing a disc of wet filter paper. This is essentially Klinker’s method (27 )j it was followed after- preliminary experiments indicated that it was satisfactory for stem slices. Pour-week-old plants were carefully selected for uni­ formity. Oxygen uptake by stem slices was determined before inoculation and at 1 , 2 , I4 , 7$ 9 , U * inoculation. 1 3 * and 1 5 days after Respiration dropped slightly 1 day after inoc­ ulation, but increased on the second day after inoculation and remained at a higher level than that of the controls throughout the rest of the experiment (Pig. 7)* The pattern is similar to that of leaves, except that striking Increases may come earlier. Results were more erratic than those ob­ tained with leaves. These experiments were conducted during the winter months when light conditions were poor and root formation was retarded, which resulted in an irregular dis­ ease response. Attempts were made to isolate the fungus from stems of inoculated plants. Stem slices taken from the same region as those used for respiratory measurements were placed on potato dextrose agar and incubated at 2$°Cm for 1 week. Isolations of the pathogen were made 2 days after inocula­ tion} however, all sections did not contain the fungus until PIG-. 7- Respiration of stem slices from diseased plants expressed as the ratio of respiration in diseased plants to that of healthy plants under the same conditions. 29 RESPIRATION .7 .6 .5 .3 RATIO OF .2 . I .0 .9 8 % o 2 4 DAYS 6 A FTER 8 10 12 INOCULATION 14 16 30 the 7 th day. Determination of respiratory quotients. Respiratory quotients of leaves from diseased and healthy plants were determined for comparative purposes. Possible differences might indicate differences in respiratory sub­ strates. Plants 3 2 days old were inoculated and respiratory quotients were determined 2 , 6 , 1 0 , and li|_ days later, using appropriate controls. 'Two flasks in each group contained alkali in the center well and two did not. Leaf discs from healthy plants and from diseased plants in all stages of disease development had a respiratory quotient of approxi­ mately 1 . 0 (Table l). Respiration quotients do not indicate a difference in substrates between diseased, and healthy plants. The effects of fusarlnic acid on respiration. Fusarinic acid, a known product of F. oxysporum f. lycopersici, was tested to see whether or not it could account for the observed increases in respiration. Leaf discs were vacuum infiltrated with solutions of fusarinic acid and M/30 potassium phosphate buffer and. oxygen uptake was determined manometrieally. In one series concentrations of fusarinic acid at 10 ~ 3 M, lO'*4- M, 10"^ M, 10 “ 6 M, 10 “ 7 M, and 10 - 8 M were used at pH 6.0. 1 0 M and at 10"^- M. Respiratory inhibition was noted at In all other concentrations fusarinic acid had little or no effect on respiration. This experi­ ment was repeated at the same pH but with slightly different concentrations: 10"^ M, 5 x 10"^* M, x 10 ^ M, 5 x 10 ^ M, 31 TABLE? 1 . - Respiratory quotients of leaf discs from from healthy and diseased tomato plants. Days after inoculation Respiratory quotients He althy Diseased 2 0.92 0.9? k 0.9? 0.96 6 0.97 0.9*4- 10 0.9? 0.93 Average of hr Warburg determinations. 32 5 x 10 7 M, and 5 x 10"® M. In this experiment fusarinic acid at 10“2 M markedly inhibited respiration. At 5 x 1 0 K there was slight inhibition, but lower concentrations were without effect* In a third series, fusarinic acid was used at 10"3 M, J x 10"^ M , 5 x 10"£ M, 10"® K, 10"? M, and 10~8 M at pH Ip* 8. Respiration was Inhibited In concentrations of 10 3 M and 5 x 10""^ M, but was not significantly affected at the other concentrations (Table 2). It Was concluded that fusarinic acid was not responsible for respiratory Increases in infected plants. Respiration of pectinase treated cuttings. Since Fusarium produces pectic enzymes, an experiment was designed to test the effect of pectolytic enzymes on res­ piration. used. Pectinase, a mixture of pectolytic enzymes, was Tomato cuttings were allowed to take up solutions of pectinase for 3 days following the procedure of Scheffer and Walker (39). Leaf discs were then cut and respiration was determined manometrically. Active pectinase in 0.5* 0.25* and 0 . 1 2 5 per cent solutions along with appropriate heated controls were used. All levels were run with 5 cuttings each, Pectinase had no effect on respiration in any of the concen­ trations used. It was concluded that pectinase was not re­ sponsible for the respiratory increases in infected plants. Effects of ethylene on respiration of leaves and stems. Ethylene is known to Increase respiration in certain fruits (2l\,), but Its effect on leaves has never been exam­ ined. Since ethylene is a known product of Fusarium infected TABLE 2. - The effect of fusarinic acid on respiration of tomato leaf discs. Experiment Ho. and pH Molar Concentration control Average (a) 1 0o/mg. dry wt./hr . 2.58 Respiratory ratio (Expressed as per cent of control) — CO 1 o H 2.29 0.88$ 10”^ M 2.50 0.96$ 10"^ M 2.29 0.88$ control 2.16 10-6 M 2.15 0.99$ 1.9l| 0.90$ 10-8 M 2.07 0.96$ control 2 .)1.5 1 - 5! 10*^ M 2.13 0 .8 7 $ 10"^ M 2. hr2 control 2.uh H S o.U5$ o 1.10 O • i H O i pH 6.0 1 .0 3 $ 5 x 10“7 M 2.25 0 .92 $ 2.72 1 .1 1 $ 0 1 H ,3 1 2.51 CO 5 x 10“^ M vn 3 1 1 pH 6.0 O 2 -- control 3-59 10 “3 H 2.5ii 0 .7 1 $ 5 X 10“^ K 3.05 c.85$ 5 x 10_£ M 3.5U 0 .9 8 $ pH U.8 2.61 2.53 0.97$ 10"7 M 2.73 l.Oli.$ H o s o i control CO I S o H 2.8L|. (a) Average reading for two flasks* 1.08$ 3b tomato plants (lip) the possibility that it may account for observed respiratory increases became apparent. The effect of ethylene on the respiration of healthy tomato plants was tested in several■w a y s • In one series of experiments cuttings from 8 to 10 inches in length were al­ lowed to take up ethylene solutions which were 1:10, 1:100, 1:5>00, and 1:1000 dilutions of a. solution saturated at l8°C. Cuttings were treated with ethylene for 3 days. The solu­ tions were changed and the bases of the stems trimmed each day. These experiments were done in the laboratory under artificial light at approximately 2\\.°C. For each concentra­ tion 5 flasks each containing 1 cutting were used. Five flasks each containing distilled water and a cutting served as a control. After 3 days treatment, leaf discs and stem slices were cut and oxygen consumption was measured raanometrically. Results showed that ethylene did not change res­ piratory rates in stems or cuttings. The respiration of leaves from 25 days old plants gassed with ethylene was determined. Plants were placed in 10 liter vacuum desiccators and concentrations of ethylene in air of 1:10 and 1:1000 were added to the partially evacuated desic­ cator by injecting a needle through a rubber serum bottle cap placed over the opening In the side arm of the desiccator. The same method was used to fill the syringe from the ethyl­ ene tank. The desiccators were allowed to come back to at­ mospheric pressure, resealed, and plants were held overnight. Results showed that oxygen uptake by leaf discs from gassed 35 plants did not differ from oxygen uptake by imtreated controls* In h. similar experiments excised, leaves from 5-weeks—old tomato plants were gassed with 1:10, 1:100, 1:500,. and 1:1000 ethylene-air mixtures. Two leaflets taken from each of 5 plants were used for each, mixture. Controls were treated the same way except that etl ylene was omitted. The leaflets were left 12 hours in the gas chambers before discs were cut and oxygen uptake determined m an ome trie ally, Hesiilts again showed no effect of ethylene on leaf respiration. Leaf discs from 35-days-old tomato plants were vacuum infiltrated with ethylene dissolved in M/30 potassium, phos­ phate buffer solutions at pH 5*9. tions were diluted 1:10, 1:100, in the solutions, Ethylene saturated solu­ and 1:1000. After 1 hour leaf discs were removed, blotted dry, and quickly transferred to Warburg flasks. Results showed no effect of the ethylene solutions on oxygen uptake by leaf discs. It was concluded that ethylene was not responsible for the respiratory increases in diseased plants. Effect of ethylene on di sense development. Inoculated and uninoculated plants of resistant and susceptible tomato varieties were trea.ted with ethylene to determine whether or not ethylene could alter the course of disease, such as is caused by ethanol and several respiration inhibitors (21). Cuttings xwere allowed to take up bud cell suspensions by trcnspirational pull• The following day cut­ tings were placed in solutions of ethylene prepared by dilut­ ing a saturated solution 1:10 and 1:100 with tap water. Cut- 36 tings were kept in ethylene solutions, with frequent changes of solution, for 3 days. Appropriate controls were used, i n ­ cluding inoculated and untreated plants, uninoculated and ethylene treated plants, and uninoculated and untreated plants. When the cuttings developed roots they were potted in sand and kept under conditions favorable for disease devel­ opment. Five plants were used in each treatment. Fourteen days after inoculation plants x^ere rated for severity of wilt and vascular browning. Results showed that disease developed more rapidly in ethylene treated, inoculated plants than in the inoculated untreated controls of the susceptible variety. The uninoculated treated controls showed non-permanent epinasty and slight stunting but no other response similar to that of diseased plants. At the end of the experiment there were no evident differences between uninoculated treated plants and uninoculated untreated plants. The effect of ethylene on expression of resistance by a resistant variety was determined. Cuttings were inoculated and treated with a saturated solution or a 1:5 dilution of ethylene for 6 days and rooted in sand. Fourteen days later inoculated treated cuttings showed typical symptoms of dis­ ease. Inoculated untreated controls gave no evidence of dis­ ease. Uninoculated treated controls showed e p m a s t y and slight yellowing of lower leaves and were slightly stunted at the end of the experiment. No other response similar to diseased plants was noted. Attempts were made to isolate the fungus from inoculated 37 treated plants by placing stem slices on potato dextrose agar. Positive isolations were obtained along the entire length of inoculated treated plants but were made only from the basal portion of inoculated untreated plants. Other possible effects of ethylene. Ethylene was reported by Hansen (25) to make pectin more soluble and Rosa (37) reported that ethylene activated pectolytic enzymes. Since pectic enzymes are involved in Fusarium wilt (1 3 ), the possibility of ethylene-pectin and ethylene-enzyme interactions was considered. To determine whether or not ethylene acted upon the pectin directly, ethylene was bubbled through a 1 per cent pectin solution for 30 minutes at l8°C. Viscosity measure­ ments were made on the pectin-ethylene mixture after stand­ ing for various periods of time (1 hr., 6 hr s., 12 hrs.,^ and 2i]_ hrs.). The viscosity measurements showed ethylene to have no direct effect upon this particular pectin under the condi­ tions of these experiments. The possible effect of ethylene on pectin methyl ester­ ase was determined. for 5 days, Fusarium was grown in wheat bran cultures after which 200 ml. of water were added to each flask and the cultures left to autolyze for 12 hours. The autolyzed wheat bran x>ras squeezed through cheesecloth and clarified by centrifugation at Ljlj.10 R.C.F. for 15 minutes. Ethylene was bubbled through the clarified solution until it was saturated and the solution was then assayed for pectin methyl esterase activity using the standard procedure. Ethyl- 38 ene had no effect on pectin methyl esterase activity* Viscosity measurements were used to determine the effect of ethylene on pectin splitting enzymes from Fusarium* The fungus was grown as outlined above and ethylene was bubbled through the clarified solution* Depolymerase activity as measured by reduction in viscosity was not affected by ethyl­ ene . Detection of ethylene * Dimond and Waggoner (111) have shown that ethylene is produced by both Fusarium-and Fusarium infected plants* Many methods used to identify ethylene are non-specific and un­ saturated compounds might give a positive reaction. Infrared spectroscopy and gas chromatography were used because they are specific and also can yield a complete spectrum of volatiles from plants. Ethylene was not positively identified as a volatile material from diseased plants due to the great difficulty in separating mixtures recorded on the infrared spectrograph. Figure 8 shows an infrared spectrograph of gases collected from diseased plants. It can be seen that some of the peaks in the unknown correspond to peaks produced by pure ethylene (Fig* 9)* One component which was positively identified by both gas chromatography and Infrared spectro­ scopy was nitrous oxide (laughing gas) (Figs. 10, ll). How­ ever, nitrous oxide was not an emanation from either the fun­ gus or diseased plant material, but apparently came from a bacterial contaminant which grew on the leaves as they col­ lapsed* FIG-. 8 . Infrared spectrograph of gases collected from diseased plants. 39 Wave length in microns to 100 to CO U O T S S T IU S U B a q . q .U G O « I 0 Wave length 00 in microns <— U 100 cO u o T ssyu xsire a q . 3.1100 a o j FIG-. 1 1 . Infrared spectrograph of pure nitrous oxide. k2 to i —i