THE TOXICITY, DISTRIBUTION AND MODE OF ACTION OF DICHLOBEI‘IIL (2,5 -DICHLOR0 * BENZONITRILE) IN PLANTS r Thesis for the Degree of Phi). MICHIGAN STATE UNIVERSITY HUGH CRISWELL PRICE 1969 H E51. IIII III “1293 10656 604 IIIIIIIIIIII III IIIII rig: . .1 LRn?Y suchigan 3mm IWL Unicrsi 37 r This is to certify that the thesis entitled THE TOXICITY, DISTRIBUTION AND MODE OF ACTION OF DICHLOBENIL (2,6-DICHLORO- BENZONITRILE) IN PLANTS presented by Hugh Criswell Price has been accepted towards fulfillment of the requirements for Ph.D. degree in Horticulture Major professor / I -- . .- , Dated/[1142, /I, /I’J7 I/ / 0-169 L . . I -. , ”It“ I‘- . I ‘\ J 2, \ .4 "”\ r1 . I l ~v , ABSTRACT THE TOXICITY, DISTRIBUTION AND MODE OF ACTION OF DICHLOBENIL (2,6-DICHLORO- BENZONITRILE) IN PLANTS By Hugh Criswell Price Dichlobenil is used as an herbicide on perennial horticultural crops. It inhibits germinating seeds and the meristematic tissues of perennial weeds, thus is best utilized as a preemergence treatment. The objectives of this research were to determine the fate of dichlobenil in plants and to ascertain the physiological processes responsible for its phytotoxicity. Carbon labeled dichlobenil was taken up by corn roots and translocated to all plant parts within A hr; lLIC-dichlobenil within the shoot Approximately 10% of the volatilized into the atmosphere. This volatilization was independent of light and dependent on temperature. The lLIC—dichlobenil readily diffused from roots of preloaded corn plants placed in distilled water or nutrient solution. This indicates that dichlobenil is not actively held within the root cells, and readily traverses intervening membranes and cell walls. Either the lipophilic nature of the molecule or its capability of disrupting the semi-permeable nature of cellular Hugh Criswell Price membranes may explain this phenomenon. The latter is sub- stantiated by the observation that dichlobenil induces the leakage of betacyanin and reducing sugars from red beet root sections. This leakage of pigment was partially alleviated by the addition of Ca++, a membrane stabilizing ion. Respiration of red beet root sections, corn roots, I} and cucumber seedlings is increased by dichlobenil as evidenced by increased 02 consumption and glucose utili- zation. However, mitochondria isolated from cucumber hypocotyls and cotyledons were not affected by lO-SM dichlobenil. The activity of the mitochondria was assayed polarographically, manometrically and by measure- ment of inorganic phosphate esterified. In contrast, the monophenolic metabolite of dichlobenil (2,6-dichloro- 3-hydroxybenzonitrile) effectively uncoupled oxidative phosphorylation at 10—5M. Cucumber seedlings treated 2“ hr with 10‘5M dichlo- benil showed an accumulation of polyphenolic compounds as determined by UV absorption of alkaline extracts, and phloroglucinol—HCl treatment of tissue sections. It is postulated that a primary mechanism of action of dichlobenil is alteration of tonOplast permeability and subsequent leakage of phenolic and other ergastic sub- stances into the cytoplasm. The phenols are thus subject to oxidation by soluble oxidase enzymes of the cytoplasm. Hugh Criswell Price The oxidation products of the phenolase enzymes polymerize readily to form polyphenols, which uncouple oxidative phosphorylation and inhibit vital enzymes. THE TOXICITY, DISTRIBUTION AND MODE OF ACTION OF DICHLOBENIL (2,6—DICHLORO- BENZONITRILB) IN PLANTS By Hugh Criswell Price A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture I969 w is\ \V: \U \u '3\ \x) ACKNOWLEDGMENTS The author would like to express sincere apprecia- tion for the guidance and encouragement received from his advisor, Dr. A. R. Putnam, throughout this research pro— ject. Thanks is also extended to Drs. S. K. Ries, D. R. Dilley, W. F. Meggitt, C. J. Pollard and D. Penner for serving on the guidance committee and for their conscien— tious review of the final manuscript. Appreciation is also expressed to Drs. S. K. Ries, D. R. Dilley and M. J. Bukovac for their cooperation in making their laboratory facilities available for this research project. For his generous advice on the isola- tion of mitochondria and use of the radiorespirometer, the author would like to thank Mr. M. L. Brenner. The writer is also appreciative of financial sup— port and isotopes provided by the Thompson Hayward Chemi- cal Company. ii TABLE OF CONTENTS ACKNOWLEDGMENTS LIST OF TABLES. LIST OF FIGURES INTRODUCTION LITERATURE REVIEW. History of Dichlobenil Physical Properties Uses of Dichlobenil . . . . . . Behavior in the Soil. . . . . . . Uptake and Translocation . . Metabolism in Plants and Animals. Physiological Effects . Morphological Studies MATERIALS AND METHODS Toxicity of Dichlobenil to Quackgrass . Uptake and Translocation Studies. Volatilization of Dichlobenil from Plants. Mode of Action of Dichlobenil. . Membrane permeability . . . Respiration of intact tissues. . Oxidative phosphorylation in isolated mitochondria Phosphate esterification and determination of P/O ratios . . . . . . . . lLIC- Glucose utilization. . . . . . Changes in free amino acids pools . Lignin- like Polyphenol Accumulation. Spectrophotometric assay . Histological test for lignin RESULTS AND DISCUSSION Toxicity of Dichlobenil to Quackgrass Uptake and Translocation Studies. . Volatilization of Dichlobenil from Plants. iii Page ii vi l\) H NKOCDNUIWNIU [.1 r: MNNI—‘l—‘l—J l—‘OONU‘IJ: N I'\) NMNNI’UN (I)\]\'IO\\J'IJ=’ [UN \O\O (JULIO Jrl-’ Mode of Action of Dichlobenil. Membrane permeability . . . Respiration of intact tissue . Oxidative phosphorylation in isolated mitochondria . . . P/O ratios determined by manometric. techniques . . . . . . . Pi esterification. . l C- Glucose utilization. Amino acid accumulation. Efiect of Dichlobenil on the Formation of Lignin- -like Polyphenols. . . Spectrophotometric determination. Reaction of dichlobenil treated tissue with phloroglucinol-HCl. SUMMARY AND CONCLUSIONS. . . . . . . LIST OF REFERENCES iv Page 37 Al A5 50 50 52 52 55 55 57 61 66 -5'.) ‘ H“""h I‘DQ’ Table LIST OF TABLES The growth of intact and clipped quackgrass plants receiving dichlobenil . . . . Effect of dichlobenil, 2,6-dichloro-3- hydroxybenzonitrile and DNP on oxidative phosphorylation by cucumber cotyledon mito- chondria. . . . . . . . . . . . Effect of 10'5M dichlobenil on the utiliza- tion of lucl and 1“C5 labeled glucose by A— day- -old cucumber seedlings. . . . . The free amino acid content of oat plants treated with dichlobenil . . . Page 31 51 5A 55 l“? ”5.0—— “ US .11“! 'A A Figure LIST OF FIGURES Apparatus for trapping l[IO—dichlobenil volatilized from corn leaves. . Effect of dichlobenil on growth of axillary buds of quackgrass . . . . . The per cent of the initial CPM in the nutrient solution, using minimal (15 ml/min) and rapid aeration (100 ml/min) . . . . Radioactivity recovered from the roots and shoot of corn plants grown in nutrient solu- tion containing luc- dichlobenil with rapid aeration (100 ml/min) (A) and minimal aera- tion (15 ml/min) (B) . . . . . . . The distribution of radioactivity in corn plants after preloading with luC— dichlo- benil for 2“ hr followed by 24 hr vapor trapping period . . . . . . The per cent of initial radioactivity re- maining in preloaded corn plants after 24 hr in the light or dark at 27 C. The per cent of initial radioactivity re- maining in preloaded corn plants after exposure to 27 C and 10 C for a 2A hr loss period . . . . . . . . . Leakage of betacyanin, expressed as per cent of control, from red beet root sec— tions treated with dichlobenil (A) and 2,A-dinitrophenol (B) . . . . . . Effect of Ca++, added at 12 hr on leakage of betacyanin induced by treatment of red beet root section with 10-“M dichlobenil (A) and lO-uM 2,A-dinitrophenol (B) vi Page 19 3O 32 33 36 38 38 no “2 Figure 10. ll. l2. 13. 14. 15. l6. 17. 18. 19. The influence of l. 6 x lO-uM CaCl on the leakage of betacyanin fgom red beet root sectionfi induced by 10 M dichlobenil (A) and 10' M 2,A-dinitrophenol (B). . Time course of the leakage of reducing sugars from red beet root sections treated with dichlobenil or 2,A-dinitrophenol. 02 consumption of red beet ro t sections incubated in solutifins of 10‘ M or lO‘5M dichlobenil and 10‘ M dinitrophenol O2 consumption of corn root tips incubated in .05M phosphate buffer (pH 6.5) containing various concentrations of dichlobenil. . Polgrographic traces showing the effect of M dichlobenil (A),5 8 x 10-5M 2 ,u— dinitrophenol (Bg, 105M 2, 6- dichlorobenzoic acid (C) and lO M 2 ,6- dichloro-3- hydroxybenzonitrile (D) on ADP dependent respiration in mitochondria (.20 mg N) isolated from cucumber cotyledons Polarographic traces showing he effect of lO-5M dichlobenil (A) and 10' M 2,6- dichloro-3-hydroxybenzonitrile (B) on oli- gomycin (150 mug) inhibited respiration in mitochondria (.20 mg N) isolated from cucumber cotyledons. . . . Time course of Pi uptake by mitochondria (.16 mg N) isolated from cucumber hypo- cotyls . . . . . . . . . Difference spectra of lignin extracts of cucumber hypocotyl (A) and root tissue (B) Difference spectra of lignin extracts of oat shoots (A) and roots (B). Comparison of the accumulation of poly- phenolic substances after A8 hr in roots of control (A, longitudinal section) (C, cross section) and lO‘SM dichlobenil (B, longitudinal section) (D, cross section) treated cucumber roots (125 x) vii Page “3 AA 46 U6 “7 A9 53 56 58 6O F— INTRODUCTION Although major advances have been made in under- standing the practical uses for herbicides, the bio- chemical basis for toxicity has not been clearly elu- r cidated for any herbicide. There is voluminous literature I on physiological and morphological abnormalities induced by herbicides. From this literature it is difficult to rank plant responses in order of occurrence or to separate secondary from primary herbicidal effects. The lack of knowledge on primary sites of action for existing herbi- cides has necessitated the empirical screening for new herbicides. Increased knowledge on the primary sites of action should lead to the development of more specific or selective chemicals. The objectives of this research were to study the toxicity, distribution and mode of action of dichlobenil (2,6—dichlorobenzonitrile) in plants. Mode of action studies were concentrated on events occurring over a relatively short time course with the lower physiologi- cally active concentrations of dichlobenil. This approach was utilized to establish a logical sequence of events after plants are exposed to the herbicide. LITERATURE REVIEW History of Dichlobenil The herbicidal properties of dichlobenil were first described in 1960 by KOOpman and Daams (33) of Philips- f” Duphar. Dichlobenil was registered by the USDA in 196A for use in cranberries (Vaccinium macrocarpon Ait.) and later registered for apples (Malus sylvestris L.), avo- cados (Persea americana Mill.), blueberries (Vaccinium s2, L.), brambles (Rubus ER- L.), cherries (Prunus cerasus L.), (E. avium L.), grapes (Vitis s2, L.), mangoes (Mangi- fera indica L.), peaches (Prunus persica (L.) Patsch), pears (Pyrus communis L.), and plums (Prunus domestica L.), (P. salicina L.) in 1966 (A). Currently, dichlobenil is being utilized for weed control in perennial horti- cultural crops such as orchards, vineyards, and nurseries. Physical Properties Dichlobenil is a white, crystalline solid with a characteristic odor (27). Its water solubility is only 18 ppm at 20 C, however, it is very soluble in most or- ganic solvents. Dichlobenil is extremely stable thermally and is not subject to degradation by ultraviolet irradia- tion. The high vapor pressure of this compound, 5.5 x 10-14 mm Hg at 20 C, is an important physical property. 2 Pate and Funderburk (A6) determined that 98% of the radio- activity from 1“ C-dichlobenil was lost from an open plan- chet in 2 hr. Based on the rate of water loss from soil, Hartley (2“) has estimated that a herbicide having about 10 times the molecular weight of water, but about one third of the diffusion rate in air, could be expected to disappear at a rate of 2A lb/A per month if its vapor E“ pressure was approximately 10"“ mm Hg. Since dichlobenil I has a vapor pressure 5.5 times greater than this, sub- stantial loss could obviously occur. Vaporization may 3 also play a significant role in the mode of action of this compound in plants (7, 11, Al). Uses of Dichlobenil Although dichlobenil is a powerful inhibitor of germinating seeds and actively dividing meristems (33, 41), it may be safely applied only to selected estab- lished crops. If roots of the crop come into contact with high concentrations of dichlobenil in the upper layers of soil, selectivity may be lost. Monocotyle- donous and dicotyledonous weeds are controlled by both inhibition of growth of germinated seeds and young seed- lings. Also, several perennial weeds, such as Pteridium aquilinum (L.) Kuhn (brackens), Equisetum EB- (horse- tail), Agropyron repens (L.) Beauv. (quackgrass), Artemisia s2. (mugwort), and Cynodon dactylon (L.) Pers. (bermuda grass) can be controlled if dichlobenil is applied prior to shoot emergence (27). Dichlobenil is much less effective in controlling weeds which are al- ready established. Fall applications of the granular formulation of dichlobenil are effective for controlling perennial weeds around fruit trees (51, 68). Applications of 6.0 lb/A f * provides 3—H months of weed control in most areas. The wettable powder formulation is less effective (51), probably due to volatility. There have been a few re— ports of injury to tree fruits (19, 52) by dichlobenil treatment. Damage is generally manifested by marginal chlorosis of leaves and decreased terminal growth. Early spring or late fall treatments of dichlobenil were found by Dana (15) to be effective for controlling several annual and perennial sedges, grasses and broad- leaved weeds in cranberry bogs. Dichlobenil at “.0 and 6.0 lb/A caused some reduction in terminal growth and fruit size. The granular formulation of dichlobenil is also effective for weed control around ornamentals and in nursery plantings (1, 17). To decrease the volatility losses of dichlobenil, Ahrens (2) found that shallow in- corporation after planting, provided improved weed control without injury to the nursery stock. Dichlobenil may also be incorporated into an organic mulch prior to applying the mulch to an ornamental planting bed (17, 3A). This not only reduces the loss by volatility, but facilitates application of the herbicide to irregular areas. In an evaluation of many herbicides for activity on the submerged aquatic weeds, Potamogeton pectinatus L. (sago pondweed) and P. nodosus Poin. (American pondweed), dichlobenil was one of two compounds which performed most effectively (21, 6A). However, postemergence applica- tions did not control rooted, submerged, aquatic plants and filamentous algae (6A). Dichlobenil is not acutely toxic to fish at herbicidal concentrations. Behavior in the Soil Massini (Al), studying the adsorption of dichlo- benil, found it to have a very high affinity for lignin (k==A00—1000) and lipids (k = 180-300). When a 6.0 ppm solution of dichlobenil was passed through a column con- taining potting soil (22% organic matter) or a sandy soil (5% organic matter), no dichlobenil could be detected in the effluent. However, sand did not adsorb dichlobenil under these circumstances. Horowitz (26) showed that organic matter had a more restrictive effect on the movement of dichlobenil than clay. The lateral spread of the herbicide near the surface was inversely proportional to the organic matter content of the soil. Dichlobenil may also move in soil as vapor (A1) but to a much greater extent in sand than in a soil high in organic matter. In moist soil, the vapor movement of dichlobenil is much less than in dry soil (26). Incorporation of dichlobenil into the soil immedi- ately after application increased persistence (2, 7, 55). Sheets (55) found that the initial loss of dichlobenil from plots treated with A0 lb/A and incorporated was very rapid, until the residue concentration drOpped to about 10%. Subsequent loss occurred at a very slow rate, which is attributed to the slow release of the remaining herbi- cide from the adsorbed phase. Barnsley (7) reported similar results and concluded that persistence and rate of loss by volatilization are drastically modified by mechanical incorporation with the soil and rainfall after spraying. Experiments carried out in the tropics showed that loss by volatilization was accentuated at the higher temperatures. This is substantiated by Hein (25) who showed under laboratory conditions, that vapor losses from soil increased with increasing temperature, the greatest loss occurring from 30 to 40 C. As the moisture level is increased from air dry to field capacity, the volatility of dichlobenil increased sharply. In cran- berry bogs, subjected to periodic flooding, dichlobenil was quite persistent in the 0-H in depth with very little leaching into the “-8 in depth (AA). However, it appeared that most of the herbicide in the 0-A in depth was ineffective due to adsorption on the organic matter. Uptake and Translocation Massini (Al) found that French dwarf beans (Phaseolus vulgaris L.) which had been exposed to a saturated at- mosphere of dichlobenil at room temperature for A days absorbed the compound almost uniformly by all aerial parts. However, when applied as a foliar spray, uptake was limited (7, A6). Dichlobenil is readily taken up from an aqueous solution through the cut petiole of a bean leaf and translocated upward (A1). The acrOpetal movement of dichlobenil is much slower than that of water which Massini attributes to the high affinity of dichlobenil for plant material. Uptake via the roots of beans from an aqueous solution gave the same type of distribution pat- tern as uptake via the petiole (Al, 6A). There is some evidence that dichlobenil is volatil- ized directly from the shoots. Verloop and Nimmo (6A) found that dichlobenil accumulates in bean roots about 3 fold the concentration detected in the shoots. How— ever, the concentration in the leaves drops to about l/A (after 1 day) to about l/20 (after 5 days) of the root concentrations indicating that the herbicide volatilizes from the leaves or is metabolized to C02. Pate and Funderburk (A6) studying the uptake of lLIC-dichlobenil in bean and alligator weed (Alternanthera philoxeroides (Mart.) Griseb.) found that the movement of lLIC-dichlobenil or its lLIC-labeled metabolites in a basipetal direction was limited, but acropetal movement occurred readily. Carbon labeled dichlobenil applied in a lanolin paste is absorbed and translocated, but only in an acropetal direction (Al, A6). Metabolism in Plants and Animals A water soluble metabolite of dichlobenil, 2,6- dichlorobenzoic acid, was found in bean, alligatorweed and four genus of fungi using thin layer and gas chromato— graphy (A6). Massini (Al) also found limited evidence that dichlobenil was metabolized in plants, but the metab— olite was not identified. Traces of 2,6—dichloro-3- hydroxybenzonitrile and its A-hydroxy analog were detected in apple and wheat (Triticum vulgare L.) plants A months after application of 2,6-dichlorothiobenzamide (chlor- thiamid) to the soil (9). Rabbits, rats and dogs (22, 59, 69) readily metabo— lize dichlobenil to 2,6-dichloro—3—hydroxybenzonitrile and its A—hydroxy analog. The monophenolic metabolites account for approximately A5% of the administered dichlo— benil (23). Other metabolites found are 2,6-dichloro- benzamide, 2,6-dichlorobenzoic acid, 2,6-dichloro—3-and- A-hydroxybenzoic acid and 6 other polar constituents. These metabolites are excreted primarily in the urine. The monophenolic metabolites are present both in the free state or as glucuronide conjugates. Flin‘ . Physiological Effects Dichlobenil is a strong inhibitor of germination and appears to have its most pronounced effects in the meri- stematic regions of established plants (33). The symptoms produced by dichlobenil on established plants are similar to those induced by boron deficiency (A3). Milborrow suggests that the macroscopic and microscopic appearance of dichlobenil treated and boron deficient plants are so much alike that in each case, the same basic process is affected. This hypothesis can be substantiated only when the role of boron in plant metabolism is elucidated. Ind“ _'A Foy and Penner (20) demonstrated an uncoupling ef— fect of dichlobenil on oxidative phosphorylation in mito- chondria isolated from cucumber (Cucumis sativa L.) cotyledons. Dichlobenil consistently stimulated succi- nate and a-ketoglutarate utilization at concentrations of A and 0.73 x 10—“ M. When inorganic phosphate 1.A5 x 10— (Pi) esterification was measured, dichlobenil had a pro- nounced inhibitory effect, providing P/O ratios of 0 to 0.5. The effect of dichlobenil on mitochondria differed from that of 2,A-dinitrophenol (DNP) at 10‘5 M, since DNP did not affect 0 consumption but did decrease Pi esteri- 2 fication. Wit and van Genderen (70) found both of the mono- phenolic metabolites of dichlobenil from animals to cause a significant increase in oxygen consumption of starved lO yeast cells incubated with a limited quantity of glucose. Dichlobenil, 2,6-dichlorobenzoic acid and 2,6—dichloro- benzamide were inactive in this assay. It has been proposed that in animals the monophenolic metabolites are the uncouplers of oxidative phosphoryla- tion and also combine with glucuronic acid and sulphuric acid to give harmless conjugates (60, 71). Over a criti- cal dose level of dichlobenil, the formation of the I — .M-O‘ct LIL”, phenolic metabolites in the liver may be great enough to uncouple oxidative phosphorylation such that insufficient ATP will be available for further conjugation of phenols. “ Since the hydroxylation of dichlobenil is independent of ATP, this process may continue. Subsequently, the concen- tration of the free phenols in the liver may rise sharply, resulting in a complete loss of ATP and the death of the cell from a lack of energy. Mann gt_§l. (38) studying the effect of herbicides on protein synthesis found that dichlobenil at 2.0 and 5.0 ppm did not greatly inhibit the incorporation of luC—leucine into protein in either barley (Hordeum vulgare L.) coleoptiles or cory (Sesbania exaltata Raf.) hypo- cotyls. These experiments were carried out with short incubation periods (3 hr) in an attempt to detect the primary effects of the herbicides being tested. Dichlo- benil does not appear to affect protein biosynthesis directly. When cory hypocotyl sections were incubated ll A.5 hr in solutions containing 10 and 20 ppm dichlo- benil, lipogenesis, measured by incorporation of 1“C from malonic acid-2—luC was inhibited 30 to A0% (39). It was not determined whether dichlobenil had a direct effect on lipogenesis or whether the inhibition was due to lowered ATP levels in the treated tissue. Since dichlobenil is an effective seed germination T inhibitor, its effect on the activity of various enzymes during germination has been investigated. Ashton e£_al. (5) found that the proteolytic activity in the cotyledons of 3-day-old squash seedlings germinated in 10-” M dichlo- i benil, was 30% of the control. However, the activity of the proteolytic enzymes isolated from control plants, was not affected by the presence of dichlobenil in the reac— tion mixture. In cotyledons from 2-day-old squash seed- lings, the inhibition of proteolytic activity induced by 10-)“1 M dichlobenil could be partially overcome by the addi— tion of 10-5 M benzyladenine to the culture solution (A8). Dichlobenil may affect the synthesis or action of the hor- mone which controls the synthesis of proteolytic enzymes. The deficiency of ATP caused by uncoupled oxidative phosphorylation, may also affect the development of pro- teolytic activity. Using intact barley seeds, Penner (A9) showed that treatment with 10-“ M dichlobenil for two days caused amylase activity to decrease to A9% of the control. This 12 inhibition was not overcome by adding 10—6 M gibberellic acid to the incubating medium. However, with de-embryonated half seeds, a similar inhibition of amylase activity was observed but could be eliminated by adding gibberellic acid to the incubation solution. Thus, dichlobenil appears to interfere with the control of amylase synthesis and not with its activity per se. In studies conducted by Funderburk and Carter (22), the first trifoliate leaf of bean plants treated with 20 ppm dichlobenil was exposed to 1“CO2 for 2A hr in con— tinuous light. Subsequent extraction and identification of labeled compounds showed that dichlobenil did not af- fect the distribution of fixed l“cog. Morphological Studies Nodal tissue of alligator weed from plants treated with 2.0 lb/A dichlobenil was sectioned and investigated by Pate et_a1. (A5). After 2A hr, the end walls of young sieve tubes had collapsed. Four days after treatment, damaged tissues were observed both within and above the nodes, however, inactive buds generally were not injured. Coagulation of protoplasm and collapsed cell walls in the phloem and surrounding parenchyma cells was also observed. Seven days after treatment, large segments of phloem and associated tissues appeared to be destroyed. The lack of injury to axillary buds was attributed to the fact that 13 vascular connection between inactive buds and the stem was not differentiated, thus no herbicide was translocated into the bud. Devlin and Demoranville (16) made the observation that cranberry bogs treated with dichlobenil produced berries with more intense red color. Berries from plots receiving spring applied dichlobenil at 3.0 and A.0 lb/A r- contained Al% and 6A% more anthocyanin respectively than control berries. Walker (69) made applications of dichlobenil at A.0 and 8.0 lb/A each year for the control of Virginia chain fern (Woodwardia virginica L.) in cranberry bogs. The first injury symptoms on the fern were observed A to 6 weeks after herbicide treatment. The initial symptom was a brittleness that caused numerous broken stems. Similar symptoms were also observed on cherry trees which had re- ceived an excessive application of dichlobenil (52). MATERIALS AND METHODS Toxicity of Dichlobenil to Quackgrass Rhizomes of quackgrass were collected from the field and cut into 2 cm single—node sections. After washing, the sections were planted in quartz sand in 11 x 7 cm plastic pots; 10 sections per pot. The pots were watered with Hoagland's No. 2 nutrient solution containing 0, 8 6 5.8 x 10' , 2.9 x 10'7, 5.8 x 10'7, 2.9 x 10- and 5.8 x 10-6 M of either dichlobenil or 2,6-dichlorobenzoic acid. Each treatment was replicated A or 5 times in a completely randomized design. Every third day, the pots were flushed with distilled water and fresh solutions added. After 2 weeks the number of buds which had initiated growth and the fresh weight of shoots were recorded. To compare the effect of dichlobenil on intact quackgrass plants vs clipped plants, single node rhizome sections were grown for 3 weeks in quartz sand and watered with Hoagland's No. 2 nutrient solution. After thinning each pot to 5 uniform plants, the shoots in one half of the pots were cut off at the surface of the sand. The pots were treated with nutrient solution containing 0, 6 5.8 x 10’ , 2.9 x 10-5 or 5.8 x 10'5M dichlobenil for a 1A 15 period of 2 weeks. The treatments were replicated A times and completely randomized on a greenhouse bench. At the termination of the experiment the shoots were re- moved, dried and weighed. Uptake and Translocation Studies Preliminary experiments with quackgrass plants indi— cated that after an initial uptake of lac-dichlobenil there was a subsequent loss of radioactivity from the shoots. Seedling corn plants (§E§.T§X§ L. cv. Harris Gold Cup) were selected for further studies, so that uni- form plants could be obtained rapidly. Seeds were germinated in vermiculite moistened with distilled water. After the first leaf emerged from the coleoptile, the seedlings were transplanted into 11 x 7 cm plastic pots containing quartz sand. The pots were watered daily with Hoagland's No. 2 solution and grown at 27 C with 2,000 ft-C light (16 hr) and at 17 C for an 8 hr night. When the plants were approximately 15 cm high, they were re—' moved from the sand and the seed coats were removed. After rinsing the roots, 8 plants were placed in each 250 ml beaker containing 200 ml of nutrient solution which was continuously aerated. The plants were sup- ported by placing the shoots through perforated aluminum foil which covered the top of each beaker. Treatments 16 were applied after the plants were allowed to equili- brate for 2A hr. Uptake of dichlobenil was studied by replacing the nutrient solution with fresh solutions containing 1 he nitrile labeled luC-dichlobenil (specific activity A.65 mc/mM) per 100 ml of solution. The concentration of dichlobenil in the solution was 2.1 x 10-6M. Because of F" the volatility of this compound, all tracer studies were carried out in a hood under a bank of fluorescent lights. At time intervals of A, 12, 36 and 72 hr, plants were re- moved from the radioactive solutions and their roots panacea aw .' ~ rinsed 5 times in distilled water. The roots were blotted between paper towels and dissected from the shoots. The tissue was cut into small sections, placed in glass vials and quick—frozen in acetone-dry ice. The tissue was main— tained in the frozen state prior to extraction. Preliminary experiments using the extraction pro— cedures of Meulemans and Upton (A2) and Pate and Funder- burk (A6) demonstrated that there were no water soluble metabolites of dichlobenil formed in corn plants within 72 hr. Therefore, a single benzene extract was ascer— tained to be sufficient to account for all lLIC-dichlobenil within corn plants at this stage of growth. The frozen samples were weighed and homogeniZed thoroughly with A.0 ml benzene in a glass hand homogenizer. The homogenate was fast-filtered through Whatman No. l 17 filter paper and the residue rinsed 3 times with 1.0 ml aliquots of benzene. The filtrate was brought to a con- stant volume and a 1.0 ml aliquot placed in a scintilla- tion vial containing 15 m1 toluene-BBOT. After counting for 10 min, the data were corrected for quenching and the results expressed as dpm per mg fresh weight. The radioactivity in the nutrient solution was also assayed at each sampling time. This was accomplished by first measuring the volume of the nutrient solution and then removing 1.0 ml which was placed in a scintillation vial containing 15 ml of toluene BBOTzTriton X-100 (10:A) (A7). The radioactivity in plant extracts and nutrient solution was identified by spotting 10-30 ul of the con- densed samples on a prepared (Kodak) silica gel G thin layer plate. Pure 1[IO—dichlobenil was also spotted on each plate as a reference. After deve10ping the chroma- tograms in an appropriate solvent system for 10 or 15 cm, they were cut into sections 1.0 cm wide and each section placed in a separate scintillation vial for counting. Dichlobenil had an Rf of 0.A0 when developed in ethanol: hexane (1:9 v/v) at 25 C and an Rf of 0.65 when developed in diethyletherzpetroleum ether (1:1 v/v) at 3 C. Volatilization of Dichlobenil from Plants Ten-day-old corn plants were placed in nutrient solutions containing 2.0 uc l[JG-dichlobenil for 2A hr. ””3? ..“JV 5 ink" - 18 At the end of the preloading period, the roots were rinsed 6 times with distilled water and 3 plants were placed in each of 3, 25 ml erlenmeyer flasks containing 20 ml of fresh nutrient solution. Duplicate plants were frozen immediately for subsequent determination of the initial l[IO—dichlobenil concentration. The top of each flask was sealed with a plug of cotton and polyethylene glycol 1500 P" which was found to be impermeable to luC-dichlobenil. The 3 flasks were placed inside the quart mason jar component of the trapping device (Figure 1). Glass tubes were inserted into the metal top to provide an air inlet II'. ‘l-g- “ I and outlet. To the outlet tube 2, 200 ml gas washing bottles with glass fritted disks were connected in series. The gas washing bottles were filled with 10 ml toluene, the trapping solvent, and maintained in an ice bath. Fil- tered, compressed air was passed through a CaCl2 moisture trap, into the jar containing the plants and then bubbled through the trapping solvent. The toluene solutions were adjusted to a constant volume, 20 ml aliquots were removed, and 80 mg BBOT was added to each aliquot prior to counting. The remainder of the trapping solvent was condensed by flash evaporation and the radioactivity identified by thin layer chromatography. Also at the termination of the experiment, the radioactivity was assayed in the roots, shoots, and nutrient solution. l9 .mo>moH choc Bonn uoNHHHpmao> Haconoanofiolo ....x. ... .. ......n...........~ . . a. . I .. . .....n........wxu...u..n ............... .. burl-r. :H wcaggmgp Mom munchmaq< .H opswfim 20 The influence of temperature on the loss of lAC_ dichlobenil from corn plants was determined by placing preloaded corn plants in separate growth chambers at 27 C and 10 C with continuous darkness. After 2A hr the plants were extracted and the results expressed as per cent of the initial dpm per mg fresh weight. Similar studies were conducted to study the influence of light on the loss of l[IO-dichlobenil. Preloaded corn plants were placed in a growth chamber at 27 C with continuous light. One—half of the plants were covered with a box which excluded all visible light. After 2A hr the plants were removed and extracted as previously described. Mode of Action of Dichlobenil Membrane permeability.-—The leakage of betacyanin from red beet (Beta vulgaris L. cv. Detroit Dark Red) root tissue was used as an assay for the effect of dichlobenil on membrane permeability. The technique adopted was modi- fied from Veldstra and Booij (63). Cylinders, A.0 mm in diameter, were taken from fresh red beet roots with a cork borer and cut into sections, 5.0 mm thick, with equally spaced coupled raxor blades. The sections were rinsed with distilled water until all pigment from dis- rupted cells had been removed. Any sections containing large amounts of vascular tissue or those which were ex— ceedingly colored were removed prior to initiating the 21 experiment. Ten root sections were placed in a 50 m1 erlenmeyer flask containing 20 ml of solution of the compound to be investigated. All treatments were pre— pared in distilled water containing 20 ppm of streptomy- cin sulfate. The flasks were stoppered with a porous plug and placed in a water bath at 30 C. After 2, A, 8, l2, l6, and 2A hr, 3.0 ml aliquots were removed from each flask and their absorbance measured at 5A0 mu on a Beckman DB-G spectrophotometer using fresh treating solu- tion as a blank. Immediately after removing the 3.0 ml l-rv- “llfl‘dk'. o2 aliquot for assay, 3.0 m1 fresh treating solution was padded to the flask. The leakage of reducing sugars into the ambient solution was also measured as an index of permeability changes. Nelson's Test (1A) was utilized on aliquots of the solution at each sampling period. All treatments were replicated 5 times and experiments were repeated at least 3 times. Respiration of intact tissues.—-The oxygen consump- tion of red beet root sections, described in the previous paragraph, was measured using manometric techniques (59). Two root sections were placed in a Warburg flask contain- ing A.0 ml of a solution of the compound being investi- gated plus 20 ppm of streptomycin sulfate. After allowing the flasks to equilibrate in a water bath at 30 C for 22 15 min, the manometers were closed and readihgs taken periodically over a 12 hr interval. At the end of each experiment, the tissue was removed from the flasks, dried, and weighed. Results were expressed as pl 02 consumed per mg dry weight. Similar experiments were carried out using roots from 5-day—old corn plants which had been grown in petri dishes. The roots were excised 3.0 cm E”' from the tip and 3 roots placed in a Warburg flask con- taining 3.0 ml of .05 M phosphate buffer (pH 6.5) to I 6 which 0, 5.8 x 10— and 5.8 x 10-5M dichlobenil had been added. Oxidative phosphorylation in isolated mitochondria.-- Mitochondria were isolated from etiolated, A-day-old cucum- ber hypocotyls or cotyledons. The seedlings were germi- nated on cheesecloth placed on hardware cloth suspended in a plastic tray containing tap water. The trays were covered with perforated polyethylene and placed in a growth chamber. All light was excluded from the chamber and the temperature was maintained at 30 C. Cotyledons and hypocotyls were excised and placed in separate beakers which were packed in ice until ex- traction. Prior to extraction, the cucumber hypocotyls were cut into segments 2 to 3 cm long and divided into 25 g lots. Each 25 g lot was macerated for 30 sec in a prechilled mortar and pestle with 50 ml of grinding medium. The cotyledons were also divided into 25 g lots 23 and ground with 50 m1 grinding medium, but the length of grinding was increased to 60 sec. All grinding and sub- sequent contrifugation operations were carried out between 0 and + A C. The mitochondria were isolated using the procedure of Ikuma and Bonner (29) which was modified by including 0.0HAN-2-hydroxyethyl piperazine—N-ethane sulfonic acid w. (HEPES) pH 7.2 in the grinding, washing and reaction mediums. The final volume of the mitochondrial suspen- sion was 1.0 to 1.A ml containing 15 to 25 mg protein as determined by micro—Kjeldahl. Oxygen uptake was measured polarographically using a Clark type oxygen electrode connected to an amplifier and recorder assembly. The electrode was encased in a lucite plunger which was placed in the sample chamber con- taining the reaction medium. Materials were introduced into the sample chamber by means of a hypodermic syringe inserted in an access groove in the plunger. The tempera— ture of the sample chamber was maintained at 25 C with a constant temperature circulating water bath. The reaction medium was thoroughly aerated prior to adding mitochondria and substrates. Respiratory rates were calculated from a recorder trace on the basis of 2A0 uM O2 (720 mumoles 02/ 3 ml) in the aerated medium as calculated by Chance and Williams (13). Oxygen consumption rates were expressed as mumoles O2/min per 3 ml of reaction medium. 2A All solutions were prepared in distilled water and sterile filtered through a 5 u Millipore filter immedi- ately before use. The concentration of ADP was measured spectrophotometrically at 260 mu on the basis of its mM extinction coefficient of lA.5. The concentration of other chemicals was measured gravimetrically. Stock solutions of dichlobenil, 2,6-dichloro-3-hydroxy- I“ benzonitrile and DNP were prepared in ethanol and added 3 to the reaction media in 5 ul aliquots. é Phosphate esterification and determination of P/O I ratios.--Time course phosphorylation experiments were conducted by placing 0.1 ml of the mitochondrial suspen- sion (0.15 mg mitochondrial N) in a 50 ml erlenmeyer flask containing 2.9 ml of the reaction medium to which 0.5 mg hexokinase and 50 mM glucose was added. After removing 0.1 ml for Pi determination, the flasks were stoppered and placed in a 25 C water bath. Aliquots were removed at 15, 30, A5, and 60 min and assayed for Pi by the Taussky and Shorr (58) method as modified by Penner (50). The Pi consumed was plotted as a function of time and the slopes compared using Student's T test. The P/O ratios were determined manometrically by placing 0.1 ml of the mitochondrial suspension (0.80 mg mitochondrial N) in Warburg flasks containing 2.7 ml of the hexokinase reaction medium. The substrate, 20 mM succinate, 820 mumoles ADP, and the inhibitor being 25 investigated were placed in the side arm in 0.3 ml of the reaction medium. After removing a 0.1 ml aliquot for Pi determination, the flasks were attached to the manometers and placed in a 25 C water bath. After allowing the flasks to equilibrate for 15 min, the manometers were closed and the substrate—inhibitor mixture tipped into the flasks. Oxygen consumption readings were taken at 10 min intervals for A0 min. The flasks were removed and immediately placed on ice prior to removing a 0.1 ml aliquot for Pi determination. The quantity of Pi esteri- fied at 0 and A0 min was determined as described above. 1A C—Glucose utilization.-—The utilization of C and 1 C6 labeled glucose by A-day-old cucumber seedlings was determined using a radiorespirometer. The device was similar to that designed by Wang (66). Single cucumber seedlings treated for 2A hr with lo‘5M dichlobenil were placed in reaction flasks and compared to controls. Each treatment was then divided and one half of the flasks received 3.0 ml of sterilized, distilled water containing 0.5 he of C labeled glucose (specific activity 3.3A mc/ 1 mM); the other half received the same quantity of C6 labeled glucose (specific activity 3.60 mc/mM). The flasks were attached to the CO2 trapping system and placed in a circulating water bath at 25 c. The 1“002 evolved was trapped in 10 ml ethanol:ethanolamine (1:1). After adjusting the air flow rate to 60 ml per min, the 26 air flow was diverted through the alternate l4002 trap containing fresh solution to initiate the experiment. The trapping solutions were sampled at 1 hr intervals for a period of A hr. At each sampling time, the ethanol: ethanolamine was removed and the chamber rinsed with 3.0 ml of ethanol. The trapping solution and washing were ad- justed to 15 ml with ethanol and a 5.0 ml aliquot removed P” for counting by liquid scintillation. At the termina- tion of each experiment, the tissue was removed from the reaction flask and oven dried. The sample was then com- a busted using the Sch6niger technique (67) and the 1“CO 2 . liberated was collected in ethanol:ethanolamine (1:1) for counting by liquid scintillation. Changps in free amino acids pools.--Oat seeds were planted in styrofoam cups containing quartz sand and watered with Hoagland's No. 2 solution. After 1 week, 6M or lo'5M dichlobenil were applied in the nutri- 0, 10- ent solution. Throughout the growth and treatment period, the plants were maintained in a growth chamber with a 16 hr photoperiodem 2A C and an 8 hr night at 18C. After 2A hr the plants were removed and all sand washed from the roots. Shoots and roots were placed in separate aluminum containers and quick-frozen in acetone-dry ice prior to lyophylization. One hundred mg of the dried tissue was ground in a mortar and pestle with 70% ethanol containing a small number of glass grinding beads. The homogenate 27 was transferred with several washings to a 50 ml poly- ethylene centrifuge tube; the total volume of the extract was 20 ml. After gentle boiling of the extract for 15 min, it was centrifuged at 1000 x g for 10 min. The supernatant was decanted and the residue extracted 2 more times with 70% ethanol. The supernatants were com— bined and condensed to dryness in a flash evaporator. F" The residue was then carefully taken up in 10% isopro- panol, 1.0 ml for root extracts and 2.0 ml for shoot ex- , tracts. The free amino acids present were determined by the ninhydrin colorimetric method described by Rosen (53). Results are expressed as umoles per mg dry weight. Lignin-like Polyphenol Accumulation Spectrophotometric assay.-—Three-day-old etiolated cucumber seedlings were placed on filter paper in petri dishes containing either 0, 10_6 or lO-SM dichlobenil. After A8 hr, the plants were removed, the cotyledons dis- carded, and the roots dissected from the hypocotyls. The tissue was oven dried and ground in a Wiley mill with a A0 mesh screen. Thirty mg of the dried tissue was ex- tracted with 3.0 ml of 0.5 N NaOH for 16 hr in a water bath shaker (70 C). Using the method of Stafford (56) the ultraviolet absorption spectra were determined on two aliquots. One was diluted with 0.05 N NaOH (pH 12.3) and the other with 0.05 M phosphate buffer (pH 7.0), the 28 difference spectrum being obtained by subtraction. Ab- sorbance was measured using a Beckman DB-G spectrOphoto- meter at intervals of 10 mp from 230 to A50 mu. Similar studies were conducted using l—week—old oat plants which had been grown in quartz sand. Histological test for lignin.--Three—day—old cucum- ber seedlings in petri dishes were treated for A8 hr with [- distilled water or lO-SM dichlobenil. The roots were I killed and fixed in FAA (A0% formaldehyde-acetic acid— 50% ethanol 5:5:90 v/v/v). After dehydration of the tissue through a tertiary-butyl alcohol series (32), it was in— L1 filtrated with paraffin and imbedded in Tissuemat. Sec- tions 30 u thick were prepared on a rotary microtome and affixed to glass slides using Haupt's adhesive. The paraffin was removed with xylene and the sections stained with phloroglucinol:HCl (5A). RESULTS AND DISCUSSION Toxicity of Dichlobenil to Quackgrass Sprouting of axillary buds was completely inhibited 6 F‘s-.1. M (Figure 2). The lethal con- , 6 by dichlobenil at 2.91 x 10— centration for these active buds was between 0.58 x 10- and 2.91 x 10-6M. Established plants are more tolerant, p)... . I however, their dry weight was reduced 50% when 5.8 x 10-6 M dichlobenil was added to the nutrient solution for ._l a 2 week period. The susceptibility of established plants was increased by removing the shoots prior to application of dichlobenil to the nutrient solution (Table 1). Growth from intercalary meristems was completely inhibited. This data confirmed field results where spring applications of dichlobenil provided better quackgrass control when the tops were removed either by mechanical mowing or applica- tion of paraquat (51). These results agree with several workers (33, Al, A6) who have found dichlobenil to be most toxic to meristematic tissue and germinating seeds. The fact that dichlobenil is less toxic to intact plants may be due to several factors; it is metabolized to a non—toxic form, it becomes complexed with plant constituents, or the concentration in the shoots is so dilute that it cannot 29 30 No. of Active Buds per Pot .. fin C>I ~J CD 4) f \ 5 DO 48 O .58 1.16 1.74 2.32 2. 1 Dichlobenil Conc. (X10'6MI Figure 2. Effect of dichlobenil on growth of axillary buds of quackgrass. 31 accumulate at its site of action. To answer these questions, uptake studies were carried out using lAC_ dichlobenil. TABLE 1.—-The growth of intact and clipped quackgrass plants receiving dichlobenil.l Dichlobenil conc Dry Wt (8) (M) Clipped Intact O .37 1.33 5.8 x 10"6 .08 .63 2.9 x 10‘5 .04 .38 5.8 x 10-5 .03 .A8 1The F value for clipped vs intact X linear re- sponse to dichlobenil is significant at the .05 level. Uptake and Translocation Studies With rapid aeration (100 ml air per min) of the 1“C- dichlobenil solutionS, 57% of the radioactivity was lost from the solution within 12 hr (Figure 3). This loss of radioactivity was not accounted for by plant uptake, which suggested volatilization directly from the nutrient solu- tion. With this aeration system, the highest concentra- tion of lLIC-dichlobenil in corn roots was attained at A hr and then continuously decreased until the experiment was terminated at 72 hr (Figure AA). The decrease in 'C' .-'t ‘. 100 o In)I an a 10 :60 .3; 50 E40 ‘ ‘ .230 E20 10 32 100 80 70 60 A Minimal Aeration 50 ° 0 Rapid Aeration 40 30 20 °. 0 10 .H'LQ1 >2: 5 IV“ ”A? J 0412 24 36 72 Figure 3. Time (In) The per cent of the initial CPM in the nutrient solution, using minimal (15 ml/min) and rapid aeration (100 ml/min). At 36 hr, the minimal aeration scheme was brought up to the original concentration by adding sufficient lAc-diohlohenil. Figure A. 33 N U! N c: d U! 10 DP Jl’ng Fresh Weight lime ( In] 50 r 50 ° I 40 ' 40 A Root mi. 0 SIM“ .u 30 s 20 ”P56! Fresh Weight ‘10 l 0 - II 24 35 72 time [hr] Radioactivity recovered from the roots and shoot of co n plants grown in nutrient solution contain- ing 1 C-dichlobenil with rapid aeration (100 ml/ min) (A) and mineral aeration (15 ml/min) (B). 3A lLIC—dichlobenil in the roots closely paralleled the de- 1A crease of C—dichlobenil in the nutrient solution. The concentration of lLIC—dichlobenil in the shoots continued to increase for 36 hr but decreased over the subsequent 36 hr (Figure AA). The data indicates that with a limited source of lac-dichlobenil, there is a net loss of radioactivity from the shoots. The observed loss may E“‘ be a result of volatilization from the leaves, a recycl- I ing and loss from the roots, or metabolism of the herbi- é cide to luCO2. I 1A ' A relatively constant concentration of C- dichlobenil in the nutrient solution was attained by reducing the aeration to a minimum (15 ml/min) and adding sufficient l[IO—dichlobenil after 36 hr to return the nutrient solution to its original concentration (Figure 3). Utilizing this procedure, both roots and shoots contained increasing amounts of radioactivity up to 72 hr (Figure AB). Under these conditions, any loss of lLIC-dichlobenil from the shoots was masked by con- tinued uptake. Volatilization of Dichlobenil from Plants Corn plants extracted immediately after the 2A hr preloading period contained 6A% of the total radioactivity in the roots and 36% in the shoots. However, after 2A hr in the vapor trapping device, only 12% and 7% of the 35 initial radioactivity was detected in the root and shoot respectively (Figure 5). Of the initial radioactivity, 10% was recovered in the toluene traps. The l[IO—compound had the same Rf value as dichlobenil on thin layer chroma- tograms utilizing two solvent systems. No radioactivity was recovered from stoppered control flasks containing nutrient solution to which 0.05 no luC-dichlobenil had been added. These experiments show that lLIC-dichlobenil is transported to the shoots and may volatilize directly from the leaves of corn plants to the atmosphere. Massini (Al) showed that vapors of dichlobenil can be readily taken up by all aerial plant parts, thus it seems logical that the reverse process could also occur. The largest quantity (71%) of the radioactivity was detected in the nutrient solution (Figure 5), indi- cating rapid efflux of dichlobenil from the roots with the reversed concentration gradient. Since the amount lost to the nutrient solution plus that recovered from the roots was greater than the initial quantity in the roots after preloading, there appeared to be limited cycl- ing of l[IO—dichlobenil from the shoot to the root. The total amount of radioactivity recovered after the 2A hr trapping period was within 5% of the initial radio- activity in the preloaded plants. 1A The efflux of C—dichlobenil from the shoots and roots of preloaded corn plants over a 2A hr period was - n’flfllbLflnj 36 no 173237 333 133 333 ‘ 90 80 70 60 50 40 30 ‘ 20 I0 'Nutrient solution gimme traps Percent of total IIPM After 24-!" AIter24-llr Preloarl flapping , Figure 5. The distribution of radioactivfity in corn plants after preloading with 1 C-dichlobenil for 2A hr followed by 2A hr vapor trapping period. 37 similar in the light and dark at 27 C (Figure 6). This supports the hypothesis that the volatilization of dichlo- benil is not primarily through open stomates but occurs directly through the cuticle. At 10 0 there was only a 6% loss in radioactivity from the shoots, however, at 27 C the shoot contained only 58% of the initial radioactivity (Figure 7). Similarly, N_ :7 the efflux from the roots was greater at the higher tem- perature. At both temperatures the efflux from the roots was much greater than that from the shoots. The rapid efflux of luC—dichlobenil from the roots E— of preloaded plants when placed in nutrient solution indi— I r cates that it is not actively held within the root cells, but readily passes through intervening membranes and cell walls. The lipOphilic nature of the dichlobenil molecule may explain the apparent ease of movement through the plasmalemma and perhaps, other membranes. Another possi- bility is that the dichlobenil molecule partitions into the lipid-bilayer causing a disruption of the specific permeability properties of membranes.' Hence, not only dichlobenil, but other soluble cell substances could be expected to leak from the treated root tissue. Mode of Action of Dichlobenil Membrane permeability.--The efflux of betacyanin 6 from red beet root sections incubated in 10- , 10-5, or Figure 6. Figure 7. Percent of initial Mt“ Fresh Weight 1L lrgllt Dark The per cent of initial radioactivity remaining in preloaded corn plants after 2A hr in the light or dark at 27 C. Percent of initial ”'9‘, fresh Weight The per cent of initial radioactivity remaining in preloaded corn plants after exposure to 27 C and 10 C for a 2A hr loss period. ‘ 39 lO-MM solutions of dichlobenil was characterized by a lag phase for the first 8 hr (Figure 8A). However, after 8 hr of incubation, the leakage was rapid and increased linearly until 2A hr when the experiment was terminated. When equimolar concentrations of DNP were used, only the lO-uM concentration caused a significant increase in betacyanin leakage into the ambient solution (Figure 8B). With lO-u M DNP, rapid leakage occurred within 2 hr of incubation and subsequent efflux of betacyanin continued over the 2A hr period. Since DNP influenced permeability more rapidly than dichlobenil, the rate of uptake of the compounds into the tissue may differ or the two compounds may affect permeability in different manners. DNP is a well known uncoupler of oxidative phosphorylation, and its effect on permeability may be due to a reduction of available energy (ATP) necessary to maintain the integrity of the tonoplast and plasmalemma. With dichlobenil, a time interval is required either to accumulate a threshold concentration necessary to affect both membranes or to induce physiological changes preceding permeability changes. An alternative hypothesis is that dichlobenil itself is inactive, and must first be metabolized to a form which is active. When Ca++, which is known to stabilize membranes (37, A A0, 61) was added as 1.6 x 10- M CaCl to the incubating 2 solution at 12 hr, it reduced the efflux of betacyanin "I Figure 8. PER CENT OF CONTROL PERCENT OF CONIROL A0 .00“ . A .10‘4M orcntoumt o 700‘ 010'5M DICHtOIENll .10"M orcutoeemr 600- 500‘ l O O A 400~ 3004 200. ’//////////////// A o%/ o 100d V I 1' U U U P 7 U 1 # T V 2 4 8 16 24 TIME (HR) 800- .ro"m 2,4-0mruomenor 010'5M 2,4-0mrraoeuenor arc-6» 2.4-0mrreoeneuor 3 700‘ ///////,/’//////// 600‘ a 0 Cl 1 . 0 ° l "3' '\ YIAIE (Hll Leakage of betacyanin, expressed as per cent of control, from red beet root sections treated with dichlobenil (A) and 2,A—dinitr0phenol (B). A1 A induced by 10— M dichlobenil (Figure 9A). However, a similar concentration of CaCl2 added to red beet root sec- uM DNP for 12 hr had no effect on tions treated with 10- subsequent leakage (Figure 9B). Again, there is an indi- cation that the two compounds are acting differently. The membranes of the tissue treated with DNP may be irre- versibly disrupted such that they cannot be stabilized :- with CaFt When 1.6 x lo-uM CaCl2 was added to the incubat- ing solution initially, it alleviated the affect of 10’5M dichlobenil and lo-uM DNP similarly (Figures 10A and 10B). The leakage of reducing sugars from red beet root 6 sections treated with 10‘ and lo'5M dichlobenil occurred in a similar manner as betacyanin (Figure 11). DNP at lO-Ll M, however, induced no leakage of reducing sugars. It may be postulated that the DNP treated tissue had metabolized all free reducing sugars due to the uncoupled respiration, hence, no reducing sugars were available for leakage into the ambient solutions. Respiration of intact tissue.-—The rate of 02 con- sumption by red beet root sections incubated in 10"6 or lO-SM dichlobenil increased in the same magnitude as with lO-Ll M DNP (Figure 12). The rate of O2 consumption of all treatments was significantly greater than the control. Thus, the difference observed in leakage of reducing sugars from red beet root sections, treated with dichlo- benil or DNP, cannot be explained by differences in :respiration. The increased 02 consumption of the 60 PER CENI OF CONIROL A2 ro"M orcertosemt _ -CoCl2 ’ / -..-- .1.oxro"M (:oCI2 TIME (Hm em: 10'434 2,4—omrrnoeusuor 3 - O .3 —— CoCl2 . 2 ---- . 1.3.10'434 CoC|2 , ,,,,,, .600 l 0 U 5 Como .- 2 III U 8 “‘300 L Figure 9. 4 a 12 16 24 YIME (HR) Effect of Ca++, added at 12 hr on leakage of betacyanin induced by treatment of red beet roo section with lo-AM dichlobenil (A) and 10- M 2, A-dinitrophenol (B). - nil..f ' my A*~‘.g‘ PER CENT OF CONTROL Figure 10. en ceur or CONIROL A3 0 orsrrueo H20 01.6110"M Cocr, o 30"» orcmoeenrt 700‘ . ro‘5M orcntoumt o r.ono"M coco, eoo« soc-4 O A 400- / A 300- 200‘ /o . 1" O / 34’, o T Y r I T ' o 4 a 12 16 20 24 rme (HR) 7 - ”I o orsrrtteo H20 . Leno ‘M CoCl2 3 600- 500-3 400- 300- 2% o ro"~r 2,4-ormreoeweuot . ro"~r 2,4—DINITROPHENOL + r.o::ro"~r Cocr, /? A O A ,/ A 100 , ____.__—————-—° 0 o 0 I I V fl I T I 0 4 l 12 16 20 24 TIME (NR) [4 .. 'The influence of 1.6 x 10' M CaClg on the leakage e of betacyanin from red beet root ctions induced by lo-5M dichlobenil (A) and lo-“M 2,A-dinitro- phenol (B). The CaCl2 was added to the incubating solution initially. AA 32 o CONTROL 0 10"M 2,4-omrraoew‘euoc as a 10'5M orcmoseurt V . 10'6M orcutosemt 24 20 . E . a I w . .... I. 12 SlJGJlRS pg REDUCING O rm: (m Figure 11. Time course of the leakage of reducing sugars from red beet root sections treated with dichlobenil or 2,A-dinitrophenol. A5 dichlobenil treated sections appears to be typical of that occurring in an uncoupled system. Similar results were obtained with corn root tips incubated in buffer containing 3 concentrations of dichlobenil (Figure 13). The experiment failed to show a significant difference between concentrations, however, the rates of O2 consump- tion for all dichlobenil treatments were significantly If. greater than the control. Since both experiments showed no differences between concentrations of dichlobenil, it ; apparently affects lg 1319 respiration at a very low con- I centration. Although the increased respiration rate induced by dichlobenil is indicative of uncoupled oxida- tive phosphorylation, this is not conclusive and must be verified with concomitant measurement of Pi esterifica- tion in isolated mitochondria. Oxidative phosphorylation in isolated mitochondria.-— When lO-SM dichlobenil was added during state A, there was no stimulation of the ADP dependent respiration (Figure 1AA) as was induced by 8 x lO-5M DNP (Figure lAB). Sub- sequent additions of ADP after dichlobenil treatment showed that the phosphorylation mechanism was coupled and typical respiratory control and ADP:O ratios were maintained. Similar results were obtained using rates as high as lO-HM dichlobenil on mitochondria isolated form cucumber hypocotyls, potato tubers and cauliflower florets. Dichlobenil at lO-EM also did not circumvent A6 "1 15‘ p- ‘9 .4 U 3 -l > 9" I a d ' O I 8.4 s t ,/’ o a. / o cournor. -l O a /a/ o 13:61. orcrrtouurt 0 3d / s .. 3/ c 10' M DICHLOIENIL R ‘ / o Ill-‘3: omrreoeueum "ME (HR) Figure 12. 0 consumption of red bee root sections incu— hEted ifl solutions of 10- or lo-5M dichlobenil and 10' M dinitrophenol. 2|- 26-1 I u ° C 12. E 9 1" ; 0 ll- ' > 3 u~ 3 w . E u- A. a I" x 3 .. n u 0 cannot c U 3‘ 0 51-16533 orcutoumt 6‘ ‘ a 53.36% orcutoumt .. 0 3.0-30"“ orcrrtouem R / f r I ‘ 0 1 2 1 4 ma tall Figure 13. 0; consumption of corn root tips incubated in .05M phosphate buffer (pH 6.5) containing various concentrations of dichlobenil. “7 T r $.13... ill. I I .SSHooE coauomon go as m you :Hs\moaos :8 CH oxmuas mo no mopon ohm moomnp on» macaw mnonasz opmcfiooSm mo coapmppcoocoo one .2: mm ma one no coaoaooo some one as a ma .mcopoahuoo nonESoSo 809m oopmHomH “Z we om.v mahoconoOpHE CH coapmnaamon pcoocoqoo ma¢ co Amy maakpficouconzxomomzrmrouoasofio to.m 2mroa one .o. oaoo oaoncooosoflnoaorm.m 2mroH ..m. Hoconoosoacaore.m zmuoa x m ..a. Hacoooanoao zmroa mo coouco I 1 I 4 I4 ...... 2:23.... - :22: -m -.....o_a-o.~ o... .o.~ : . no _ vn . _ 82.3.5 :- s... ...: ...... .... o r a 9.3. on nu_olgsounn.o ...... 2 .23.... -2:..E.1.n K 8.. v... =. 3. Ba _. .o. 3.. _ ....- ..E ...... . ...... .. on» mcflzonm moomnp canamnwonmaom ’ Qua° 3. mad 8. cm.— A: 8. 86 = no. OK.— ..3 ...: ...: can. u- !3 :2... $8.35-?“ K .:H opswfim .21.. n 3... glows"... um. mo.— mo.— on. ....- 93¢ __..:_o.._ua no.— ,— no.~ :— 3...“ = 3.. _ .3.- at: u- 93.... n =... .... 8h... U8 oligomycin inhibited O uptake (Figure 15A) as is typical 2 of most uncoupling agents (30). These results conflict with those reported by Foy and Penner (20) who found dichlobenil at 0.73 x lO-uM to be an effective uncoupler of oxidative phosphorylation as determined by manometric techniques. This discrepancy in results may be due to the concentration of dichlobenil used, however, concentrations which increased in Kilo oxygen consumption did not un— couple oxidative phosphorylation in mitochondrial prepara- tions. One metabolite of dichlobenil, 2,6-dichlorobenzoic acid, which has been found in plants (46), also had no effect on ADP-limited respiration (Figure 14C). However, 2,6-dichloro-3—hydroxybenzonitrile was found to stimulate state U respiration and eliminate subsequent respiratory control with ADP at lO-SM (Figure 14D). It also circum- vented oligomycin inhibited O uptake (Figure 15B) at the 2 same concentration. This compound at 3.“ x lO-SM was found by Wit and van Genderen (71) to cause a signifi- cant rise in 0 consumption of starved yeast cells incu- 2 bated with a small amount of glucose. It also induced ATPase activity in isolated, intact rat liver mitochondria. Dichlobenil had no effect in either of these assays indi- cating that the monophenolic metabolite is the toxic form in animals. Currently this metabolite has been reported in plants only after extended periods of treatment with a i Figure 15. 0.: 720 I p Iolgs II Mint. “9 {a =720 II '5'": I: mm | Cycle nu. Iatio -| L36 ..94 l|~ 2.15. 1.08 “P licjlobuil .IIII. A A- A A A A L It “'80 I Cult In“. In“. I 1.61 1.08 H 2.47 1.26 Oil". Polarographic traces sh wing the effect of lO-SM dichlobenil (A) and 10' M 2,6-dichloro-3- hydroxybenzonitrile (B) on oligomycin (150 mug) inhibited respiration in mitochondria (.20 mg N) isolated from cucumber cotylendons. The concen-” tration of succinate is 8 mM and each addition of ADP is 55 uM. Numbers along the traces are rates of O uptake in mu moles/min per 3 ml of reaction mgdium. 50 related herbicide, 2,6—dichlorothiobenzamide (chlor— thiomid) (9). Since plants do contain many hydroxylase and mixed-function oxidase enzymes,the formation of the monophenolic metabolite from dichlobenil seems feasi- ble. P/O ratios determined by manometric techniques.-- The quantity of Pi esterfied and O consumed was not $- I 6M, 2,6- I 2 affected by 2 x 10‘5M dichlobenil or 2 x 10‘ dichloro-3—hydroxybenzonitrile, therefore, their P/O ratios are similar (Table 2). The monophenolic metabolite at 2 x lO-SM reduced the 0 consumption slightly and re- 2 duced the Pi esterified by nearly 70%, consequently lower- ing the P/O ratio to 0.42. Although DNP did not influence the 02 consumption, Pi esterification was reduced but not as greatly as with the monophenolic metabolite. The P/O ratios substantiate the polarographic determination that dichlobenil at these concentrations does not uncouple oxidative phosphorylation. The suppression of 02 con- sumption by 2 x lO-BM 2,6-dichloro-3-hydroxybenzonitrile was evident only after the first 10 min of the experiment, consequently it would not have been detected in the polaro- graphic determinations where the O2 utilization was mea— sured for only 2 to 3 min after adding the compound. Pi esterification.--The rate of Pi esterified by cucumber hypocotyl mitochondria incubated in a reaction medium containing 10‘5M dichlobenil was not significantly 51 TABLE 2.--Effect of dichlobenil, 2,6—dichloro-3-hydroxy- benzonitrile and DNP on oxidative phosphorylation by cucumber cotyledon mitochondria.l Conc Pi esterified O consumed P/O Inhibitor (Mx2) mole/40 min uatgméuo ratio None 16.5 a2 u7.73 1.03 a2 Dichlobenil 10"5 19.1 a 50.u 1.16 a 2,6-dichloro- 3-hydroxy- -6 , benzonitrile 10 15.6 a “9.5 0.96 ab , 2,6-dichloro- ' 3-hydroxy- _5 benzonitrile 10 5.1 c 36.0 0.42 c DNP 10‘5 9.8 b uu.9 0.66 bc 1 20 mM succinate utilized as substrate. 2Within columns, means followed by the same letters are not significantly different at the .01 probability level as determined by Duncan's multiple range test. 3F value for comparison of control with 2 x 10-5M 2,6-dichloro-3-hydroxybenzonitri1e significantly different at the .05 probability level. 52 different than the control (Figure 16). An equimolar concentration of the monOphenolic metabolite, however reduced the rate of Pi esterification compared to both the control and dichlobenil treatments. lac-Glucose utilization.--The utilization of both 01 and C6 labeled glucose by cucumber seedlings was doubled by treatment with 10‘5M dichlobenil for 2h hr (Table 3). e. The ratio of C6 to Cl glucose utilized was not signifi- cantly altered by the dichlobenil treatment. The smaller amount of radioactivity remaining in the dichlobenil treated tissue at the termination of the experiments is also indicative of increased glucose utilization. In- creased glucose utilization is expected,based on previous observations of increased 02 consumption by intact tissue. Increased catabolism may be due to uncoupling of oxidative phosphorylation induced by dichlobenil or alleviation of a limiting factor in the respiration mechanism. Since there was no change in the C6/Cl ratio, the same pathways- of glucose metabolism appear to be operative in the treated tissue, albeit at a faster rate. Amino acid accumulation.--Within 2“ hr after treating 6 oat seedlings with 10' or lO'SM dichlobenil there was an increase in the total free amino acid content of both shoots and roots (Table 1). This accumulation may be due to reduced incorporation of the amino acids into protein 53 700 o CONIROL ‘o1o‘5M DICHLOBENIL o . 10'5M 2.6-DICHLORO-3- vuvoaoxvasnzounnus m / / 600 400 300 A pg? CONSUMED 200 100 ' O 15 ' 3O 4 YINQE (II!!!) «a 0' ‘0 Figure 16. Time course of Pi uptake by mitochondria (.16 mg N) isolated from cucumber hypocotyls. 54 TABLE 3.--Effect of 10‘5 dichlobenil on the utilization of 1401 and 1406 labeled glucose by 4-day-old cucumber seedlings.l 14 14 F C02 Evolved C-Remaining Dichlobenil C5/C from fresh tissue in dry tissue Conc R til (opm/mg) (opm/mg) (M) a 0 C1 C6 C1 C6 0 .62. 158 a 79 a 6394 a 7811 a 10‘5 .40 373 b 140 b 2813 b 3615 b 1 Within columns, means followed by the same letters are not significantly different at the .01 probability level as determined by analysis of variance. Data are means of 16 observations. 55 because oxidative phosphorylation is uncoupled and the necessary ATP is unavailable. Also, as noted earlier, respiration in the treated tissue is increased, hence, amino acids may be produced at a faster rate without con— comitant incorporation into protein, or more protein may be hydrolyzed. TABLE 4.-—The free amino acid content of oat plants treated with dichlobenil. Dichlobenil conc uMoles/mg dry Wt I“: ..'~.("-' (M) Shoot Root 5 0 .173 a . .089 a 5—5 10'6 .211 b .oau a 10'5 .227 c .107 b 1 Within columns, means followed by the same letters are not significantly different at the .01 probability level as determined by Duncan's multiple range test. Effect of Dichlobenil on the Formation of Lignin-like Polyphenols Spectrophotometric determination.—-The difference spectra for cucumber hypocotyl and root alkaline extracts are shown in Figures 17A and 17B, respectively. Aulin- Erdtman (6) has shown that the peak at 280-300 mu rep- resents the non-conJugated phenols, while phenols with large conjugated side chains such as hydroxycinnamic acid derivatives will account for peaks at wave lengths greater 56 .200- ‘ oCONTlOI. .10'°M oncuiounu era's» DICHLOIENIL .1004 200 300 320 340 360 300 400 420 ‘40 460 WAVE lENGTH (mp) .200. a '1 oCON‘rIOl no“M DICHLOIENII. .10'5M DICHLOIENIL .1004 ‘fi’\_ '\ x»,- .050 200 300 320 340 360 300 400 4 20 440 460 WAVE [ENGYH (my) Figure 17. Difference spectra 0f lignin extracts of cucum- ber hypocotyl (A) and root tiSsUe (B). Each ml of solution in the curvette contains an aliquot of a lignin extract equivalent to 1.5 mg dry wt root tissue, diluted either with 0.05 M phos- phate buffer at pH 7.0 or with 0.05 M NaOH at pH 12.3. - 57 than 300 mu. Based on these difference spectra, extracts 6 and 10‘5M from cucumber hypocotyls treated with 10' dichlobenil have a significantly higher peak at 350 mu than that of the control. However, the AD (350 mu) of the extract from treated root tissue was not different from the control. Only the 10-5M dichlobenil concentra- tion significantly increased the AD (350 mu) of extracts m- from oat shoots (Figure 18A), however, both concentra— 5 tions of dichlobenil significantly increased the AD at this wave length in the root extracts (Figure 18B). Dichlobenil causes an increase of lignin-like sub- 111‘.."". ’.~- ‘ .' ‘ stances in both cucumbers and oats within 48 hr after treatment. Reaction of dichlobenil treated tissue with phloroglucinol-HCl.--Mounted sections from cucumber roots treated with 10'5M dichlobenil for 48 hr showed extensive red coloration of the epidermal and outer cortical cells when stained with phloroglucinol—HCl (Figure 19). This constitutes a positive test for lignin, however, Jensen (31) points out that tannins and other polyphenolic com- pounds related to lignin will react similarly with phloro- glucinol-HCl. The lignin-like substances detected in these sections generally appeared as amorphous masses both within the cells and in the intercellular spaces. The stained area was localized in epidermal cells and parenchyma cells of the outer region of the cortex. Sections from 58 s«> A oCONtIOl £00 ' .no"u'oucuioneunt IIO'3M~D|CHlOIENIl 320 330 340 350 360 370 300 “IA V E l E N‘Gr? H (tau) I o C ODN T [‘0 l 010'6M oncwtouuu .10'5M DICHtOIENUL a .m . o/°\o : O .500 .000 320 330 340 350 360 370 300 WAVE lENGYH(nfl Figure 18. Difference spectra of lignin extracts of oat shoots (A) and roots (B). Each ml of solution in the cuvette contains an aliquot of the lignin extract equivalent to 1.0 mg dry wt shoot tissue or .5 mg dry wt root tissue diluted either with 0.05 M phosphate buffer at pH 7.0 or with 0.05 M NaOH at pH 12.3. 59 untreated roots showed only the normal lignification associated with the secondary thickening of the xylem vessels. A 'V 2"); ’ m ( Ire—o" 60 I. ’I“ 11-..'.‘ )In. to»...- ‘ f ' - Figure 19. Comparison of the accumulation of polyphenolic substances after 48 hr in roots of control (A, lon itudinal section) (C, cross section) and 10' M dichlobenil (B, longitudinal section)(D, cross section) treated cucumber roots (125 x). Sections are 30 p thick and stained with phloroglucinol-HCl. SUMMARY AND CONCLUSIONS Dichlobenil is rapidly taken up by the roots of corn plants from an aerated nutrient solution and trans- located to all plant parts via the xylem. The unaltered compound may be volatilized directly from the leaf into the atmosphere. Volatilization from the leaf is tempera- ture dependent and light independent. Due to the lipo- philic nature of dichlobenil, it has the capability of rapidly traversing membranes, and cuticle layers. In the leaf, which is subject to relatively high temperatures, a portion of the dichlobenil may be present in the vapor phase and thus be subject to volatility loss. Under circumstances of limited dichlobenil uptake by the roots, it does not accumulate in the shoots due to the concomi- tant volatility loss from the leaves. The relatively unrestricted movement of dichlobenil in plant tissue is also evident in the root, where it readily diffuses from preloaded corn roots when placed in fresh nutrient solution. Dichlobenil also induces the leakage of betacyanin and reducing sugars from red beet root sections, indicating a disruption of the tonoplast and plasmalemma. It may be postulated that dichlobenil 61 62 partitions into the lipid bilayer of the membranes and causes them to be more permeable to cell constituents. + , however, the membranes are In the presence of Ca+ stabilized and either the dichlobenil within the membrane has a reduced effect on permeability or inhibits dichlo- benil from penetrating the membrane. DNP induces the leakage of betacyanin more rapidly than dichlobenil, but does not influence the leakage of reducing sugars. Since DNP is a known uncoupler of oxidative phosphory- lation, its effect on permeability may be due to a lack of ATP necessary to maintain membrane structure. y Differences in the rate and reversibility of DNP and dichlobenil induced permeability changes indicate that their initial site of action may be different. Red beet root sections and corn roots when incu- 6 bated in 10‘ to 10‘5M solutions of dichlobenil exhibit increased 02 consumption which is similar to that pro- 4 duced by 10' M DNP. Cucumber seedlings treated with lO-SM dichlobenil for 24 hr also show increased respira- tion as evidenced by the 2-fold increase in the amount of C and C6 labeled glucose utilized. Although this indi- l cates uncoupled respiration, isolated mitochondria were unaffected by 10'5 to 10-4 M dichlobenil when assayed using the oxygen electrode, manometric techniques, or Pi con- sumption assays. The monophenolic metabolite of dichlo- benil (2,6-dichloro-3-hydroxybenzonitri1e), however, did 63 act as an uncoupler of oxidative phosphorylation in all 3 assay techniques. The free amino acid content of roots and shoots of oat plants increased within 24 hr after treating with lO-SM dichlobenil. This accumulation may be due to re- duced incorporation of amino acids into protein because oxidative phosphorylation is uncoupled and the necessary ATP is unavailable. Also, amino acids may be formed at a faster rate because of the increased glucose utiliza- tion. The oxidation products of endogenous phenolic com- pounds have been shown to be uncouplers of oxidative phosphorylation in mitochondria isolated from potato tubers (57) and sweet potato roots (35). These phenolic compounds, which are found in nearly all higher plants (10), are generally located within the vacuole (18) and thus spacially separated from the phenol oxidizing enzymes. Dichlobenil may destroy this compartmentation by altering the permeability of the tonoplast. Subsequently, the phenols would become available for oxidation by phenol- oxidase, peroxidase or other oxidizing enzymes. The quinones and polyphenols formed by non-enzymatic polymeri- zation reactions could inhibit oxidative phosphorylation. Spectrophotometric assay of alkaline extracts of cucumber and oat seedlings indicated that lO-SM dichlo- benil induced the formation of lignin-like polyphenols 64 within 48 and 72 hr respectively. This would lend evi- dence that the oxidation products of phenols released from the vacuole are being conjugated into polyphenolic com- pounds. Histological evidence for this was also obtained. Cucumber root sections upon staining with phloroglucinol- HCl, manifested red amorphous masses around the periphery of the root. The polyphenolic compounds are specifically indicated by this procedure. The phenol oxidizing en- zymes generally utilize molecular oxygen as an electron acceptor, but the concentration of oxygen required for half maximal activity is very high compared to that of respiration (3). Thus, it is only around the outside of the root that the partial pressure of oxygen is high enough to support phenolase activity. A similar mechanism might also be postulated to explain the coagulation of cytoplasm in the phloem and surrounding parenchma of the nodal tissue of dichlobenil treated alligator weed noted by Pate and Funderburk (45). Polyphenols react rapidly to form covalent bonds with protein, thus causing denaturation (36). It is by this mechanism that polyphenols are thought to inhibit enzymes and subcellular organelles (3, 28). LIST OF REFERENCES LIST OF REFERENCES Ahrens, J. F. 1966. Presistence in soil of dichlo— benil and EPTC applied for quackgrass control in ornamentals. Proc. NEWCC. 20:630-631. 1968. 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