l‘ H ‘i ‘ M H} “’| ll (1 l‘l ‘1‘} yTl‘i H“ ‘ \ wls “ “‘3 H ’ ‘ “I M > w M y H 1‘ 1 M ‘ t ‘1 e; W. ‘4 i.‘ W11: ‘» M L ‘V ml ‘1‘ I I‘ w‘ \M ‘ ~13 I ‘ 7 THE INFLUENCE OF OILS ON THE TOXICH'Y 0F 3 - AMlNO- l, 2, 4- TRIAZOLE 0N QUACKGMSS, (AGROPYRON REPENS (L) BEAUV.) Thesis for the Degree of M. S. MICHIGAN STATE UNEVERSITY FREDERICK ONOUNURAUETE AYA 1967 IL. -4 _tv in ’J) - 3‘!“ - - A... I,’ !‘ LIB-R R Y Liichigan State ‘ UnLversity ‘L D "WI. ABSTRACT THE INFLUENCE OF OILS ON THE TOXICITY OF 3-AMINO-l,2,4-TRIAZOLE ON QUACKGRASS (AGROPYRON REPENS (L.) BEAUV.) by Frederick O. Aya The combination of 3-amino-l,2,4-triazole (amitrole) with ammonium thiocyanate (NH SCN) in equimolar proportions has been re- 4 ported to effectively control the top growth of quackgrass (Agropzron repens (L.) Beauv.), a formidable primary noxious weed in North America. The need for a more effective and economic control of the weed led to the inclusion of certain mineral oils in a number of herbicide sprays both in the field and under greenhouse conditions. These were non-phytotoxic paraffinic and naphthenic type oils which had proven effective in enhancing the activity of insecticides, fungicides and miticides. In field trials, the oils enhanced the effectiveness of amitrole and amitrole—T on quackgrass when included in the herbicidal sprays at the rate of 2.0 gpa, buttuntaconsistently increased the toxicity of foliar-applied dalapon, diuron, paraquat, simazine or terbacil. Under greenhouse conditions, a paraffinic mineral oil (11E) en- hanced the activity of amitrole on quackgrass to a greater degree than NH4SCN. The application of a solution of 84 ppm of nitrogen as NH4N03 to the quackgrass also increased the effectiveness of amitrole-T in combination with the oil. Frederick Aya - 2 In studies with isotopes, the inclusion of a paraffinic mineral oil (llE) in herbicidal treatments at the rate of 6% (v/v) increased the absorption of 14C amitrole (8h%) and amitrole-T (26%) by quack- grass, #8 hours following foliar applications. The greater portion of the applied radioactivity moved in an acropetal direction initially in each case, but while the rate of this movement decreased with time, the rate of basipetal translocatlon increased. The oil enhanced both the amount and the rate of 1“C amitrole and amitrole-T translocated from the treated leaf to the shoot and roots, this being more pronounced with amitrole (twelvefold) than with amitrole-T (twofold). These studies suggest that the observed enhancement of amitrole and amitrole-T activity on quackgrass by the oils is due to an increase in the amount of the herbicide absorbed and translocated by the plant. THE INFLUENCE OF OILS ON THE TOXICITY 0F 3-AMINO-l,2,h-TRIAZOLE 0N QUACKGRASS, (AGROPYRON REPENS (L.) BEAUV.) BY Frederick Onounuraijete Aya A THESIS Submitted to Michigan State University _ in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture I967 ACKNOWLEDGEMENTS The author is indebted to Dr. S. K. Ries for his guidance and suggestions throughout this study and in the preparation of this thesis. Thanks are also due to the members of the examining com- mittee: Drs. D. R. Dilley, C. M. Harrison, A. E. Mitchell and A. R. Putnam. Sincere gratitude Is hereby expressed to Messrs. A. 0. Rewane and S. J. Okudu for their encouragement and financial assistance at a most trying period of my academic career and to both the Nigerian Institute for Oil Palm Research and the United States Agency for International Development for their scholarship which made this study possible. Special thanks are also due to D. Carlson for his assistance with field studies and to Dr. T. M. Chen for his invaluable suggestions on experimental technique. ACKNOWLEDGEMENTS . LIST OF TABLES . . . . LIST OF FIGURES . . . INTRODUCTION . REVIEW OF LITERATURE . TABLE OF 0 The Quackgrass Menace NitrOgen Fertilizers and Quackgrass Control Herbicides and Quackgrass Control Characteristics of Amitrole Amitrole Within the Plant CONTENTS Plant Surfaces and Herbicide Entry . The Use of Adjuvants in Herbicidal Characteristics of Oils. Summary MATERIALS AND METHODS Greenhouse Studies Laboratory Experiments RESULTS AND DISCUSSION Field . . . . Greenhouse . Isotope Studies Summary LITERATURE CITED . Sprays O‘C‘U‘I l3 I6 17 l8 28 3o 35 A2 A3 LIST OF TABLES Table Page I. The influence of oils (nga) on the effectiveness of foliar-applied herbicides on quackgrass in the fie]d . O O O O O O O O O O 0 O O O O O O O O 28 2. The effect of oils on the toxicity of foliar-applied herbicides to quackgrass in the field . . . . . . 29 3. The influence of oils on the effectiveness of amitrole and amitrole-T on quackgrass in the field . . . . 30 A. The effect of oil (2 gpa) on the activity of amitrole and amitrole-T (2 Ib/A) on quackgrass at two levels of nitrogen . . . . . . . . . . . . . . . 3i 5. The influence of oils on the absorption of ILIC amitrole and amitrole-T by quackgrass . . . . . 3l IA 6. The influence of oils on the translocation of C amitrole and amitrole-T by quackgrass . . . . . . 37 IA 7. The effect of oils on the rate of C amitrole and amitrole-T translocation in quackgrass. . . . . . Al Figure 3a. 3b. LIST OF FIGURES The technique used to Taunt quackgrass leaves for treatment with -labeled amitrole and amitrole-T. . . . . . . . Set-up of the apparatus for translocated ]“C de- termination . . . . . . . . . . . . . . . . . Comparative phytotoxicity of amitrole + oil, amitrole-T and amitrole-T + oil on quackgrass twenty-five days after spraying in the green- house. I: control, 2: amitrole-T alone, 3: amitrole-T + oil, A: amitrole + oil. . . The effects of amitrole, amitrole + oil, amitrole- T and amitrole-T + oil on quackgrass fourteen days after application as a single droplet at a concentration of Shoo ppm in the laboratory. a: amitrole alone, b: amitrole-T alone, c: ' amitrole + oil, d: amitrole-T + oil . . . . . . Thelfiffect of oils on the percent absorption of C amitrole and amitrole-T by quackgrass leaves. The influence of oilsuon the rate of acropetal translocation of C amitrole and amitrole-T in quackgrass leaves. . . . . . . . . . . . Page 2] 25 32 32 35 39 INTRODUCTION Quackgrass (AgrOpyron repens (L.) Beauv.) is widely acclaimed as one of the most notorious perennial weeds in the north central and northeastern states. Its noxious attributes transcend the primary competion by most other weeds with crops for moisture, nutrients and light. Once established, quackgrass overruns whole farms by its ag- gressive growth habit. It also excretes toxic substances which hinder the germination and growth of subsequent crops (29, Al, #2, #9). Mechanical control of this weed is expensive and often ineffective. Herbicides offer the best control measures but most chemicals now used are only effective at rates which injure crOp plants or leave unde- sirable residues in the crops. Amitrole has proven a valuable weapon against quackgrass. Its rapid inactivation in the soil obviates the problems of soil persistence and herbicide carry-over to the next crop. However, amitrole is not effective at the desirable low doses and must be enhanced with adju- vants to be of practical use. Such an adjuvant was found in ammonium thiocyanate but the search still continues for a more effective chemical. The objective of this study was to investigate the influence of. certain mineral oils On the effectiveness of various herbicides on quackgrass and to determine the mechanism of herbicidal enhancement, if any, by the oils. REVIEW OF LITERATURE The Quackgrass Menace Quackgrass is a perennial grass weed widely distributed in Western Europe and first observed in New England about I770 (38, 39). It is also called wheatgrass, couchgrass, wiregrass and witchgrass (39, 65). In this country quackgrass is found in eVery state north of Florida and Arizona but poses a serious weed problem mainly north of the Ohio and east of the Missouri rivers (39). The weed is so well established in Minnesota that some farms have been abandoned to it (2). Quackgrass propagates both by seeds and creeping rhizomes. The seeds contribute to its noxious classification and can initiate an in- festation but play only a minor role in the persistence of the weed once established. Quackgrass seed may be di5persed by man in seed and feed grain, baled hay, animal manure and by farm machinery (2, 65). Seeds may germinate before they are mature and the seedlings assume perennial characteristics in 2 to A months (2). The rapid Spread of quackgrass through an infested field is due to an elaborate system of ramifying rhizomes which also serve as storage organs for food reserves (3, SI). Most of the rhizomes are found in the tOp h inches of the soil and growth is prolific under optimum conditions (2h, 38). In a heavily infested field, 6 tons per acre of new rhizomes may be produced in a single growing season (2). The tips of the rhizomes or rootstocks are so sharp that they can penetrate potato tubers (65), thus, complicating the storage problems of potato farmers. An important fact in the greenhouse cul- ture of quackgrass is the effect of gravitational orientation on the rhizome. By placing the rhizomes in the soil with their tips pointing vertically upwards so that the force of gravity acted along the shoot in a basipetal direction instead of across it, Palmer (50), prematurely halted the subterranean phase of their development and induced the plant to produce aerial shoots. Pointing the tips vertically downwards did not induce the change in the natural habit and dormancy of the rhizomes. There is no easy or rapid method of destroying quackgrass. Mechanical eradication of the weed from infested areas entails persis- tent mowing, plowing, disking and harrowing (2, #5, SI, 60). Arny, in ISIS (2), estimated the cost of such operations at $8.l0 per acre which with present costs would be prohibitive. Apart from the high cost, mechanical measures are often ineffective. Rhizomes broken or cut into sections containing at least one node during tillage can grow into new plants, propagate independently and start new centers of in- festation (60). Thus, ordinary tillage practices tend to aid the Spread of quackgrass rather than limit it. Quackgrass has other attributes which are reSponsible for its de- signation as a prohibited noxious weed. By virture of its relation to wheat, it can serve as a host for black stem rust of wheat (2) and per- petuate the disease. Farmers have often noted drastic reductions in the yields of crops grown in competition with quackgrass (flfi. Some experienced great difficulties in establishing new stands of alfalfa on newly plowed quackgrass sods (AI). In field trials Kommedahl _£ El. (Al), noted that both the dry weight of plants and the stands of flax, alfalfa, wheat, barley, and cats were greatly diminished when sown on soils previously infested with quackgrass. Water soluble extracts of quackgrass leaves and rhizomes reduced the dry weight of alfalfa and the germination of wheat grains in greenhouse tests. Le Tourneau _£_§l, (AA), found that aqueous extracts of quackgrass rhizomes inhibited the root growth of wheat and pea seedlings. The inhibitor was soluble in methanol, ethanol, dialyzable, non-volatile and could not be removed from solution by anion and cation exchange resins. Ohman _g gl. (#9), obtained several stunted and chlorotic alfalfa stands with a 92%.reduction of dry weight when grown in competition with established quackgrass. Oats showed similar symptoms with a h5% reduction in dry weight as compared to pure stands. This effect was virtually overcome when the concentration of the Hoag- land‘s nutrient solution was increased fivefold, which indicated that quackgrass was more capable of withdrawing available nutrients from culture media than crOp plants. Only hot water extracts of quackgrass produced any deleterious effects on the oats and alfalfa plants. Cold water extracts had no toxic effects. Helgeson (29) and Lastuvka (42) noticed that while concentrated aqueous extracts of quackgrass rhizomes were toxic to young seedlings, dilute solutions of the extracts caused a stimulation of early seedling growth. The undesirability of quackgrass in a farmland is thus well established making its elimination a necessity, particularly for cultivated cr0ps. Nitrogen Fertilizers and Quackgrass Control The effect of nitrogen fertilizers on the growth habits and per- sistence of quackgrass has been investigated by a number of workers. Dexter (l5), observed that quackgrass stands receiving a high level of ammonium sulfate produced more top growth and a greater number of new rhizomes than controls but that such new rhizomes had a lower dry matter content. Later reports (l6) indicated that an abundant nitrogen supply promoted luxuriant top growth at the expense of food reserves. The grass was more susceptible to clipping treatments at a higher than a lower level of nitrogen nutrition. McIntyre (#6), observed no change in root- stock dry matter at nitrogen levels of 2.6 and 2l0 ppm, although the shoot dry weight at the lower level was only 25%.of that grown at the higher level. McIntyre also noted that tiller development was sup~ pressed at nitrogen levels below 2.6 ppm, while bud dormancy was eli— minated at 2lO ppm of nitrogen. Ries (Sh),found dalapon more effec- tive on quackgrass plots treated a month prior to application with l2l lb/A of NH4N03 than on those receiving 333 lb/A of a l2-l2-12 fertilizer formulation and concluded that the nitrogen was the nutrient responsible for the increased effectiveness, since the addition of A0 lb/A of P205 and K20 did not further increase the control ratings. Lowe _£ 21- (#5), also noted the tendency of quackgrass infestations in plowed and disked plots to decrease as the rate of application of NHhNO3 increased from O to 600 lb/A. Herbicides and Quackgrass Control The control of quackgrass with herbicides is a welcome departure from mechanical cultivation. In his appraisal of herbicides, Shaw (59), pointed out that chemical methods of weed control greatly reduced energy requirements on farms. Herbicides, he noted, reduced the manpower, machine hours and machine horsepower demands of crop production, and provided a new dimension for improving efficiency and lowering pro- duction costs. Raleigh (53), found it relatively easy to control over 80%.of quackgrass infestations with chemicals but observed that varying inter- vals of time (depending on the herbicide) were necessary between treat- ments and the ensuing crop in order to avoid injury. Soil applied herbicides have the disadvantage of being required in amounts above the necessary doses because of several factors which inacti- vate them in the soil. These high rates of application lead to undesirable herbicide toxicity on crops. Vengris (63), applied 50 lb/A of TCA and dalapon to the soil for the control of quackgrass but used only l0 lb/A of MH as a foliar Spray for the same purpose. Characteristics of Amitrole Amitrole (3-amino-l,2,A-triazole) is a fine, white, crystalline pow- der with a faintly disagreeable odor and a melting point of l56-l57 C. It is readily soluble in water and ethanol but insoluble in benzene and acetone (#0). Amitrole reacts with acids, bases, aldehydes and ketones, forms complexes with several metals and numerous metabolites within treated plants but Is not subject to breakdown under ordinary atmospheric conditions (A, l3, 23, A8). Schweizer and Rogers (58), while Investigating the physi6logical activity of amitrole and eleven other closely related compounds, found that the -NH2 radical on the triazole ring must be in a reactive form for greatest physiological activityand that the substitution of the -MH2 with a chloro or a mercapto radical resulted in less activity. The triazole ring was necessary for the full range of amitrole activity. Amitrole may exert Its effects on plants In several ways. The most typical symptom of amitrole injury is an almost pure white coloration in the newly formed leaves of susceptible species due to the failure of pig- ment development (4, Al, #8). In lethal doses the terminal meristem dies first, followed by necrosis down the stem. Other amitrole effects include: a reduction In starch and oil content of corn kernels (#0), loss of geo- tropism and abnormal root hair development In cotton seedlings emerging from amitrole-treated seeds (l0), defoliation of cotton plants (A), and a sharp decline of cytochrome C and catalase activity in beans (#8). Amitrole is a translocated systemic herbicide best sprayed on plant foliage for Optimum results. It is readily absorbed by plant roots (4) but rapidly disappears from warm moist soils. Ercegovich and Frear (23) could not detect any amitrole in corn plants of any age grown in pots treated with 8 lb/A one day before seeding the corn. They showed that the rate of amitrole recOVery from soils Is a function of temperature, pH, soil moisture and microbial activity. An increase or decrease in pH from neutrality hastened the disappearance of amitrole from a Hagerstown silt loam. The pH of foliar sprays has no apparent influence on amitrole or amitrole-T activity between pH h.6 and 9.6 on quackgrass (l7). Amitrole decomposition in soils is not closely related to the soil type but is more rapid in organic soils and X-ray defraction techniques indicate some absorption of the chemical by montmorillonite (23). The rate of depletion in autoclaved soils and soil samples treated with metabolic poisons is greatly reduced (23). Soil microorganisms appear to be the chief agents of amitrole degradation in the soil as this occurs most rapidly under conditions favoring rapid microbial growth (23). The mixture of amitrole and NHASCN is called amitrole-T. The addition of NHASCN enhances the translocation of amitrole (h, l9). There is, however, an Optimum concentration above which translocation is retarded because of the rapid killing of the treated portion of the plant as evidenced by the appearance of necrosis (4). Amitrole-T is superior to amitrole alone on several grass species (A, I3, 40), 2 lb of the mixture being equivalent to 8 lb of amitrole against quackgrass (I3). Amitrole-T is also more effective on Bermudagrass, bentgrass and nutgrass. Amitrole Within the Plant The fate of amitrole within the plants is still subject to con- troversy. Most research workers agree, however, that the herbicide is translocated to and accumulated primarily in the meristematic regions (I, no). Autoradiographs of Zebrina Sp. treated with Inc-amitrole showed that after h days the radioactivity had bypassed all mature leaves except the treated one and there was accumulation in both the shoot and root tips (h). Rogers (56), reported that amitrole is translocated throughout plants in several forms, depending on the plant species. Part of the transported material is pure amitrole but a large fraction ls metabolized. Soybeans appear to translocate only the metabolized form. A metabolite of amitrole with a low Rf value found in susceptible soybeans and Canada thisle is not found in Johnsongrass, which is tolerant. Rogers also noted that amitrole could be translocated from one thistle to another connected by a rhizome. Bondarenko g£_gl, (6), reported that maximum radioactivity from labeled amitrole was detected in all parts of Canada thistle within #8 hours after treatment. Hull (35) noted that movement was both acropetal and basipetal and that only acropetal movement could traverse a cotton stem girdle. In bean and four-o'clock (35), the translocation of amitrole was ID to 60 times faster than that of its hydroxy analog, 3-hydroxy-l,2,A-triazole. It was the hydroxy-amltrole which accumulated In young leaves whereas amitrole accumulated in all parts of the plant. Movement in nutgrass was rapid, reaching the roots in 2 hours from a single leaf treatment. Andersen (I), linked amitrole movement in nutgrass with photosynthetic activity. He observed that when the treated leaf was exposed to light with the rest of the plant in the dark, amitrole quickly moved out of the leaf and into the rest of the plant. With the treated leaf In the dark and the rest of the plant I. light, the labeled amitrole did not move out of the leaf. Apparently, the movement of photosynthates into the leaf from the rest of the plant had carried the herbicide towards the tip of the leaf. Herrett (30), made an observation neglected by others. He observed that there was a time lag between the application of amitrole to a plant's leaf and its movement out of the leaf into the rest of the l0 plant. He reported that amitrole does not move out of treated leaves within l2 hours following application (A). During this time a trans- locatable product is formed. If the synthesis of this product is prevented by applying sodium fluoride to the treated leaf within the lag period, the leaf withers but no further amitrole symptom occurs. Movement in the xylem is not subject to this lag so that transport of the herbicide towards the tip of the treated leaf is almost immediate. Herrett g5_§l, (3i), isolated three unidentified metabolites from Canada thistle and found that while two of these were relatively in- active, the third was herbicidally more potent than amitrole and would also translocate In the absence of light, a condition unfavorable for amitrole translocation. The absorption of amitrole is greatest during the first A hours following application but its translocation increases with time up to 96 hours (l9), with symptoms appearing in cotton in 9 days (h). Crafts (l3), expressed the belief that amitrole moves principally in the phloem and seldom, if ever, escapes either into the xylem or into the culture medium but found some transfer from the phloem to the xylem of barley seedlings (ll). Clor _£Hgl. (9), reported that amitrole was rapidly translocated in the phloem at a rate similar to sucrose trans- port in cotton and oak seedlings and concluded that the herbicide was not metabolized by these plants. Hill _£_gl. (32), noted that the translocation of amitrole In yellow nutsedge, was primarily acropetal and was not Influenced by the maturity of the plant. Donnalley g£,gl. (l8), reported that a labeled substance was translocated to and accumu- lated In the tubers of nutgrass following an application of labeled amitrole to the foliage. The viability of the intact tubers was greatly t al, (69), reported reduced by the accumulated substance. Yamaguchi that the amitrole was absorbed by xylem tissues and thought that this could limit the amount of the substance free to move. The tolerance of plants to amitrole is probably due to differential absorption and men tabolism. Tolerant bindweed absorbs amitrole more slowly than Canada thistle but metabolizes it more rapidly (30). The area of greatest controversy is in the mode of action of ami- trole, which is probably due to the variety of reactions in which the substance can be involved. A popular but unacceptable hypothesis is that concerning the structural similarity between amitrole and the pyrrole rings of chlorophyll. This gave rise to the suggestion that owing to their similarity, amitrole molecules were substituted for some pyrrole rings during the synthesis of chlorophyll, resulting in the absence of color from the new leaves (4). The critics of this postulate have accumulated considerable evidence to di5prove it. The first doubts came with the observation that the lhC—activity in isolated pigments from leaves treated with labeled amitrole was extremely low (l3), indicating that the amitrole was not entering directly into the structure of the pigments. Amitrole, it was further contended, has the same decolorizing effect on carotenoids which have no pyrrole rings (A, 67). The effects on plant pigments thus seemed secondary to an effect on the plastid itself. In experiments designed to determine what stage of chlorophyll synthesis was affected, Naylor (#8), found that amitrole did not interfere with the conversion I2 of holochrome, a protochlorophyll, into chlorophyll. Thus, although amitrole prevented the Synthesis of new protochlorophyll, it would not hinder the formation of chlorOphyll from preformed holochrome. Wolf (67), reported that microscopic evidence showed a clear interference of amitrole with plastid development and refuted the suggestion that amitrole inhibition of chlorophyll synthesis in higher plants was due to its simi- larity in structure to the pyrrole rings of chlor0phyll. Alternative explanations have been given for the mode of action of amitrole in plants. Hilton (33) observed the growth inhibition of baker's yeast by amitrole and noted that the addition of lO'ZM L-histidine to the culture medium necessitated a 3000-fold increase in the amount of amitrole required to reduce the growth of the yeast by 50%. He concluded that L-histidine represents the product of some metabolic pathway which can be inhibited by amitrole. This antagonism between L-histidine and amitrole does not occur in algae or higher plants. Castelfranco _t._L. (7) and Wolf (68), countered the inhibitory effect of amitrole on the growth of algae with adenine and several other purines and suggested that amitrole is either an inhibitor of purine biosynthesis or a competi- tive inhibitor of purine utilization. Wolf's hypothesis is perhaps the most plausible, although the answer to the question of amitrole action on plants is still incomplete. He stated that amitrole inhibits purine synthesis and interferes with the formation of RNA, which is essential for chlorophyll replication. It is now known (A) that free or me- tabolized amitrole can disrupt purine synthesis by reacting with glycine in the growing points, interfere with enzymes and structural l3 proteins needed for growth and the formation of chloroplasts, producing the chlorotic plants so typical of amitrole injury. Plant Surfaces and Herbicide Entry The leaf cuticle is the primary barrier to the entry of herbicides into plants since it appears to line the entire leaf surface and the substomatal cavities as a continuous membrane (36). To enter the symplast, foliage applied chemicals must traverse the cuticle and epidermal walls, or in the case of stomatal penetration, the walls of the substomatal chambers (h). The nature of the cuticle and the underlying epidermal cells has been discussed by Danielli (62), Crowdy (36) and Frey-Wyssllng (h, 25) in relation to the penetration of foreign molecules. The cuticle contains long-chain polymerized alcohols and acids that have their exposed terminals containing unsaturated linkages. Cuticle also contains waxes which are short-chain esters and alcohols lacking reactive end-groups. They are relatively inert and hydrOphobic but can dissolve lipoidal compounds and hence are important with respect to the partition of fat soluble herbicide molecules. The epidermal cell walls are composed inainly of pectins and cellulose impregnated with cutin waxes. The pectin and cellulose are hydrOphilic. This combination of cutin waxes, pectin and cellulose makes up a unique layer which constitutes the outer epider- lnal wail of the plant. This surface presents the greatest barrier to the absorption of foliar applied herbicides. The failure of herbicidal effectiveness may be due to lack of pene- tration. Normally, both surfaces of plant leaves can absorb foreign mole- cules but the lower epidennis is more permeable. Preferential areas of IA foliar absorption are veins, glandular trichomes, Open stomata, ruptures caused by mechanical injury and cuticular imperfections (In, 25). En- try is via aqueous and lipoidal routes. There is no organized system for the transport of substances from the cuticle to the conducting channels. Currier and Dybing (IA), showed that the time taken by 2,A-D to move from the cuticle to the phloem was greater than that needed for normal transport from the leaf to the roots. Stomatal entry is often insignificant because of complex factors which influence their opening. Open stomata are readily penetrated by gaSes, vapors, oils and to varying degrees by aqueous solutions containing surfactants but not by water and aqueous solutions of polar substances. Completely closed stomata can exclude all fluids (IA, 25). Another component of foliar absorption Is metabolism. This component enables the "loading" of a chemical into plant cells. A QIO value of 2 for the foliar uptake of 2,h-D (l6) sug- gests a limiting metabolic reaction. Such a process can effectively increase the concentration gradient from the cell to the surface and enhance entry. Spray retention by plant surfaces depends upon the characteristics of the spray and the plant species (22). The repellent tendency of leaves to aqueous solutions can be overcome by the inclusion of suitable surfac- tants in the formulations. The entry of herbicides into plant leaves is thus a complex process involving a system comprising the chemical, the carrier and the leaf surface. Modifications of any of these will affect the entry of the chemicals. Surfactants may enhance herbicide entry through a function of their concentration, structure or physico-chemical prOperties.(36). 15 The Use of Adjuvants in Herbicidal Sprays An adjuvant is a non-toxic material added to a pesticide to improve its physical or chemical characteristics (2i). Most adjuvants are of the surface active type and are commonly used to improve the depositing and weathering properties of pesticide sprays and dusts. Water, the usual carrier of pesticides, has a high surface tension and tends to be repelled by smooth or waxy surfaces like leaf lamina so that aqueous sprays often fail to penetrate plant surfaces because of their incompatibility with water. As early as l890, the damage to foliage by insecticidal arsenicals was found to be increased when applied in strong soapy solutions or flour- paste suspensions (37). Jansen (36), could detect 80% of amitrole applied in a micro droplet containing a surfactant within the leaf tissues 6 hours after application whereas 80%.of the same chemical applied as a pure a- queous solution remained on the surface after h days. The concept of the reduction of surface tension and contact angle of aqueous sprays by sur- factants has often has: used to explain their mode of action (27, 36, 37). Surface active agents exert a great influence on the wetting, spreading and sticking preperties of pesticide sprays on plant surfaces. There is now evidence that the action of adjuvants is more than a simple modifica- tion of the physical characteristics of aqueous sprays. Several workers (h, 2i, 26, 36, #0) have reported that maximum sur- factant enhancement of herbicidal sprays occurs at concentrations con- siderably higher than those necessary for the greatest possible reduction of the surface tensions of the aqueous mixtures. Jansen g; 31. (37), l6 found no penetration of soap solutions through insect tracheae if the insect was first killed with KCN and suggested that some vital forces were involved in the penetration of living membranes. Both Jansen _£._l- (37) and Foy gt g1. (26), found no clear relationship between herbicidal enhancement and the surface active properties of surfactants. A suitable adjuvant may: promote the wetting and coverage of sprayed surfaces, solubilize the plant cuticle, regulate the Spray retention and the herbi- cide penetration, reduce volatility or aid as herbicide co-solvents and humectants (lh, 36, 6i). Dybing and Currier (20), noted that dye solu- tions containing Vatsol OT, a surfactant, penetrated Zebrina 5p. leaves in 5 minutes while aqueous solutions of the dye alone did not. Characteristics of Oils There are three classes of oils: mineral or petroleum, animal and vegetable oils (34). inneral oils may be paraffinic, olefinic, aromatic or naphthenic hydrocarbons (28, 3h, 62). The aromatics rank highest in phytotoxicity followed by naphthenes and olefins, while the saturated paraffins are the least toxic. Havis (30), investigated oil toxicities in relation to their boiling point ranges and found that toxicity to plants was associated with a boiling point range of lSO-27S C; those with boiling points on either side of this range were less toxic. Hough and Mason (34), worked out a comprehensive relationship be- tween oil phytotoxicity and some of their physical and chemical properties. These are the Saybolt viscosity, the unsulfonated residue (UR) and the distillation range. The Saybolt viscosity is the number of seconds taken by 60 ml of the oil at lOO F to pass through a standard orifice. Toxicity 17 is associated with Saybolt viscosities of less than 70 seconds. 'The UR is an index of saturation and represents the percentage of the product that will not react with hot concentrated H250“. The injury level is usually less than 90%“ The distillation range is the tempera~ ture at which 5-90%.of the oil distills off. Toxicity is associated with a boiling point range of l50-275 C (28). Before l930, no special weed oils were manufactured (62). Unal- tered diesel oil was employed to kill stubborn weeds. Present-day weed oils are selected for specific purposes. Oils penetrate the crown of grasses where-the growing tissues are located, and also open stomata. Once inside the leaf, oils solubilize the lipoids of cell membranes (ho). This process makes thesemi-permeable membranes more permeable and the cell sap leaks out into the intercellular spaces. Oil movement inside the plant is slow but its direction may be down, up, radial or tangen- ,tial. Toxic oils usually provide poor kill because of phloem inactivation. Oil components of oil-water Sprays_aid wetting and act both as cuticular and Stomatal penetrants (lh). Summary Quackgrass is a noxious weed reSponsible for sizeable farm losses. its growdlcharacteristics render mechanical control measures ineffective and expensive. Herbicides which promise a major breakthrough towards a solution of the problem of quackgrass control are only effective on the weed at rates which also injure crop plants or leave undesirable residues in the crops. Adjuvants, by enhancing the effectiveness of herbi- . cides, enable weed control at lower herbicide levels which can then be employed with a greater margin of safety. MATERIALS AND METHODS Chemicals The oils employed in this study are mineral in origin and are designated by the symbols: 7E, llE and 92E. They are miscible with water and are paraffinic except92£ which is naphthenic. They have Saybolt viscosities of 7i, lOS and 209, unsulfonated residues of 93, 92 and 73 and distillation temperatures of 59,'l23 and l6h, respectively. The addition of a dispersing agent to the oil formulation is indicated by "D." The herbicides were selected on the basis of their effects on quackgrass (52). Commercial formulations of herbicides and experimental samples of oils were employed in all field and controlled environment tests. Field Studies Field trials were conducted on established quackgrass sods at East Lansing from May to September, l966. The treatments were applied on randomized plots each h ft wide and 25 ft long with a carbon dioxide- pressurized small plot Sprayer (55),utilizing a quart bottle as the herbicide container and applying the Spray at 36 gpa. All_treatments were repliCated three times in a randomized block design. The rate of herbicides used was expressed as lb/A of active ingredient and oils as gpa unless otherwise specified. Visual ratings were obtained on the field and greenhouse plots on the conventional l to 9 scale (28), with l indicating no control, 6 acceptable commercial control and 9 complete kill. To eliminate possible bias, ratingswere obtained without reference to the differential treat- ments. The data were statistically evaluated by analysis of variance and comparisOns of treatment means made by the Least Significant Difference l8 l9 (LSD) test. figeenhouse Studies For the greenhouse experiment, quackgrass rhizomes were harvested from the field, cut into sections and prOpagated in quartz sand contained in h—inch earthenware pots with drainage holes at their bottoms. The pots were placed in slightly wider plastic vessels to serve as reservoirs for the nutrient solutions. To water the plants, the sand in each pot was rapidly flooded with nutrient solutions until the plastic reservoirs were nearly full. This treatment was repeated as often as needed to en- sure good growth of the plants. Each pot containing several quackgrass shoots constituted a plot. The plots were divided into two groups, one maintained with tap water (representing low nitrogen), and the other with a solution of 8h ppm nitrogen as NHhNO3. The temperature regime during the period of the experiment was 73- 80 F in the day and 68-70 F at night. The daylight was supplemented with cool white fluorescent light from tubes supplying about 700 ft-C which enabled a maintenance of l6 hours light and 8 hours darkness throughout the experiment. The quackgrass was lO-l8 inches high when the treat- ments were applied. Herbicide-oil combinations were applied in the greenhouse with a system using compressed air as a source of pressure. Plant containers were passed under a flat fan nozzle which applied Spray at the rate of no gpa. .The plant containers were moved by a conveyor whose speed was adjusted to a constant 3 mph. After Spraying, the pots were placed on a greenhouse bench in a major split with the herbicide treatments re- plicated four times. 20 Laboratory Experiments Absorption and translocation experiments were conducted in the laboratory where the temperature was maintained at a constant 72 F i 2. Quackgrass plants for these studies were grown from single- node rhizome sections. These sections were first planted in quartz sand in plastic flats and later tranSplanted into test tubes con- taining half-strength Hoagland's solution when the Sprouts were A to 6 inches high. A light cycle of l6 hours day and 8 hours night was maintained using a fluorescent light source with an intensity of 700 ft-C. Amitrole and amitrole-T labeled with I4C in the 5-position of the triazole ring at a concentration of SAOO ppm of the active in- gredient were utilized in the absorption and translocation studies. The radioactivity in the treatment drOplets was equivalent to 0.05 uc. The oil used was llE and when present constituted 6% by volume of the total treatment liquid. Uniform plants were selected for each repli- cate and the leaves were taped in a horizontal position to ensure re- tention of the treatment droplets (Fig. l). A faint point was marked on the adaxial surface of the taped leaf with a liquid-tip black marking pen midway between the apex and the ligule. A lO ul droplet of the treatment liquid was placed on the mark, using a 10 U1 syringe. Each treatment was replicated four times. The Spread of the droplets, if any, was not hindered but its final limit was marked. When the drOplets were dry, the taped leaves were restored to their normal positions. At the end of the treatment periods which were 2h, #8 and 96 hours, each leaf was excised at two Figure l. 2! The technique used to mount quackgrass leaves for l4 treatment with C-labeled amitrole and amitrole-T. 22 1 Figure 23 points along its longitudinal axis just beyond the margin of the drop- lets. The unabsorbed herbicide was washed off by allowing the leaf section containing the drOplet to stand in 5 ml of ethanol for 5 minutes in a counting vial and swirling it for one minute.in four changes of the same amount of ethanol. Ten ml of toluene - BBOT counting fluid were added to each vial and counted for l0 minutes in a Liquid Scintillation Spectrometer equipped with external Standardization. For the treatments with oils, the washing solution was a mixture of 5 ml ethanol and 10 ml toluene-BBOT counting fluid. All other procedures were the same, except that no further counting fluid was added to these vials. The method proved efficient and reproducible, giving negligible counts by the third or fourth washing. Background subtraction was made for all counts and the sum of the net counts in the four vials represented the unabsorbed herbicide in that treatment. The difference between the applied radio- activity and the recovered amount was considered absorbed. A quench series was prepared for the labeled amitrole by adding 0, X, 2X, 3X, and Ax quantities of the quenching material to a constant volume of the alcohol solvent in counting vials. Ten ul dr0plets of labeled amitrole containing a known radioactivity were added to each vial and assayed with external automatic standardization. The quench curve was drawn by plotting the percent counting efficiency against the external standard count (52). From.the curve, counts per minute (cpm) were converted to disintegrations per minute (dpm). A gain of l6%.and a window setting of 50-950 were em- ployed for all counts. To determine the amount of translocated Ib’i: amitrole or amitrole-T each treated leaf was removed at the ligule and the portion apical to the 2% limit of the treatment droplet was assayed for acrOpetal translocation. The remainder of the plant was partitioned into root and shoot with each part assayed separately. These samples were placed in 50-ml beakers and freeze dried. The radioactivity in each sample was assayed by a modification of the Schoniger combustion method (8, 6h). Each sample was placed in a black, ashless, sample paper wrapper and enclosed in a platinum basket attached to the ignition head of a Schoniger or Lehner flask. The ignition head also provided a gas-tight stopper for the flask. A stream of oxygen was passed into the combustion flask to flush out the air, closed and placed within the safety shield housing (Fig. 2). Combustion was achieved by momentarily focussing an external infrared beam from a Thomas-Ogg Safety Oxygen Flask lgnitor Assembly on the sample wrapper. After ignition the carbon dioxide in the combustion‘ flask was absorbed in a 20-ml mixture of 2:1 ethanol-ethanolamine, allowing 30 minutes for complete absorption. At the end of this period, a 5-ml aliquot of the solution was pipetted into a counting vial, l0 ml of scintillation fluid were added and the sample was held in the dark for 30 min before counting (68). Background subtraction was made for all samples so that the counting rates reported were net cpm. To determine the efficiency of this system, the Schoniger combus» tion and sample recovery were repeated using a l0 ul aliquot Of the uC-amitrole solution as the sample. After counting, 100 Hi of toluene- 7-IQC with a Specific activity of 87.h dpm/ul were added to the same vial and counted a second time. Using the net counts before and after the addition of the internal standard, the counting efficiency was 25 Figure 2. Set-up of the apparatus for translocated ll‘C deter- mlnations. 26 ‘ fCAUTlOl l O “1. 9 Oyi l i. ‘ _ unmet; .3 N 1" :mmm \\ T3 2%,. _ Figure 2 27 calculated. From this efficiency the net cpm for each experimental sample were converted to dpm. The leakage of I“ C herbicide from quackgrass roots was investi- gated by making up the solution in each culture test tube to 50 ml and counting O.l ml aliquot with 5 ml alcohol and lO ml scintillation fluid for l0 min. RESULTS AND DISCUSSION Field In the first field study, none of the oils tested Significantly increased the activity of all herbicides on quackgrass (Table l). However, some of them enhanced the effectiveness of amitrole-T, making further trials worthwhile. In a test to find the most suitable level of oils to combine with amitrole-T, there was no difference between 2 and h gpa, and the rate of 2 gpa was arbitrarily employed in subsequent studies. Table l. The influence of oils (2 gpa) on the effectiveness of foliar- applied herbicides on quackgrass in the field. QCR after 3h days Rate SLSD ' Herbicide (lb/A) None 7E llE 92E 5% 1% None l.O dluron 4 2.7 3.7 #.O 3.7 0.7 NS simazine 4 2.3 3.0 3.7 2.0 0.7 l.h paraquat l 6.7 6.7 7.3 7.7 0.7 NS amitrole-T 2 h.7 4.3 6.7 6.3 0.7 l.# Data from another field study indicate a striking enhancement of amitrole-T activity on quackgrass by all the oils tested but none increased the effectiveness of the other herbicides Significantly (Table 2). This finding agrees with reports that the enhancement of herbicidal activity by adjuvants is related to the specific affinities between the herbicides and adjuvants (l3, 26, 36, 40). The presence of the dispersing agent in the 28 29 oil formulations did not augment the effects of herbicide-oil com- binations on quackgrass. Table 2. The effect of oils on the toxicity of foliar-applied herbi- cide to quackgrass in the field. QCR after 20 days Rate LSD Herbicide (lb/A) None 7E 7ED llE llED 5%. l% None l.O amitrole-T 2 5.0 7.0 7.3 8.0 7.0 l.2 diuron h h.3 5.3 5.0 5.3 “.7 NS dalapon 5 6.3 6.7 6.3 6.3 7.0 NS terbacil A 5.3 5.3 5.7 5.7 6.0 NS The oils alone were not toxic to quackgrass at the rate of 2 gpa (Table 3). This result is in agreement with the known relationship between the phytotoxicity of oils and their physical and chemical characteristics (3%). The data also indicate a greater enhancement of amitrole activity than of amitrole-T by the oils on quackgrass. The importance of this result can only be appreciated when the relative effectiveness of amitrole and amitrole-T as herbicides is considered. Amitrole-T is considered about four times more active than amitrole as a herbicide (l3). 30 Table 3. The influence of oils on the effectiveness of amitrole and amitrole-T on quackgrass in the field. QCR after 20 days Oils (2 gpa) None amitrole amitrole-T None 1.0 I 2.0 4.3 7E0 l.0 5.0 5.3 llED l.0 4.7 4.7 928 l.3 “.7 5.0 LSD at 5% NS 0.7 LSD at 1% 1.0 l.0 Greenhouse In a greenhouse test to compare amitrole-T and amitrole + oil, the latter gave a greater measure of quackgrass control at two levels of nitrogen (Table A, Fig. 3a). This suggests that the oil is a better adjuvant than NHuSCN for enhancing amitrole phototoxicity. The effect of amitrole-T + oil was greater at the higher level of nitrogen. This agrees with a finding by Ries (Sh) that a high level of nitrogen nutrition predisposes quackgrass to herbicidal control. 31 Table 4. The effect of oil (2 gpa) on the activity of amitrole and amitrole-T (2 lb/A) on quackgrass at two levels of nitrogen. Herbicide-oil (llE) 05“ after 2‘ days LSD combination Low N (tap water) High N (84 ppm N) 9% None l.25 1.50 NS amitrole-T 3.75 4.25 NS amitrole-T + oil 5.50 7.00 1.45 amitrole + oil 7.00 7.25 NS LSD at 5% 1.68 1.68 LSD at l% 3.09 3.09 Table 5. The influence of oils on the absorption of 17C amitrole and amitrole-T by quackgrass. Herbicide-oil (llE) Duration I‘ic Absorbed %.of that combination (hr) (dpm x 103) applied amitrole 24 6.7 6 amitrole + oil 24 91.7 82 amitrole-T 24 64.0 57 amitrole-T + oil 24 74.l 66 amitrole 48 9.2 8 amitrole + oil 48 99.1 88 amitrole-T 48 49.5 4“ amitrole-T + oil 48 98.3 88 amitrole 96 12.8 ll amitrole + oil 96 105.2 95 amitrole-T 96 74.8 67 amitrole-T + oil 96 103.2 93 LSD at 5% ll.4 10 LSD at 1% 15.3 13 Figure 3a. Figure 3b. 32 Comparative phytotoxicity of amitrole + oil, amitrole-T and amitrole-T + oil on quackgrass twenty-five days after spraying in the greenhouse. 1: control, 2: amitrole-T alone, 3: amitrole-T + oil, 4: amitrole + oil. The effects of amitrole, amitrole + oil, amitrole-T and amitrole-T + oil on quackgrass fourteen days after application as a single drOplet at a concentra- tion of 5400 ppm in the laboratory. 3: amitrole alone, b: amitrole-T alone, c: amitrole + oil, d: amitrole-T + oil. 33 Figure 3a Figure 3b 34 Isotope Studies To explain the observed enhancement of the herbicidal activity of amitrole and amitrole-T by the oils, two hypotheses were explored: that the mineral oils accentuate the wetting of quackgrass and in- crease the absorption of the herbicides; and that they increase the amount of herbicide translocated by the quackgrass. In the isotope Studies, the absorption of both amitrole and amitrole-T was significantly increased by the llE mineral oil (Table 5). The increase was very pronounced in the case of amitrole alone where the amount absorbed was augmented by about 55 to 84%.and was great enough to offset the decline in amitrole-T absorption between the first 24 and 48 hours (Fig. 4). in all replicates, the treatment droplets with oil Spread over a large portion of the leaf surface while those without the oil remained as tiny beads on the treated Spots. This indicates that the increased absorption is due to a greater area of contact between the herbicides and the leaf and is probably a component of the herbicidal enhancement observed in field and greenhouse studies. A large proportion of the 'hc amitrole and amitrole-T applied to quackgrass was translocated in an acropetal direction (Table 6). This movement of amitrole-T was increased by the oil over a 48 hour duration and declined thereafter for all treatments. The finding agrees with previous reports (1, 30). Anderson (1), referred to the initial acropetal movement as a passive one assisted by the move- ment of food materials into a temporarily non-functioning leaf. 35 14 Figure 4. The effect of oils on the percent absorption of C amitrole and amitrole-T by quackgrass leaves. lSiVflHilll Z 36 PEHEENI "ill '09 F, no : awmme '— — =— amume * 1—alnmm'— '— ' -' “a i 1-I|llll!llll!‘_ --—v 37 Table 6. The influence of oils on the translocation of Ib’C amitrole and amitrole-T by quackgrass. Herbicide-oil (11E) Duration Translocated Inc (dmp/mq dry wt) combination (hr) AcropetalI Shoot Root amitrole 24 108 20 12 amitrole + oil 24 ' 228 312 94 amitrole-T 24 1869 12 10 amitrole-T + oil 24 989 100 8 amitrole 48 8O 4O 20 amitrole + oil 48 455 529 168 amitrole-T 48 2275 17 7 amitrole-T + oil 48 6909 37 13 amitrole 96 23 40 19 amitrole + oil 96 121 383 98 amitrole-T 96 1362 21 16 amitrole-T + oil 96 1063 42 12 LSD at 5% 2652 53 22 LSD at 1% NS 71 30 IDesignates the portion of the leaf apical to the treatment droplet. Herrett (4, 30), suggested that amitrole translocation is preceded by its conversion into a form which is more innocuously translocated in the phloem and that acrOpetal movement was not subjected to that condition. While the rate of acrOpetal movement of amitrole was not affected by the oil in these studies, that of amitrole-T was sharply increased 38 during the first 48 hours and there was an interaction between time and the rate of the acropetal translocation (Fig. 5). Both the rate and the amount of basipetal translocation of I“C amitrole and amitrole-T were increased by the all (Tables 6 and 7), resulting in a greater intensity of radioactivity in the shoot and roots of plants treated with herbicide - oil combinations. This effect was more pronounced with amitrole than amitrole-T. The in- creased mobility of the herbicides is probably another component of the observed herbicidal enhancement of amitrole and amitrole-T activity on quackgrass by the oils in the field and greenhouse studies. Thus, the observed greater activity of amitrole + oil on quack- grass (Figs. 3a and 3b), is probably due to the differential absorption and translocation of the herbicide combinations, which were greater with amitrole than with amitrole-T. The increased accumulation of radioactivity from the amitrole - oil combination in quackgrass roots could lead to long term effects of the herbicide on quackgrass rhizomes. This possibility was not investigated in this study but the indication, if confirmed, would be a valuable step toward meeting the quackgrass challenge. H in these studies, there was no difference between the translocation of amitrole and amitrole-T (Tables 6 and 7). This is contrary to earlier findings (19, 21) but does not contradict them. Australian workers (6) have reported that NHASCN can only enhance the translocation of amitrole up to a certain concentration beyond which the translocation is impeded by injury to the living phloem tissues. At the concentration of 5400 ppm of the herbicides applied, amitrole-T was visibly more injurious to the quackgrass leaves as evidenced by the appearance of necrosis on Figure 5. 39 The influence of oils on the rate of acropetal trans- location of leaves. 14 C amitrole and amitrole-T in quackgrass 1111 [HIM/1111 11111111l11111 llifl - 1111 1 ‘. \ e - mimic . .— .— —. animluul 1211 . ...—... allillil-l _ i \ ‘_..._‘ ammle-Inll . ,/ ‘.\ I“ r |K\\i// ‘\\\\ 5" ' :/\\ 111 ' / \‘\ ‘-. 21 r \x '7 + ~ ~ —. = 11 l 2 4 4O lllli 1111181 Figure 5 41 the Spots treated with amitrole-T at the end of four days. Under such conditions the translocation of the herbicide would be hindered. 14 Table 7. The effect of oils on the rate of C amitrole and amitrole-T translocation in quackgrass. Rate of 11‘1C translocation Herbicide-oil (115) Duration 1 (dpm/m9 d'Y “t/h') combination (hr) AcrOpetal Basipetal amitrole 24 5 l amitrole f oil 24 10 17 amitrole-T 24 78 l amitrole-T + oil 24 41 5 amitrole - 48 2 l amitrole + oil 48 9 15 amitrole-T 48 47 'l amitrole-T + oil 48 144 l amitrole 96 O l amitrole + oil 96 l 5 amitrole-T 96 14 O amitrole-T + oil 96 ll 1 LSD at 5% 69 3 LSD at 1% 92 ‘v 42 Summary In both field and controlled environment studies, paraffinic and napthenic oils increased the herbicidal activity of amitrole and amitrole-T on quackgrass. The oils had little or no effect on the activity of foliar-applied dalapon, diuron, paraquat, simazine and terbacil. The enhancement of amitrole by the non-toxic oils was more than that of amitrole-T and was associated with increased absorption and translocation of 140 amitrole or its metabolites. 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