MSU RETURNING MATERIALS: Place in book drop to LJBRAfiJES remove this checkout from ._c—. your record. FINES will be charged if book is returned after the date stamped be10w. fut-fifflp: 1.5.9 4’4" 'W a . , EFFECTS OF INHIBITORS AND DIVERTERS OF PHOTDSYNTHETIC ELECTRON TRANSPORT ON HERBICIDE RESISTANT AND SUSCEPTIBLE NEED BIDTYPES By Eugene Patrick Fuerst A DISSERTATION Submitted to Michigan State University in partia] fuifiilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soi1 Science 1984 ABSTRACT EFFECTS OF INHIBITORS AND DIVERTERS OF PHOTOSYNTHETIC ELECTRON TRANSPORT ON HERBICIDE RESISTANT AND SUSCEPTIBLE NEED BIOTYPES By Eugene Patrick Fuerst Herbicide cross resistance was evaiuated in triazine resistant biotypes of four species. Triazine resistant smooth pigweed, common iambsquarters, common groundsel, and rapeseed were aiso resistant to bromacii and pyrazon, moderateiy cross resistant to buthidazoie, and more susceptible to dinoseb. Atrazine resistant smooth pigweed was aiso resistant to cyanazine and metribuzin, moderately cross resistant to Tinuron and desmedipham, and not resistant to diuron, bromoxyni], benta- zon or dicamba. Resistance at the whole piant levei was high‘ly correiated with resistance at the ievei of photosynthetic electron transport. The physioiogical basis for the increased susceptibility to dinoseb in triazine resistant smooth pigweed was studied. The increase in sus- ceptibiiity to dinoseb couid not be attributed to either a photosynthetic mechanism or to differences in carbohydrate ieveis in the two biotypes. Numerous herbicide treatments were evaiuated for controi of triazine resistant common iambsquarters and pigweed (Amaranthus sppJ infestations. Satisfactory control of triazine resistant common lambs- quarters was obtained with preemergence treatments of pendimethaiin or postemergence treatments of dicamba, bromoxynii, or bentazon. Satisfactory control of pigweed was obtained with preemergence treatments of alachlor or postemergence treatments of dicamba. bromoxynil, or 2,4-0. The basis for alachlor protection of corn from buthidazole injury was studied. .Alachlor protection was attributed to reduced uptake of buthidazole. Two sites of inhibition of photosynthetic electron transport were characterized for buthidazole, buthidazole metabolites, and ioxynil. Buthidazole and ioxynil inhibited primarily at a site similar to atrazine or diuron. The secondary site of action of both herbicides is on the oxidizing side of photosystem 11. Most buthidazole metabolites inhibited photosynthetic electron transport, but all were less active than buthidazole. The basis for paraquat resistance in Conyza linefolia was studied. Resistance was not due to an altered site of action. I vivo chlorOphyll fluorescence measurements indicated that paraquat was excluded from the site of action. Exclusion from the site of action was not due to differences in cuticular penetration. Autoradiograms of leaves fed 140- paraquat through the petiole suggested that resistance may be due to adsorption to the extracellular matrix and thus exclusion from the protOplast. ACKNOWLEDGMENTS I wish to thank the following individuals: Donald Penner and Charles Arntzen, for guidance and financial support. Michael Barrett, Alan Putnam, and Matthew Zabik, for serving on my advisory comni ttee. Herb Nakatani, Frank Roggenbuck, and Janet Natson, for assistance with my research. My parents, family, and friends, for moral support. Robin, my wife, for hugs that brightened each day. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES CHAPTER 1: Herbicide Cross Resistance in Triazine Resistant Biotypes of Four Species .............. ABSTRACT ......................... INTRODUCTION ....................... MATERIALS AND METHODS ................... RESULTS AND DISCUSSION .................. REFERENCES ........................ CHAPTER 2: Studies on the Basis for Differential Tolerance to Dinoseb in Triazine Resistant and Susceptible Smooth Pigweed (Amaranthus hybridus) ........ ABSTRACT ......................... INTRODUCTION ....................... MATERIALS AND METHODS ................... RESULTS AND DISCUSSION .................. REFERENCES ........................ CHAPTER 3: Chemical Control of Triazine Resistant Common Lambsquarters (Chenopodium album) and Two Pigweed SpeciengAmaranthus sppT. . ...... ABSTRACT ...... . .................. INTRODUCTION ....................... MATERIALS AND METHODS ................... RESULTS AND DISCUSSION .................. REFERENCES ........................ 21 23 23 24 25 27 33 35 35 36 37 38 47 CHAPTER 4: Protection of Corn (Zea mays) From Buthidazole Injury with Alachlor .......... ABSTRACT ......................... INTRODUCTION ....................... MATERIALS AND METHODS ................... RESULTS AND DISCUSSION .................. REFERENCES ........................ Chapter 5: Characterization of Two Sites of Action for Buthidazole, Buthidazole Metabolites, and onynil . ABSTRACT ......................... INTRODUCTION ....................... MATERIALS AND METHODS ................... RESULTS AND DISCUSSION .................. REFERENCES ........................ CHAPTER 6: Studies on the Mechanism of Paraquat Resistance in Conyza linefolia ................ ABSTRACT ......................... INTRODUCTION ....................... MATERIALS AND METHODS ................... RESULTS AND DISCUSSION .................. REFERENCES ........................ iv 49 49 49 49 so 52 53 53 54 54 56 69 71 71 72 76 80 LIST OF TABLES TABLE CHAPTER 1 1. Whole plant resistance ratios and I values (kg/ha) for atrazine, bromacil, pyrazon, buiaidazole, and dinoseb ......................... 2. Whole plant resistance ratios and 150 values (kg/ha) in smooth pigweed for the herbicides indicated ..... 3. ChlorOplast 15 values (M) for atrazine, bromacil, pyrazon, buthigazole, and dinoseb in the four species studied. ........................ 4. ChlorOplast 15 values (M) in smooth pigweed for the herbicides indicated .................. CHAPTER 2 1. Plant tolerance to dinoseb in triazine resistant and susceptible biotypes of smooth pigweed under various light conditions described in the text ......... 2. Glucose, sucrose, reducing sugar, starch, and total carbohydrate (sucrose + reducing sugar + starch) levels in triazine resistant and susceptible smooth pigweed prior to illumination ("morning") or after 8h of illumination ("afternoon") . .............. 3. Effect of exogenous sucrose on desiccation by dinoseb in excised leaves of triazine resistant smooth pigweed. . . CHAPTER 3 1. Triazine resistant pigweed control with various chemical treatments ....................... 2. Triazine resistant common lambsquarters control and corn injury with various chemical treatments. . . . ..... PAGE 10 11 12 13 28 31 32 39 41 CHAPTER 4 1. Shoot fresh and dry weight response and 14C-buthidazole present in corn shoots after various root zone and shoot zone treatments. . ................... 51 CHAPTER 5 1. Concentrations of buthidazole and ioxynil which cause 50% inhibition (150) of photosynthetic electron transport by two assays ................. 61 2. Reversibility of inhibition of SiMo photoreduction. (Activities are as a % of a 10 uM diuron control treatment) .......... . ............ 61 3. Inhibition of SiMo photoreduction by a wide range of herbicides at a concentration of 300 uM. . . . . . . . . 63 4. Variable fluorescence (F M-F0)/F, after various herbicide and chemical reatments ........ 64 and 180 values for buthidazole and its megabolites measured by DCPIP photoreduction . . . . . . 66 6. Inhibition of SiMo photoreduction by buthidazole and its metabolites. All compounds were tested at 300 pM. . 67 7. Time for 50% inhibition, using concentrations which caused 80% inhibition at 15 min. (Table 5) measured by DCPIP photoreduction . . . . . ..... . ..... . 68 CHAPTER 6 1. Estimated concentrations which cause 50% injury by reduction of in vivo chlorophyll fluorescence, % moisture or % of leaf area remaining green. The resistance ratio was estimated by dividing the I50 for the resistant biotype by the 150 for the susceptible biotype. . . . . . . . . . . . . ..... 89 (‘0 O Cuticular penetration of 14C paraquat. . . . . . . . . 89 3. 14C-paraquat in the cushion of the sucrose density gradient ...... . . . . . . . ....... . . . 100 vi FIGURE CHAPTER 1 1. CHAPTER 5 1. LIST OF FIGURES Response of common lambsquarters to atrazine: A. growth reduction, measured as fresh weight, as a preemergence treatment in seedlings; 8. inhibition of photosynthetic electron transport, measured as photoreduction of DCPIP ........ . . . . . . . . Comparison of resistance ratios measured as whole plant injury (solid bars) or inhibition of photo- synthetic electron transport (hollow bars) for atrazine, pyrazon, bromacil, buthidazole, and dinoseb. A = Amaranthus hybridus; C = ChenOpodium album; B = Brassica napus; S = Senecio vulgaris . . . . ..... Comparison of resistance ratios measured as whole plant injury (solid bars) or inhibition of photo- synthetic electron tranSport (hollow bars) averaged across species. Means followed by the same letter are not significantly different at the 5% level by Duncan' 5 multiple range test ......... . Comparison of resistance ratios measured as whole plant injury (solid bars) or inhibition of photo- synthetic electron transport (hollow bars) in smooth pigweed ........................ Simplified scheme of photosynthetic electron transport. Sites of reduction, oxidation and inhibition by various compounds are indicated. P680 is the photosystem II reaction center. P700 is the photosystem I reaction center ......... Effect of length of incubation on inhibition of SiMo photoreduction by buthidazole and ioxynil. ...... PAGE 14 16 18 57 59 CHAPTER 6 1. Structures and redox potentials of the 3 bipyridinium herbicides tested ................ . . . Effect of bipyridinium herbicide concentration on photosystem I-mediated electron tranSport in resistant (R) and susceptible (S) biotypes, using TMPD as the electron donor .................... Response of % leaf area remaining green (Open symbols) and % moisture (closed symbols) to bipyridinium herbicide concentration in excised leaves of resistant (R) and susceptible (S) biotypes ........... Response of in vivo fluorescence to bipyridinium herbicide concentration in excised leaves of resistant (R) and susceptible (S) biotypes ........... Response of in vivo fluorescence transients to paraquat in exc1se3 leaves of resistant and susceptible biotypes ................. Autoradiograms (A 12d 8) of excised leaves (C and 0) fed a solution of C paraquat pH 7 ......... Autoradiogramz (A-D) of leaves (E-H) of leaves fed a solution of 1 C paraquat pH 7, and then trans- ferred either to water (A,C,E,G) or to a solution of 24 mg/ml paraquat ................... Autoradiograms (A fad B) of excised leaves (C and 0) fed a solution of C paraquat pH 3 ......... viii 74 81 83 85 87 93 95 97 CHAPTER 1 Herbicide Cross Resistance in Triazine Resistant Biotypes of Four Species ABSTRACT The cross resistance of triazine resistant biotypes of smooth pigweed (Amaranthus hybridus LJ, common lambsquarters (Chen0podium album 1"), common groundsel (Senecio vulgaris LJ, and rapeseed (Brassica napus LJ to a selection of herbicides was evaluated in greenhouse studies. The triazine resistant biotypes of all four species showed a similar pattern of cross resistance. The four triazine-resistant biotypes showed resistance to injury from atrazine [Z-chloro-4-(ethylamino)-6- (iSOprOpylamino )jsftriazine], bromacil (S-bromo-3-sggfbutyl-6- methyluracil), and pyrazon [S-amino-4-chloro-Z-phenyl-3(2H)-pyridazinone] and showed a slight resistance to buthidazole {3-[5-(1,1-dimethylethyl)- 1,3,4-thiadiazol-2-ylJ-4-hydroxyl-1-methyl-2-imidazolidinone}. The triazine-resistant biotypes were distinctly more susceptible to dinoseb (2—gggfbutyl-4,6-dinitr0phenol). The triazine resistant smooth pigweed was studied in greater detail, and showed resistance to cyanazine {2-[(4- chloro-6-(ethylamino)j§7triazin-Z-yl)aminOJ-Z-methylpr0pionitrile} and metribuzin [4-amino-6-tgrtfbutyl-3-(methylthio)jg§7triazin-5(4H)-one], with slight resistance to linuron [3-(3,4-dichlorophenyl)-1-methoxy-1- methylurea] and desmedipham (ethyljmghydroxycarbanilate carbanilateL There was little or no resistance to diuron [3-(3,4-dichlor0phenyl)-1,1- dimethylurea], bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), bentazon 1 [3—i50propyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide], or dicamba (3,6-dichlorojgfanisic acid). Resistance was highly correlated whether evaluated as herbicidal injury or as inhibition of photosynthetic electron transport. This observation supports the widely held viewpoint that atrazine, cyanazine, metribuzin, pyrazon, bromacil, linuron, desme- dipham, and buthidazole cause plant injury by inhibiting photosynthesis. This observation also supports the widely held viewpoint that in vivo triazine resistance and cross resistance is due to a decreased sensi- tivity at the level of photosynthetic electron transport. Subsequent studies indicated that dinoseb does not inhibit photosynthesis i vivo (Chapter 3). No conclusion can be made, based on these observations, concerning the cause of injury from diuron, bromoxynil, bentazon, or dicamba. INTRODUCTION Biological organisms which devel0p resistance to one biocide may simultaneously show resistance to other biocides to which they have never been exposed. This phenomenon, cross resistance, is well documented for insecticides (Brown, 1971) and fungicides (Dekker, 1976). It is reason- able to pr0pose that the triazine resistant weeds may be cross resistant to herbicides, since so many herbicides inhibit photosynthesis at a similar site (Moreland, 1980). Cross resistance to herbicides has been measured in isolated chloroplast membranes of triazine resistant weed biotypes (Arntzen 21.21:: 1982; Pfister and Arntzen, unpublished dataL In these studies, resistance was reported in members of the triazine, triazinone, pyridazinone, quinazoline, and uracil families. There was little resistance to certain members of the phenylurea and thiadiazolyl families. There was an increase in susceptibility to certain members of the nitrOphenol, nitrile, benzothiadiazinone, and benzoxazinone families. (Such negatively correlated cross resistance has been reported previously in cases of insecticide resistance (Brown, 1971) and fungicide resistance (van Tuyl, 1977)). Moreover, the pattern of cross resistance was strik- ingly similar across several species, suggesting that the genetic basis for resistance was very similar in all species tested. Resistance to herbicides in plants is a relatively recent develOpment (Ryan, 1970). Numerous Species have shown resistance to triazines, and this resistance has been attributed to the alteration of the triazine binding site (Arntzen §t_§l:, 1982). A chloroplast membrane localized polypeptide of about 32 kilodaltons (k0) has been shown to contain the atrazine binding site (Pfister et_al:, 1981; Steinback gt 31:, 1981). A change in a single amino acid, from serine to glycine, is the mutation conferring resistance (Hirschberg and McIntosh, 1984) in both A; hybridus and Solanum nigrum. Corn (gga_mays L.) contains this 32 k0 polypeptide and exhibits a natural and extreme tolerance to atra- zine. Detoxification is the basis for this tolerance (Shimabukuro 3; 31,, 1971; Shimabukuro et 21., 1970) in corn but detoxification has not yet been shown to be a primary basis for resistance in triazine resistant weeds. The terms "resistance" and "tolerance" should be defined in a manner similar to the definitions used and accepted with reference to insecti- cide resistance (Brown, 1958). Thus, biocide or pesticide resistance is the capacity of a biological or pest organism to survive doses of toxicants which would be lethal to the original or normal p0pulation of the same species. "Resistance", as currently used in the entomology and phytopathology literature, refers to a stable and heritable trait. "Tolerance" is characteristic of species in which the original or normal p0pulation can survive large doses of toxicants which prove lethal to other species (e.g. corn) (Shimabukuro gt _a_l., 1970). "Tolerance" also refers to cases of phenotypic adaptation, where an increased ability to survive toxicants may be acquired through environmental conditioning. For example, exposure to sublethal herbicide concentrations has been shown to increase the tolerance of corn to atrazine (Jachetta and Radosevich, 1981). One objective of this investigation was to determine the extent of herbicide cross resistance in four triazine resistant species at the whole plant level. Herbicides were selected to represent a diversity in herbicide chemistry. A second objective was to determine whether the resistance measured in isolated chlor0plast membranes is correlated with resistance in VTVO. MATERIALS AND METHODS In vivo studies. Seeds of resistant and susceptible biotypes of smooth pigweed and common groundsel were obtained from Nashington, common lambs- quarter from Michigan, and rapeseed from Ontario, Canada. Seeds were sown in 170 ml cups in greenhouse mix soil (sand:sandy loamzpeat moss, 1:1:1, pH 6i”. Commerical formulations of atrazine, bromacil, buthida- zole, cyanazine, dicamba, diuron, linuron, metribuzin, and pyrazon were applied preemergence in a spray volume of 150 L/ha and pots were gently watered from the top with approximately 1 cm of water after application. Pots were subirrigated individually and plants were thinned to five per pot (or four per pot for rapeseed) after emergence. Plants were grown in a greenhouse at 26‘:_4°C with supplemental lighting 14/10h, light/dark, supplied by sodium vapor lamps with a photosynthetic photon flux density of 100—200 uEPm‘z'sec'l. Shoot fresh weights were determined at the two to three-leaf stage of the control treatment. Bentazon, bromoxynil, desmedipham, and dinoseb were applied postemergence in a spray volume of 150 L/ha with(LS% v/v alkylaryl polyoxyethyleneglycol-free fatty acid- i50pr0panol surfactant (X—77 Spreader) at the two leaf plant growth stage. Dinoseb was applied no more than 2 h after the onset of illumina- tion. Shoot fresh weights were detennined 2 days after application. For each herbicide, at least five herbicide rates were applied; these rates were on a logarithmic scale. Rates were selected based upon preliminary experiments. There were six replications of each treatment. Herbicide rates required for 50% injury (I50, measured as reduction in fresh weight) were estimated using linear regression of logit-transformed ob- servations (Finney, 1979). The sigmoid dose-response curves shown (Figure 1) were modeled by this logit regression method. Resistance ratios were then estimated by dividing the 150 for the resistant biotype by the 150 of the susceptible biotype. Thus, the resistance ratio is a measure of the degree of resistance. Each experiment was repeated; the values are reported with two significant digits in Tables 1 and 2. Com- bined analysis across species (Figure 3) was done with log-transformed data, using each species as one replication. In vitro studies. ChlorOplast thylakoid membranes were isolated from susceptible and resistant biotypes and rates of photosynthetic electron transport determined over a wide range of herbicide concentrations by monitoring photoreduction of dichlor0phenol-ind0phenol (DCPIP) (Paterson and Arntzen, 1982). The assay mixture was preincubated with the herbi- cide for 10 minutes prior to assay, since some herbicides achieve maximal activity slowly. Herbicide concentrations which caused 50% inhibition (150) and resistance ratios were computed as previously described and are reported with two significant digits in Tables 3 and 4. RESULTS AND DISCUSSION In vivo resistance. The four triazine resistant Species were extremely resistant to atrazine and showed cross resistance to bromacil, pyrazon, and buthidazole (Figure 2). I vivo resistance ratios for pyrazon and bromacil were smaller than the resistance ratios for atrazine, but larger than the resistance ratios for buthidazole (except in rapeseed). In- creased susceptibility to dinoseb (negatively correlated cross resistance) was observed in all four Species (Figure 2). Cross resis- tance to cyanazine, metribuzin, linuron, and desmedipham was observed in triazine resistant smooth pigweed (Figure 4). The resistance ratios for linuron and desmedipham were much smaller than resistance ratios for cyanazine or metribuzin. Resistance to cyanazine and metribuzin was expected since they are members of the triazine and triazinone families, respectively, and are structurally related to atrazine. 150 values for the herbicides evaluated (Table 1) were generally far lower than rates which provide weed control in the field. It would be misleading to assume that injury to these species would occur at equivalent rates under field conditions. The relatively large degree of resistance to pyrazon and bromacil (Figure 2) suggests that weed control problems may be anticipated if these herbicides are used where the triazine resistant weed species are present. Also, it seems reasonable to test the value of these herbicides as alternatives to the highly persistent triazines in the newly developing triazine resistant cr0ps, such as the rapeseed studied here. The increased susceptibility to dinoseb implies that this compound would be ideal for control of triazine resistant weeds if sufficient cr0p tolerance existed. Field studies have examined this potential in common lambsquarters and two pigweed Species (Chapter 3). However, problems with using dinoseb for control of triazine resistant weeds include high mammalian toxicity (Anonymous, 1979) and limited label clearances for selective weed control in corn (Anonymous, 1981). Also, the efficacy of dinoseb on triazine resistant smooth pigweed (and presumably other Spe- cies as well) was greatest when applied in the early morning (Chapter 2) on sunny days (personal observation). However, dinoseb and other herbi- cides showing negatively correlated cross resistance (Arntzen £3 31,, 1982; Pfister and Arntzen, unpublished data) may provide as useful models for the future develOpment of safe, highly selective new herbicides targeted at the triazine resistant weeds. Correlation in vivo and in vitro resistance ratios. There was generally a close correlation of resistance ratios observed in vivo and in vitro (Figures 2-4). This correlation indicates that inhibition of photo- synthetic electron transport is probably the primary biochenical basis for herbicidal injury from atrazine, cyanazine, metribuzin, pyrazon, bromacil, linuron, desmedipham, and buthidazole. Another mechanism appears to be involved in the case of dinoseb (Chapter 2%. This correla- tion also indicates that resistance to these herbicides is due to a decreased sensitivity at the level of photosynthetic electron tranSport. However, this correlation alone is not direct proof that inhibition of photosynthesis is the primary or sole Site of action. The resistance ratios observed in vivo were consistently closer to 1 than the the resis- tance ratios observed in vitro and were significantly smaller in the case Figure 1. Response of common lambsquarters to atrazine: A. growth reduction, measured as fresh weight, as a preemergence treat- » ment in seedlings; B. inhibition of photosynthetic electron transport, measured as photoreduction of DCPIP. 100 Fresh Weight ( % of Control ) Rate of DCPIP Photoreduction ( % of Control ) 100‘ 50 ‘\ \ \ \ \ ‘ N \ A q s \ \ \ \ \ \ A \ \‘~ I 1 1 n n 0.01 0.1 1.0 10 Atrazine Rate (Kg/Ha) l L l l l if 10 10'7 6 ’5 '4 10- 10 10 Atrazine Concentration ( M ) 10 om.o 4.m 4a.: 4N.o «33.3 eqc.c am.o $4.0 mN:.o q_.c mm.c m_.: m m4.o mq.o «m3.o 00.3 mq.c m.o 94.3 cm.o omo.c am.o mn:.o Nm:.c 4 numcc4o 4.m mco.c m~.o m.m m4.c no.3 m.4 m~.o n~.o o.H nmc.o :qo.o m o4o~mc o.~ mmo.o Aco.c 4.c am:.o x4.o q.4 amo.o mmo.o o.~ mmc.o N4.o _ lfizuzm m4 owc.c 4.4 a.» mm.c xm.c m.m mm.c ~@.o o4 «43.: mm.o N m.n mac.c mm.o :4 qq:.: mq.o m.q 4m3.o mmo.o mm o~c.3 «0.0 4 44055044 om Om.o m4 m.q 4.c Cm N4 «.4 ON mm 44.o ~.m m q.q 4c.o ~.m 4.x m.4 c.9 m4 cm.o m.m q.m oq.o o.m 4 camcmxm 004.4 oc~.c 04m o~4 m~.c me On n~o.o n.m one nmo.o 44 N com._ Smc.c mm can mm.o 24m as «no.0 ~.~ 3mm 340.9 m.m 4 mcflnmta< 11Am2\w4 Om44111 11Am2\mx :mHVIII llam:\mx Om~vlll IIAm:\wx cnavlll 044mm maxuo4m waxuowm ougmx waxuo4m wazuowm o4umx maxuofim maxuoum 044mm mazuowm waxuofim ucms4 ocwufinuuz wucmu 6444“ Cam“ been“ @424“ .cmu oucmu o4nfiu ucmu mucmu 04244 acmu (Cwaxm 1m4mwm iamumzm 1m4mox imamwx immumzm imwmmm Imummx lemumam imumum imwmmx lemomam lmwmmm .4 m4umm43> owumcam .4 mama: mowmncum .4 E24~m Saavomccmzo .4 mzvflpnhz mzzucmume< .nomocfiv new .m~o~mv4:u:n .commuza .H4omeo»4 .mcwumuum new Am£\wxv mm3~m> Onm new meaumu mucmumwmmu ucmfia macs: .H mHLMH 11 Table 2. Whole plant resistance ratios and 1 values (kg/ha) in smooth pigweed for the herbicides in icated. Amaranthus hybridus L. Resistant Susceptible Resistance Herbicide Experiment Biotype Biotype Ratio ------- (150 kg/ha)—----- Cyanazine 1 3.4 0.054 63 2 4.3 0.028 160 Metribuzin 1 0.62 0.016 39 2 0.55 0.042 13 Linuron l 0.26 0.062 4.1 2 0.026 0.010 2.4 Desmedipham 1 0.12 0.079 1.51 2 0.19 0.044 4.32 Dicamba 1 0.029 0.021 1.4 2 0.042 0.041 1.0 Bromoxynil 1 0.10 0.097 1.1 2 0.29 0.27 1.1 Diuron 1 0.081 0.097 0.84 2 0.030 0.029 1.0 Bentazon 1 0.39 0.44 0.90 2 0.21 0.23 0.88 12 .Nnr4 .._4. Wu :LN4:L< 22 44:15; :4 w:4U::;nLcc:u .:u~ucu<._;; >2 1:44azzn 54:22 .cuzn4_::;:: .:o~4:h< 3:: 4;.m4CL1 N.23., x1:_x:.q clc4zc._ x1c4xa.c 21:44:.a nic4x_.r clslxc.4 ~-c_x_.4 221::4: A-c_xa.u ciesxc.4 muc4xm.o ~134xm.x x-:.xm.m muc~x34 21:4x4.o cic_xn.4 c42~1u4;szn Lmlcix:._ ea-:_xq.4 no1c_xm.m 24-:4xw.4 z-:_xm.c c-:_xc.m aaic4xn.~ acuo.xa.m .4uzscta m-:_zm.~ 41:_xm.4 m1c4xm._ Slolxq.m c-24xc.m elosxm.o onc_xa.s 4-:_xq._ ::~=.x; Lq-c4xo.s Larc4zn.s cauc4x:.~ saio4x4.m “-04xn.4 «-34xn.4 anno_xc.m selc4\ azawcts< ........ A: :m4411111111 1-11-1114: sm4411111111 1-111-114: an4411111111 1-1-1-114: cm441111111- 34:44:00m3m .:cum4rux 9~L4gzvun3m u:c4m4muz 24444;;sm3m 4cnun4mux zqswuaycmzm accum4muz 31404243: .4 m441w42> 34uucmm .4 mass: wwwnmngm .4 5:44m Esmvcmdcuzo .4 mzcprN: maggsmuze< .mmficsum mowuoam 4:0u mzu c4 numOCHU can .MHOvaqsuan .CONmuxa .Hfiumsohn .wcfinmuum new sz m034m> H ummaa0u0450 .m wanna Table 4. ChlorOplast I herbicides in 13 values (M) in smooth pigweed for the 1cated. Herbicide Amaranthus hybridus L. Resistant Susceptible Cyanazine Metribuzin Linuron Desmedipham Dicamba Bromoxynil Diuron Bentazon 3.4x10‘5 5.4x10‘5a 2.7x10‘7 2.5x10'7 >10"3 5.1x10‘6 8.1x10‘8a 3.4xio‘5a ‘(150 M) 7.3x10‘8 2.1x10‘7a 7.8X10'8 6.5x10‘8 >1O‘3 8.7x10‘6 6.0x10'8a 5.0x10‘5a anister and Arntzen, unpublished. 14 Figure 2. Comparison of resistance ratios measured as whole pl ant injury (solid bars) or inhibition of photosynthetic elec— tron tranSport (hollow bars) for atrazine, pyrazon, bromacil, buthidazole, and dinoseb. A = Amaranthus Eybridus; C = Cheno odium album; B = Brassica napus; S 8 enec o vulgaris.a 3Determined by Pfister and Arntzen, unpublished. 1 Plant lnnury Inhibiiion oi PhOIOSYTlH'leSIS . 490 Atrazme AF: 1,ooo so _l__:]1.300 Cl C ”A,” 3H3“ (anew \~ mm _1500 S Jasoa 18 Pyrazon A”. 329° Obi—flaw Cl 0 6 5 B ‘ J 55 NH, \ ,flfl N \:_/ 14 F 8:116 . 23 Bromacul —2oa 88 H C“: N 0 7.2 F \f/ h—l 31,, Br \n/N\.CH-CZH5 0 CH3 1108 Buthidazole OH N'——"|N l cw,-—c w w—cn3 ( )3 J\S Y O Dinoseb OH wo,/ ,/P+Cz”s | CH, N0, 0'1 11- 1b 7 16057050 Resistance Ratio Figure 3. 16 Comparison of resistance ratios measured as whole plant injury (solid bars) or inhibition of photosynthetic elec- tron transport (hollow bars) averaged across species. Means fol lowed by the same letter are not significantly different at the 5% level by Duncan's multiple range test. 17 - 640a Atrazine H9708 13c Pyrazon i—jsw . llcd Bromacnl i171 706 Buthidazole 3'2“ 4.8cd Dinoseb 0-328 0.11e fl I 1 1 1 0.1 i 10 100 1000 Resistance Ratio 18 Figure 4. Comparison of resistance ratios measured as whole plant injury (solid bars) or inhibition of photosynthetic electron transport (hollow bars) in smooth pi gweed. aDetermined by Pfister and Arntzen, unpublished. bDicamba did not inhibit photosynthetic electron transport. 19 Amaranthus Cyanazine “0 470 Metribuzin m 2608 Linuron 3.3 3.4 Desmedipham 2.9 3.3 Dicamba Bromoxynil O. 59 Diuron 0 94 Bentazon o 89 oooa T— T T— T T O. i 1 10 100 1000 Resistance Ratio 20 of bromacil and pyrazon (Figure 3). This suggests that these compounds act at additional sites other than inhibition of photosynthesis, or that the resistant biotypes exhibit a generalized increase in susceptibility to herbicides, or that herbicide detoxification rates, relative to the amount of pesticide applied, are lower in the resistant biotypes. That is, if the rates of detoxification in both biotypes are similar, the rate in the resistant biotype may be of relatively little consequence due to the much higher herbicide rates applied. Pyrazon caused a greater degree of chlorosis and a more delayed necrosis than other herbicides, particu- larly at high rates in the resistant biotype (personal observation). This, together with the large discrepancy between 1 vivo and in vitro resistance ratios for pyrazon (Figure 3) suggests that there may be a second site of action for pyrazon. Inhibition of carotenoid biosynthesis is a possible second site since this appears to be the primary site of action by the closely related compound, norflurazon [4-chloro-5- (methylamino)-2-Ca,a,a-trifluorojmftolyl)-3(2H)-pyridazinone] (Bartels and Watson, 1978; Vaisberg and Schiff, 1976). However, our observations agree with previous reports (Eshel, 1969; Hilton 33 313,1969) that inhi- bition of photosynthesis is the primary site of action in the susceptible biotypes. Resistance ratios were close to one for dicamba, bromoxynil, diuron, and bentazon (Figure 4). I vivo resistance ratios for dalapon and acifluorfen were 0.81 and 1.07 respectively (data not shown). Since resistance ratios were close to one, no statement can be made regarding the cause of injury from these herbicides, based on our observations. However, inhibition of photosynthesis is a mechanism of action for diuron (Izawa, 1977), bentazon (Suwanketnikom gt al., 1982) and bromoxynil (Table 1) but not dicamba. There was no inhibition of electron transport 21 by dicamba (Table 4) and there was no in 1119 resistance to this com- pound. This was expected since the mechanism of action of dicamba is interference of growth regulation (Ashton and Crafts, 1981). This obser- vation indicates that there is no cross resistance to compounds which do not affect photosynthesis. REFERENCES Anonymous, 1979. Herbicide Handbook of the NSSA, pp. 173-177. Anonymous, 1981. Premerge 3 specimen label. Dow Chemical Co. Arntzen,C«Jn K.Pfister,auuiK.E.Steinback. 1982. The mechanism of chlorOplast triazine resistance: Alterations in the site of herbicide action. In: Herbicide Resistance in Plants, HJH. LeBaron and J. Gressel, Eds. John Wiley and Sons, Inc., New York. pp. 185-214. Ashton, FkM. and ACS. Crafts. 1981. Mode of action of herbicides, 2nd ed. John Wiley and Sons, Inc., New York. Bartels, P.G. and CM. Natson. 1978. Inhibition of carotenoid biosynthesis by fluridone and norflurazon. Need Sci. 265198-203. Brown, A.N.A. 1958. Insecticide resistance in arthrOpods. N.H.O. Monograph Series, No. 38. 240 pp. Brown, AJLA. 1971. Pest resistance to pesticides. In: Pesticides in the Environment, R. White-Stevens, Ed. Marcel DéEker, Inc., New York, pp. 457-552. Dekker, J. 1976. Acquired resistance to fungicides. Ann. Rev. PhytOpathol. 14:405-428. Eshel, Y. 1969. Effect of pyrazon on photosynthesis of various plant species. Need Res. 9:167-172. Finney, Du]. 1979. Bioassay and the practice of statistical inference. Int. Stat. Rev. 41:1-12. Hilton, J.L., A.L. Scharen, J.B. St. John, D.E. Moreland, and K.H. Norris. 1969. Modes of action of pyridazinone herbicides. Need Sci. 11:541-547. Hirschberg, J. and L. McIntosh. 1984. Molecular basis of herbicide resistance and Amaranthus hybridus. (SubmittedL Izawa, S. 1977. Inhibitors of electron transport. ‘13: Photosynthesis I. Photosynthetic Electron Transport and PhotOphosphorylation. A. Trebst and M. Avron, Eds. Springer-Verlag, New York, pp. 266-282. 22 Jachetta, J.J. and S.R. Radosevich. 1981. Enhanced degradation of atrazine by corn (233_mays). Need Sci. 29537-44. Moreland, D.E. 1980. Mechanism of action of herbicides. Ann. Rev. Plant Physiol. 31:597-638. Paterson, D.R. and C.J. Arntzen. 1982. Detection of altered inhibition of photosystem II reactions in herbicide-resistant mutants. In: Methods in Chloroplast Molecular Biology. M. Edelman, R.B. Hal—Flick, and N.H. Chua, Eds. Elsevier Biomedical Press, Amsterdam, pp. 109- 118. Pfister, K., K.E. Steinback, G. Gardner, and C.J. Arntzen. 1981. Photoaffinity labelling of an herbicde receptor protein in chlorOplast membranes. Proc. Nat. Acad. Sci. U.S.A. _7__8_:981-985. Ryan, G.F. 1970. Resistance of common groundsel to simazine and atrazine. Need Sci. 18:614. Shimabukuro, R.H., D.S. Frear, H.R. Swanson, and N.C. Nalsh. 1971. Glutathione conjugation. An enzymatic basis for atrazine resistance in corn. Plant Physiol. _4_7_:10-14. Shimabukuro, R.H., H.R. Swanson, and N.C. Nalsh. 1970. Glutathione conjugation. Atrazine detoxication mechanism in corn. Plant Physiol. 4_6_:103-107. Steinback, K.E., K. Pfister, and C.J. Arntzen. 1981. Trypsin-mediated removal of herbicide binding site within the photosystem II complex. Z. Naturforsch. 365598-108. Suwanketnikom, R., K.K. Hatzios, D. Penner, and 0. Bell. 1982. The site of electron transport inhibition by bentazon (3-isOpr0pyl-1H-2,1,3- benzothiadiazin-(4)3H-one 2,2-dioxide) in isolated chlor0plasts. Can. J. Bot. 69:409-412. Vaisberg, A.J. and J.A. Schiff. 1976. Events surrounding the early development of Euglena chlorOplasts. 7. Inhibition of carotenoid biosynthesis by t e erbicide SAN 9789 (4-chloro-5-(methyl-amino)-2- (a,a,a-trifluor-m-tolyl)-3(2H)pyridazin one) and its develOpmental consequences. Plant Physiol. 57:260-269. van Tuyl, J.M. 1977. Genetics of fungal resistance to systemic fungicides. Medelingen Landbouwhogeschool, Nageningen 77-2:1-136. CHAPTER 2 Studies on the Basis for Differential Tolerance to Dinoseb in Triazine Resistant and Susceptible Smooth Pigweed (Amaranthus hybridus) ABSTRACT A triazine resistant biotype of smooth pigweed (Amaranthus hybridus LA showed less tolerance than the susceptible biotype to dino- seb (Z-SEE-butyl-4,6-dinitr0phenol) applied early in the morning. This same decrease in tolerance was observed in isolated chlorOplasts, suggesting that dinoseb injury may be related to the inhibition of photo- synthesis. However, dinoseb was actually slightly more active in the dark than in the light, indicating that dinoseb did not cause injury by inhibition of photosynthesis. Furthermore, the reduced tolerance of the triazine resistant biotype to dinoseb was also observed in the dark. ‘To evaluate whether carbohydrate levels in the plant might be related to dinoseb injury, the levels of glucose, sucrose, reducing sugars, and starch were determined in leaves of the two biotypes in the morning and afternoon. Levels of glucose and sucrose were low in all cases. Starch levels were higher in the afternoon than in the morning in both biotypes. This is consistent with an hypothesis that reducing power obtained from hydrolysis and oxidation of starch may protect plants from dinoseb in- jury. However, starch and total carbohydrate levels were not lower in the triazine resistant biotype than in the triazine susceptible biotype in the morning, indicating that carbohydrate levels cannot eXplain the decreased tolerance of the triazine resistant biotype. Carbohydrate 23 24 levels were lower in the triazine resistant biotype than the triazine susceptible biotype in the afternoon. This was consistent with previous reports that the triazine resistant biotype had a lower photosynthetic efficiency. Finally, exogenous sucrose supplied to excised leaves via the petiole did not protect the leaves from dinoseb injury. Thus carbo- hydrate levels do not appear to be the basis for the differences in dinoseb tolerance observed. INTRODUCTION Dinoseb may affect many physiological process including ribonucleic acid synthesis, protein synthesis, photosynthesis, lipid synthesis and respiration (Ashton gt 21,, 1977). A single biochemical mechanism for herbicidal injury from dinoseb has not been shown, but effects on ATP synthesis have been studied (Aston and Crafts, 1981). The symptoms of injury include rapid desiccation which is more active in the dark than the light (Meggitt gt 11” 1956; Mellor and Salisbury, 1965L The tria- zines and phenylureas are active almost solely in the light, and cause reduced growth and gradual chlorosis, followed by necrosis. Thus, symp- toms of injury from the classical photosynthetic inhibitors are distinct from those of dinoseb. It was surprising to observe that resistance ratios for dinoseb were positively correlated in whole plant and isolated chlorOplast studies in 4 species (Chapter 1) since this suggested a photosynthetic site of action. This cannot be explained by a general increase in susceptibility to herbicides, since resistance ratios for dicamba, dalapon and acifluorfen were very close to Il)(Chapter IL These herbicides do not inhibit at the triazine site of action. The differential response of the two biotypes at the whole plant level to dinoseb was clearly seen when applied in the morning on sunny 25 days, but not when applied in the afternoon (personal observation). One objective of this study was to verify this under controlled conditions. A second objective was to examine a possible photosynthetic site of action by determining the efficacy of dinoseb on the two biotypes in dark vs. light. Several lines of evidence from this experiment suggested that carbohydrate levels would be correlated with dinoseb efficacy. Therefore carbohydrate levels were assayed and the effects of exogenous sucrose was tested. MATERIALS AND METHODS Injury response to dinoseb. Smooth pigweed was grown and thinned to a uniform stand as described previously (Chapter 1) but in a growth room. Plants were grown at constant 25°C and 90% relative humidity, with 450 uE m'2 s"1 photosynthetic photon flux density with 14h/10h light/dark cycle. Dinoseb was applied at the two leaf stage either prior to illumin- ation ("morning") or after 8 h of illumination ("afternoon") and plants were returned to the same chamber in light or dark conditions. Dinoseb was applied at the two leaf stage at several rates on a logarithmic scale as in Chapter 1, with 0.5% v/v alkylaryl polyoxyethyleneglycol-free fatty acid-iSOpropanol surfactant (X-77 Spreader). Shoot fresh weights were determined 24 h after treatment. The data reported are the means of two experiments with six replications per experiment. Resistance ratios were calculated by dividing the herbicide rate that caused 50% injury (150) in the triazine resistant biotype by the I50 in the triazine susceptible biotype. Due to the nature of these data, analysis was conducted using each experiment as one replicate of a completely randomized design. Carbohydrate analyses. Details of the methods used were previously described by Haissig (1982a, 1982b) except that a different starch 26 hydrolysis enzyme was used (Mylase-IOO; GB Fermentation Industries, Inc; Charlotte, NC 28224). Plants were grown as in the previous experiment and harvested either prior to illumination ("morning") or after 8 h of illumination ("afternoon"). Dinoseb treatments were made to whole plants at these times to verify that differences in tolerance were as reported in Table 1 (data not shown). Leaf material was frozen as it was harvest- ed, ly0philized, and ground in a Niley mill to pass a 40-mesh screen. A 25 mg sample was analyzed. Soluble sugars were extracted three times in methanol-chloroform-water (12/5/3, v/v/v) and combined. Pigments and lipids were removed from the chloroform phase after the addition of water. The insoluble material was saved for starch analysis. Glucose was assayed spectrOphotometrically by the glucose oxidase/horseradish peroxidase assay using o-dianisidine as the color reagent (Sigma Chenical C0. Technical Bulletin No. 510, 1982) using glucose standards. Sucrose was hydrolyzed at 100°C in HCl, neutralized, and assayed by the same method as glucose. Initial glucose levels were subtracted to obtain true estimates for sucrose. Sucrose standards were hydrolyzed and assayed in the same manner; Reducing sugars were assayed spectrOphotometrically by the dinitrosalicylic acid method (Clark, 1964) and initial glucose levels were again subtracted. Glucose was used as the standard. Starch was enzymatically hydrolyzed for 48 h with 10 mgmil'1 Mylase-IOO in 100 mM acetate (pH SIM using potato starch as the standard. The hydrolyzed product was assayed by the same method as glucose. Mylase-IOO is a mixture of <1-amylase and other glucosidases, and is free of cellulase and hemicellulase activity OLE. Haissig, personal communicationL This enzyme was dialyzed against distilled water for 24 h to remove soluble sugars before use. Data reported are the means of two experiments with six replications per experiment. 27 Effect of exogenous sucrose. ‘Triazine resistant smooth pigweed plants were grown as described above. Leaves were excised and placed with petioles in 0, 0.5, 1, 2, or 5% sucrose. Leaves were placed in the dark for 10 h to allow uptake of sucrose and then sprayed with115 kg/ha dinoseb. Leaves were then placed in the illuminated growth room described above, and fresh and dry weights were determined 24 h later. RESULTS AND DISCUSSION Injury response to dinoseb. Both triazine resistant and susceptible biotypes had lower 150 values in the dark than in the light when treated in the morning (Table 1). This confirms previous reports of enhanced activity of dinitrophenolic compounds in the dark (Meggitt gt _13, 1956; lhellor and Salisbury, 1965), and contradicts a hypothesis that dinoseb affects photosynthetic electron transport. Resistance ratios observed for morning treatments were close to the value of 0.27 previously report- ed for smooth pigweed (Chapter 1). The resistance ratio of less than 110 in the morning treatments cannot be attributed to effects on photosynthe- tic electron transport since a Similar and slightly lower ratio was also observed in the dark treatment. 150 values were higher for both biotypes in the light in the afternoon than in the morning (Table 1). Furthermore, there was little difference in the 150 values for the two biotypes as shown in the resistance ratio. This documents previous unpublished observations with greenhouse experiments. Based on this observation, dinoseb may be more effective in the field if applied early in the morning; this is eSpecially true if dinoseb is used for control of triazine resistant weed species. 28 Table 1. Plant tolerance to dinoseb in triazine resistant and susceptible biotypes of smooth pigweed under various light conditions described in the text. Morning Afternoon Dark Light Light 150 Resistant Biotype (kg/ha) 0.045 b 0.091 b 1.05 a I50 Susceptible Biotype (kg/ha) 0.19 b 0.32 b 1.08 a Resistance Ratio 0.26 b 0.35 ab 0.98 a aMeans among the 150 values or among the resistance ratio values followed by the same letter are not significantly different at the 5% level by Duncan's multiple range test. 29 The hypothesis that endogenous carbohydrates are responsible for the observed differences in dinoseb efficacy can be pr0posed to explain the differences in dinoseb efficacy reported in Table 1. Carbohydrates may supply ATP and reducing power from glycolysis and oxidative phosphoryla- tion (if active in the presence of dinoseb) to maintain glutathione, carotenoids, ascorbate, a-toc0pherol or other celliilar protective mechan- isms in a reduced state. Several arguments support this hypothesis: 1) The triazine resistant biotype, which was more susceptible in both morn- ing treatments, fixes C02 more slowly than the susceptible biotype UDrt ._t__l” 1983; Ahrens and Stoller, I983); lower levels of carbohydrate may persist in the resistant biotype even after the dark period; 2) Dinoseb was more active in the dark than in the light; thus photosynthetic elec- tron transport and C02 fixation may have provided protection; and 3) Both biotypes showed much greater tolerance to dinoseb in afternoon treat- ments. The accumulation of photosynthetic products such as starch may have provided protection from injury. It can be proposed that the reason that the two biotypes did not differ in their tolerances in the afternoon was that there is some maximal level of protection by carbohydrate and that carbohydrate levels in both biotypes exceeded the capacity of the protective mechanism. Therefore, according to this hypothesis, it can be predicted that carbohydrate levels would be lower in the resistant bio- type than the susceptible biotype in the morning, and that that carbohydrate levels would be much higher in both biotypes in the afternoon. Carbohydrate analyses. Glucose and sucrose levels were generally much lower than reducing sugars or starch (Table 2). In no instances were carbohydrate levels lower in the resistant biotype than the susceptible 30 biotype in the morning. This contradicts the hypothesis stated. Several carbohydrate levels, most notably starch, were higher in the afternoon than in the morning. Nhile this correlative evidence is consistent with the stated hypothesis, it was also obviously anticipated. It is of interest to note that total carbohydrate levels were significantly higher in the susceptible biotype than the resistant biotype in the afternoon. This is consistent with previous reports of lower C02 fixation rates in the resistant biotype (Ort, 1983; Ahrens and Stoller, 1983) and ultra- structural studies on starch quantities in several Species (Burke gt al., 1982; Vaughn and Duke, 1983). Effect of exogenous sucrose. Sucrose or glucose has been shown to protect plants from injury by photosynthetic inhibitors (e41, Shimabukuro gt_al,, 1976). However, sucrose did not protect triazine resistant smooth pigweed from dinoseb injury (Table 3). At higher suc- rose levels, desiccation by dinoseb was actually enhanced. Therefore it is difficult to support the hypothesis that dinoseb tolerance is related to carbohydrate levels. The differential response of the two smooth pigweed biotypes cannot be attributed to differences in the sensitivity of the photosynthetic membranes, nor can it be attributed to differences in photosynthetic efficiency. However, it is known that chlorOplast lipids are less satu- rated in the resistant biotype (Pillai and St. John, 1981; Burke 33111” 1982). The possibility remains that dinoseb is more active in the resis- tant biotype for this reason. However, the increased tolerance of both biotypes to dinoseb in the afternoon remains unexplained. Finally, our observations do not contradict the hypothesis that dinoseb acts primarily 31 Table 2. Glucose, sucrose, reducing sugar, starch, and total carbohydrate (sucrose + reducing sugar + starch) levels in triazine resistant and susceptible smooth pigweed prior to illumination ("morning") or after 8h of illumination ("afternoon"). Carbohydrate levela Carbohydrate (umol/g dry weight) assayed Biotype Morning Afternoon Glucose Resistant 6.5 bc 7.8 b Susceptible 5 4 c 11 0 a Sucrose Resistant 11.1 a 6.4 b Susceptible 9.2 a 9.7 a Reducing sugar Resistant 97.8 b 100.0 b Susceptible 92.2 b 137.8 a Starch Resistant 17.3 b 142.6 a Susceptible 12.2 b 187.0 a Total carbohydrate Resistant 132.7 c 256.9 b Susceptible 119.0 c 345.5 a 3Means for each type of carbohydrate with the same letters are not significantly different at the 5% level by Duncan's Multiple Range Test. 32 Table 3. Effect of exogenous sucrose on desiccation by dinoseb in excised leaves of triazine resistant smooth pigweed. Dinoseb Sucrose Leaf Moisturea (kg/ha) (%) (%) 0 0 87.4 a 1 0 68.4 ab 1 0.5 63.1 bc 1 1 42.5 bc 1 2 52.7 cd 1 SD 26.8 d aMeans with same letters are not significantly different at the 5% level by D.M.R.T. bLeaves showed injury, due to plasmolysis, before dinoseb treatment. 33 within the mitochondria, as has been discussed by others (Mellor and Salisbury, 1965). ACKNONLEDGMENT I would like to express appreciation to Dr. Bruce Haissig of the U.S.D.A. Forestry Laboratory in Rhinelander, NI for technical advice and for supplying the Mylase-IOO enzyme. REFERENCES / Ahrens, N.H., and E.N. Stoller. 1983. Competition, growth rate and C02 fixation in triazine-resistant and -susceptible smooth pigweed (Amaranthus hybridus). Need Sci. _3_1:438-444. Ashton, FZM. and ANS. Crafts. 1981. Mode of Action of Herbicides. John Niley & Sons, New York, 525 pp. Ashton, F.M., 0.T. deVilliers, R.K. Glenn, and 14.8. Duke. 1977. Localization of metabolic sites of action of herbicides. Pestic. Biochem. Physiol. 1:122-141. Burke, J.J., R.F. Nilson, and J.R. Swafford. 1982. Characterization of chloroplasts isolated from triazine-susceptible and triazine- resistant biotypes of Brassica campestris L. Plant Physiol. _7_9_:24- 29. Clark, J.M., Jr. (ed). 1964. Experimental biochemistry. N.H. Freeman and Co., San Francisco, 228 pp. Haissig, B.E. 1982a. Activity of some glycloytic and pentose phosphate pathway enzymes during the develOpment of adventitious roots. Physiol. Plant. 55:261-272. Haissig, B.E. 1982b. Carbohydrate and amino acid concentrations during adventitious root primordiun devel0pment in Pinus banksiana Lamb. cuttings. Forest Sci. 28:813-821. Meggitt, N.F., R.J. Aldrich, and N.C. Shaw. 1956. Factors affecting the herbicidal action of aqueous sprays of salts of 4,6-dinitro- ortho-secondary butyl phenol. Mellor, R.S. and F.B. Salisbury. 1965. Influence of light regime on the toxicity and physiological activity of herbicides. Plant Physiol. 40: :506-512. Ort, D.R., N.H. Ahrens, B. Martin and E.N. Stol ler. 1983. Comparison of photosynthetic performance in triazine-resistant and susceptible biotypes of Amaranthus hybridus. Plant Physiol. 12:925-930. 34 Pillai, P., and J.B. St. John. 1981. Lipid composition of chlor0plast membranes from weed biotypes differentially sensitive to triazine herbicides. Plant Physiol. flz585-587. Shimabukuro, R.H., V.J. Masteller, and N.C. Nalsh, 1976. Atrazine injury: relationship to metabolism, substrate level, and secondary factors. Need Sci fiz336-340. Vaughn, K.C., and 8.0. Duke. 1983. The triazine-resistance syndrome. Plant. Physiol. 72 (suppl.):No. 994. CHAPTER 3 Chemical Control of Triazine Resistant Common Lambsquarters (Chen0podium album) and Two Pigweed Species (Amaranthus spp.) ABSTRACT Various chemical treatments were evaluated over two growing seasons for control of triazine resistant common lambsquarters (Chen0podium album LJ and for control of a triazine resistant infestation containing both redroot pigweed (Amaranthus retroflexus L3) and Powell amaranth (A; powellii S. NatsJ. Triazines, atrazine [2-chloro-4-(ethylamino)-6- (iSOprOpylamino)-§-triazine], cyanazine {2-[14-chloro-6-(ethylamino)-§- triazin-Z-ylJaminol-Z-methylpr0pionitrile}, and metribuzin [4-amino-6- .tgrt-butyl-3-(methylthio)-a§-triazin-5(4H)-one], provided unsatisfactory control of these species. Satisfactory control of lambsquarters was obtained with preemergence applications of pendimethalin [Ny(1- ethylpropyl)-3,4-dimethyl-2,6-dinitrobenzenamine] or dicamba (3-6- dichloro-g-anisic acid), postemergence applications of dicamba, bromoxy- nil (3,5-dibromo-4-hydroxybenzonitrile), or bentazon [3-i50proyl-IH; 2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide], or various directed late postemergence treatments. Satisfactory control of pigweed was obtained with preemergence applications of alachlor [2-chloro-Z'JY-diethyl-N- (methoxymethyl)acetanilide] or postemergence treatments of dicamba, bromoxynil, or 2,4-0 [(2,4-dichlor0phenoxy)acetic acid]. 35 36 INTRODUCTION LeBaron (1984) has recently reviewed the extent and implications of herbicide resistance in weeds. Resistance to six chemical classes of herbicides occurs in 46 species. Resistance to triazine herbicides occurs in 37 species in 12 countries. Infestations are most often seri- ous but localized. Herbicide resistance is likely to become a more serious problem in the future since trends toward decreased tillage and closer row spacings require greater dependence on herbicides. Fortunate- ly, in most cases, alternative cr0pping and/or weed control systems are available to provide control of the resistant biotypes. Triazine resistant common lambsquarters was observed in Huron County, Michigan in 1980 on land which had been in continuous corn for at least 7 years and treated solely with atrazine over this period. Two triazine resistant pigweed species (Powell amaranth and redroot pigweed) were observed in Oceana County, Michigan in 1981 on land which had been in continuous corn for 5 years and treated with atrazine and butylate (S-ethyl diisobutylthiocarbamate) plus R-25788 (N,N-dtallyl-2,2- dichloroacetamide). The two species were distinguished by the criteria of Ahrens gt al, (1981). The objective of this study was to determine chemical methods for control of these triazine resistant weeds where cr0p rotation was not desired. Treatments were selected based on chemicals known to control these species (Barrett and Meggitt, 1983), on a knOw- ledge of cross resistance (Chapter 1), and on reports on control of triazine resistant species (Anonymous, 1981c; Ritter, 1982; Parochetti 33 al., 1982). Buthidazole has been shown to inhibit photosynthesis at two sites (Hatzios 21;.2131 1980; York gt_al,, 1981). Cross resistance to buthidazole is low (Chapter 1), perhaps for this reason. Therefore this 37 compound was evaluated with and without acetanilide herbicides which have been shown to protect corn from buthidazole injury (York and Slife, 1981). MATERIALS AND METHODS All experiments were performed on plotsllOSm by 9J4n1arranged as a randomized block with CL76m row spacing. Treatments were applied with 90 L/ha water at a pressure of 210 kPa. Data were arcsine-transformed before analysis. Control of 80% or more of the weeds was considered “satisfactory." Triazine resistant pigweed. Preemergence treatments were applied 1 day after planting (DAP), on May 15, 1982 and May 14, 1983. Postemergence treatments were applied when the pigweed was 1.5 to 2.5 cm tall; this was 22 DAP in 1982 and 38 DAP in 1983. Corn was at the three to four-leaf stage, approximately 15 cm tall, in 1982 and was at the four-leaf stage, approximately 15 cm tall, in 1983. Treatments were visually evaluated 71 DAP during tasseling in 1982, and 62 DAP, prior to tasseling, in 1983. The two pigweed species were not evaluated separately. The pigweed population was ISO/m2 at 62 DAP in 1983 and was Similar in 1982. There were four replications. The soil was a sandy loam, 5.9% organic mat- ter, pH 6.5. The corn variety was Pioneer 3901 in 1982 and Supercross 1940 in 1983. Triazine resistant common lambsquarters. Preplant treatments were incorporated with two passes of a field cultivator set to a 13 cm depth. Preemergence treatments were applied on the same day (May 22, 1981; May 25, 1983), following planting. Postemergence treatments were applied when the common lambsquarters was 1,5 to 2.5 an tall. This was 19 DAP in 38 1981 and 29 DAP in 1983. Corn was at the two to three-leaf stage, approximately 10 cm tall, in 1981 and was at the six-leaf stage, approxi- mately 17 cm tall, in 1983. Directed late postemergence treatments were applied 44 DAP both years when the common lambsquarters was 20 to 30 cm tall. Dr0p nozzles directed sprays at the base of the corn plants. Corn was 45 to 60 cm tall in 1981 and 60 cm tall in 1983. Treatments were visually evaluated 62 DAP in 1981 and 66 DAP in 1983 during the tasseling stage of corn. ‘The lambsquarters p0pulation was 200/1112 at 66 DAP in 1983 and was similar in 1981. There were three replications. The soil was a sandy clay loam, 3.7% organic matter, pH 5.7. The corn variety was Payco 342 both years. RESULTS AND DISCUSSION Triazine resistant-pigweed. The triazines, atrazine, cyanazine, or metribuzin, provided unsatisfactory control of pigweed as preemergence treatments (Table 1L Alachlor was the most effective preemergence herbi- cide and provided significantly higher levels of control than metolachlor (Table 1L. Control with metolachlor [2-chloro-N-(2-ethyl-6- methylphenyl)-N;(2-methoxy-1-methylethyl)acetamide] was unsatisfactory. .Alachlor and metolachlor have previously been reported to be equally effective on redroot pigweed (Barrett and Meggitt, 1983) and on triazine resistant smooth pigweed (A; hybridus LJ (Ritter, 1982). The relative efficacy of alachlor and metolachlor is dependent upon tillage system and soil type (Strek and Neber, 1981; 1982). Alachlor plus atrazine applied preemergence in sequence with dicamba or bromoxynil applied postemergence gave satisfactory control in 1983 (Table 1). However, control was not significantly better than dicamba or bromoxynil alone postemergence. 39 Table 1. Triazine resistant pigweed control with various chemical treatments. Rate % Controla Treatment (kg/ha) 1982 1983 Preemergence Atrazine 1.1 6 4 m Atrazine 11 - 15 klm Cyanazine 1.1 0 1 8 m Metribuzin + atrazine 0.28 + 1 1 0 i 10 lm Alachlor 2.8 85 abcde 78 efg Alachlor + atrazine 2.8 + 1.1 91 abc 86 def Metolachlor + atrazine 2.8 + 1.1 77 def 56 ghij Pendimethalin + atrazine 1.7 + 1.1 45 h 42 ijkl Pre- and postemergence Alachlor + atrazine (preemergence) and 2.8 + 1 1 dicamba (postemergence) 0.28 - 100 a Alachlor + atrazine (preemergence) and 2.8 + 1.1 bromoxynil (postemergence) 0.33 - 98 abc Postemergence AtraZTne + c.o.c.b 2.2 + 2.3 L/Ha 16 1 16 klm Dicamba 0.28 94 a 97 abcd Dicamba 0.56 95 a 99 ab 2,4-0 0.56 88 abcde 91 bcde Dicamba + 2,4-0 0.28 + 0.56 95 a 100 a Bromoxynil 0.28 69 fg 92 bcde Bromoxynil 0.33 81 bcdef 88 cdef Bromoxynil 0.56 77 ef 92 bcde Dinoseb 1.1 69 fg 38 jklm Dinoseb 2.2 82 bcdef 45 hijk Dinoseb 4.5 88 abcde 71 fgh Buthidazole 0.14 58 gh 31 jklm Buthidazole 0.28 76 ef 51 hij Buthidazole + dicamba 0.14 + 0.28 94 a 99 ab aMeans within columns with same letters are not significantly different at the 5% level by Duncan's multiple range test. bc.o.c. = cr0p oil concentrate. 40 Satisfactory pigweed control was obtained with postemergence dicamba, 2,4-0, and the two combined (Table 1). Bromoxynil provided significantIy less control than dicamba in 1982 but not in 1983. Control with dinoseb (Z-seg-butyl-4,6-dinitr0phenol) tended to increase as rates increased from 1.1 to 4.5 kg/ha. The highest dinoseb rate caused slight corn injury (5%) in 1983 (data not shown). It is possible that more effective pigweed control would have been obtained with morning treat- ments of dinoseb (Chapter 2), since our treatments were made near midday. Buthidazole {3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2yl]-4- hydroxy-l-methyl-2-imidazolidinone} did not provide satisfactory post- emergence control at the rates tested and showed slight injury (5%) at the higher rate in 1983 (data not shown). Buthidazole in combination with dicamba gave satisfactory control, but not significantly more effective than dicamba alone. Triazine resistant common lambsquarters. The triazines, atrazine, cyanazine, or metribuzin, gave unsatisfactory control of this common lambsquarters biotype in various preplant, preemergence, and postemergence treatments (Table 2). EPTC (S-ethyl dipr0pylthiocarbamate) plus R-25788 provided the highest level of control of the preplant treatments both years, but not significantly greater than butylate plus R-25788 (Table 2L Decreased control in 1983 by the two treatments can be attributed to desorption and leaching of the herbicide as a result of a prolonged cool wet period after treatment. Buthidazole preplant treatments in 1983 provided 100% control, but cr0p injury was unacceptable. However, cr0p injury was somewhat less in the presence of the acetanilides alachlor or metolachlor as was previously reported (York and Slife, 1981). 41 o o 44zmcme NN seasons mm 4.4 + N.N + Ne.o 6=4NNLSN + 404guc4oume + unam04o o o :mcouun NN canons as N. 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Dicamba applied in various combinations provided satisfactory control. The preemergence efficacy of dicamba is dependent on soil type (Anonymous, 1981b). Dino- seb provided satisfactory control at the recommended rate of 10.1 kg/ha. Significant levels of control at 0.25 times the recommended rate may be due to the increased susceptibility to dinoseb of this biotype (Chapter 1L Buthidazole at(134 kg/ha provided satisfactory control with low levels of cr0p injury. However, severe injury to corn occurred at 1.1 kg/ha buthidazole. Insufficient protection was obtained by addition of metolachlor. Preemergence pendimethalin in combination with postemergence dicamba gave satisfactory weed control, but control was not significantly greater than when this same combination was applied preemergence, or when dicamba was applied alone postemergence (except(128 kg/ha dicamba in 1983) (Table 2L Postemergence applications of dicamba, 2,4-0, or the combination provided satisfactory control except in 1983 at00 00 004 40.400000000 0040000000000 400 000 0004404 0000 004 00 00304400 0000400 000403 000020 ‘I'TI' l'."l"" 'I' .I' ".I‘""‘.'!' ll..'1 0 000.00 0 000.0 0 000.00 00 N0 0 0N 000000000000 + 0000000< . 0 000.N0 0 00N.N 0 000.00 0 00 0 00 000000000400 - - - - 0 00 00 00 0040004< . 0 000.00 0 000.0 0 000.40 0 0N 00 0N - 004000000000 + 0040004< 0 000.00 0 000.0 0 000.00 0 N0 0 0N - 004000000000 - - - 0 00 00 00 - 0040000< . - u 0 004 0 000 - n 0400002 000 0400003 00000 0040000 0\E000 00004000 00 00 04004000 00 00 000~ 4000i 000~ 40000 0 000 0000 0 000 0000 400003 000 40000: 00000 400040004 0000000: lxu0>0000 004 0.0400040004 000~ 40000 000 000~ 4000 000000> 00400 040000 0000 00 4000000 040N00004znuue4 000 00000000 40000: 000 000 00000 40000 .4 04000 52 treatments (Chapter 2). More 14C was present in treatments with buthidazole alone than with buthidazole plus alachlor. Therefore alachlor provides protection from buthidazole by reducing the amount reaching the leaves, and this is probably due to reduced root uptake. Bucholtz and Lang (1978) reported that alachlor caused reduced uptake of the photosynthetic inhibitors atrazine [2-chloro-4-(ethylamino)-6- (iSOprOpylamino)j§-triazine], cyanazine {2-[E4-chloro-6-(ethylamino)j§f triazin—Z-yl]amino]-2-methylpr0pionitrile} and diuron [3-(3,4- dichlorOphenyl)-1,1-dimethylurea]. The reduced uptake was attributed to root pruning. However, these experiments were done on a susceptible species, oats (Avena sativa L.), and alachlor was used at rates which caused 46% decrease in dry weight and more than 70% decrease in root length. However, our experiment involved the use of corn, a tolerant species, and the rates of alachlor used caused only slight decreases in corn shoot weight. York and Slife (1981) used alachlor at similar rates and corn was protected from buthidazole injury. However, there was no effect on corn root weights. Therefore, the decreased root uptake may not be due to root pruning; it is possible that an altered uptake mechanism may be involved. REFERENCES Bucholtz, 0.13. and T.L. Lang, 1978. Pesticide interactions in oats (Avena sativa L. 'Neal'). J. Agric. Food Chem., 335520-524. Hatzios, KJC, and D. Penner. 1980. Potential antidotes against buthidazole injury to corn (Zea mays). Weed Sci. g§:273-276. York, A.C., and F.W. Slife, 1981. Interaction of buthidazole and acetanilide herbicide. Weed Sci. 29:461-468. CHAPTER 5 Characterization of Two Sites of Action for Buthidazole, Buthidazole Metabolites, and onynil ABSTRACT Buthidazole {3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4- hydroxy-l-methyl-2-imidazolidinone} and ioxynil (4-hydroxy-3,5,- diiodobenzonitrile) inhibit photosynthetic electron transport at two sites in chloroplast thylakoid membranes isolated from pea (Pisum sativum LA. The primary site of inhibition by both herbicides is at the secon- dary quinone acceptor, 08, the site of action of atrazine [2-chloro-4- (ethylamino)-6-(i50pr0pylamino)-s-triazine] or diuron [3-(3,4- dichlorOphenyl)-1,1-dimethylurea]. The secondary site of action was measured as inhibition of silicomolybdate (SiMo) photoreduction. Onset of inhibition at the secondary site of action was relatively rapid for ioxynil but slow for buthidazole. This inhibition was reversible for ioxynil but not buthidazole. Buthidazole did not quench in vitro chloro- phyl I fluorescence although ioxynil did. The secondary site of action of buthidazole and ioxynil is on the oxidizing side of photosystem II. Buthidazole may have reduced the rate constant of an electron carrier. onynil inhibited at an electron carrier which was required for electron donation by NHZOH or diphenylcarbazole. Most buthidazole metabolites had potential herbicidal activity in_ .11359, but were less active than buthidazole. Therefore, metabolism of 53 54 buthidazole is a detoxification process. The potential contribution of buthidazole metabolites to plant injury is discussed. INTRODUCTION Buthidazole inhibits photosynthetic electron transport at two sites including a primary site similar to atrazine or diuron, and a secondary site on the water oxidizing side of photosystem II (Hatzios 23.5fl3’ 1980; York 33 31:, 1981). onynil inhibits at the triazine binding site (Vermaas and Arntzen, 1983) as well as on the water oxidizing side of photosystem II as measured by delayed luminescence (Van Assche, 1982). The objectives of this study were: 1) to determine the relative importance of the two sites of action of buthidazole and ioxynil; 2) to ‘determine the herbicidal potential at the secondary site by a large number of herbicides; 3) characterize the reversibility of inhibition, delay of inhibition and localization of the secondary site of action; and 4) evaluate the herbicidal activity of buthidazole metabolites. MATERIALS AND METHODS Photosystem IIgpartial reactions. Chlor0plast thylakoid membranes were isolated from 2-3 week old pea seedlings and rates of photosynthetic electron transport were determined by monitoring the photoreduction of dichlorOphenol-indOphenol (DCPIP) as previously described (Paterson and Arntzen, 1982). SiMo accepts electrons from DA, the primary quinone acceptor of photosystem II (Izawa, 1980; Girault and Galmiche, 1974). Rates of photosystem II electron transport to SiMo were measured under saturating light by continuous recording of oxygen evolution using a water-jacketed Clark-type oxygen electrode maintained at 20°C. The assay buffer contained 25 0g chlorOphyll'ml'l, 20 mM Tricine-NaOH (pH 7.8), 100 mM sorbitol, 5 mM NaCl, 1011M diuron,(15 mM FeCN, and 200 uM SiMo. 55 Herbicides were preincubated in the assay buffer for 15 min and SiMo was added immediately prior to assay. Rates in control samples were approxi- mately 50 umol 02' mg chl‘l'h‘1 and were stable for at least 10 min. All data reported are the means of two replications. Partial reactions using diphenylcarbazole (DPC) or NHZOH as artificial donors to photosystem II were unsuccessful because these compounds directly reduced SiMo in the dark (personal observationL Reversibility of inhibition. Thylakoid membranes (500 mg chlor0phyll°5 ml'l) were incubated in 1 mM buthidazole, 300 pM ioxynil or 10 uM diuron (control) in a buffered solution with 10 mM Tricine NaOH (pH 7.8), IOmM NaCl, 5 mM MgCl2, and 100 mM sorbitol for ISIWHL Aliquots were removed to measure inhibition of electron transport to SiMo. The thylakoid suspensions were diluted in 30 ml of the same buffer which included 5 mg'ml‘1 bovine serum albumin (BSA). Thylakoid membranes were centrifuged at 1000 g for 5 min and resuspended in the buffer with BSA. This centrifugation/washing process was repeated three more times, with a 15 min incubation in the buffer each time. Aliquots were then removed to measure electron transport to SiMo. All steps except the assay were carried out at 4°C. Localization of the secondary site of action. In vitro<fl1lor0phyll fluorescence transients were recorded with a Nicolet Explorer digital oscil losc0pe with assay conditions as previously described (Paterson and Arntzen, 1982). Herbicides were preincubated with the assay buffer 15 min prior to assay. Various chemical treatments were added to modulate the fluorescence maximum, including 500 “M opc, 20 mM NHZOH, or 1 mg-mi’1 dithionite. Tris washing or heat treatment inactivates water oxidation. Tris washing was done by incubating 200-400 ug chlorOphyll'ml'1 in 04314 56 tris buffer pH 8.0 at 0°C for 20 min. Heat treatment was done by heating a thylakoid suspension in a preheated tube for 3 min. at 50°C and then cooling immediately to 4°C. The initial fluorescence value (F0) and the maximal fluorescence valLK3(FM) were determined and the variable fluores- cence value, (FM - F0)/F0, is reported. The sites of action of the various compounds and treatments used in these experiments are shown in Figure 1. All data reported are the means of two replications. RESULTS AND DISCUSSION Buthidazole and ioxynil inhibited DCPIP photoreduction at lower concentration than SiMo photoreduction (Table 1). The activities of buthidazole and ioxynil were compared over time at this secondary site (Figure 2). Concentrations of the herbicides were used which previously caused 50% inhibition after a 15 min incubation (Table 1). Buthidazole had no initial activity and 30-60 min. were required to reach maximal activity. In contrast, ioxynil had relatively high initial activity and reached maximal activity by 7 min. Therefore the action of these two herbicides at the secondary site differs with reSpect to the degree of delay in action. The reversibility of inhibition at the secondary site was examined (Table 2). Inhibition by buthidazole decreased slightly but not signifi- cantly after washing. In contrast, inhibition by ioxynil was reversible to a significant extent. Therefore, the reversibility of inhibition was greatest for the herbicide for which the delay in action was least (Figure 1). Thus, the action of these two compounds can also be distinguished with respect to the reversibility of inhibition. 57 Figure 1. Simplified scheme of photosynthetic electron transport. Sites of reduction, oxidation and inhibition by various compounds are indicated. P680 is the photosystem II reaction center. P700 is the photosysten I reaction center. 58 oowua oasc. aoJ D ““S S a: 20 >5 X 0 0 7 15 3O 60 Length of Incubation minutes 61 Table 1. Concentrations of buthidazole and ioxynil which cause 50% inhibition (150) of photosynthetic electron transport by two assays. [50 Values (uM) DCPIP SiMo Herbicide Photoreduction Photoreduction Buthidazole 0.11 150 onynil 0.70 43 Table 2. Reversibility of inhibition of SiMo photoreduction. (Activities are as a % of a 10 uM diuron control treatment.) % Inhibitiona Herbicide Before Washing After Hashing Buthidazole 75b 68b onynil 82b 32a aMeans followed by the same letters are not significantly different at the 5% level according to Duncan's multiple range test. 62 A wide range of herbicides were evaluated at 300 uM concentration for their ability to inhibit SiMo photoreduction (Table 3L Nitrofluor- fen [Z-chloro-l-(4-nitrophenoxy)—4-(trifluoromethyl)benzene], swep [methyl -N—(3,4-dichl or0phenyl )-carbamate] , karsil (Z-methyl -val eric-3,4- dichloroanilide), and JNP-867 (a 2-cyanoacrylic acid ester derivative) were more active than ioxynil or buthidazole. Nitrofluorfen andTJNP-867 caused 100% inhibition at 30 pH (data not shown). These observations suggest that there is herbicidal potential at this secondary site. Diuron was a very active inhibitor. The response of variable fluorescence to various herbicide concentrations was also evaluated (Table 4). 'The concentrations of ioxynil and buthidazole tested previously caused inhibition at the secondary site (Table 1). Treatments which inhibit photosynthetic electron transport on the water-oxidizing side of photosystem II, such as Tris-washing or heat treatment, cause a quenching of variable fluorescence (Table 4). Diuron at 10 UM is assumed to inhibit solely on the reducing side of photosystem II. Diuron at 1 mM and buthidazole at 300 uM or 1 mM did not quench variable fluorescence compared to diuron at 10 pM. This indicates that no inhibition was occurring at the immediate electron donor to P680“ The secondary site of buthidazole action cannot be at 0A, since variable fluorescence would not be observed if inhibition occurred at that site. It is possible that buthidazole is reducing the rate constant of a rate limiting electron carrier on the oxidizing side. The reason that vari- able fluorescence is not quenched may be that charge separation at P680 may be rate limiting under the nonsaturating light conditions used for the fluorescence experiments. onynil quenched variable fluorescence, in contrast to buthidazole. This suggests that it inhibits on the water oxidizing side of photosystem 63 Table 3. Inhibition of SiMo photoreduction by a wide range of herbicides at a concentration of 300 pM.a Herbicide % Inhibition Nitrofluorfen 100 a Swep 100 a Karsil 100 a JNP-867 100 a onynil 94 a Buthidazole 83 b Diuron 61 c Metribuzinb 49 d Tebuthiuronc 35 e Propanild 29 ef Bromoxynile 28 ef Bromacil 27 ef Desmediphamg 25 fg Dinoseb 18 gh Atrazine 15 h Pyrazon), -2 i BentazonJ -7 i aMeans followed by the same letters are not significantly different at the 5% level by Duncan's multiple range test. b4-amino-6-tgrtfbutyl-3-(methylthio)-g§7triazin-5(4H)-one. cN-[S-(1,1-dimethylethyl)-1,3,4-thidiazol-2-yl]-N-N'-dimethylurea. d3',4'-dichloropr0pionanilide. e3,5-dibromo-4-hydroxybenzonitrile. fS-bromo-B-sggfbutyl-6-methyluracil. gethyl m—hydroxycarbinilate carbanilate (ester). h2-§_e-_c_;-butyl-4,6-dinitrOphenol. 1.5-amino-4-chloro-Z-phenyl-3(2H)-pyridazinone. j3-isopr0pyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide. 64 Table 4. Variable fluorescence (FM-FO)/F0, after various herbicide and chenical treatments.a (FM 'FO )/F Subsequent addigions: b Pretreatment DPC NH20H Dithionite Experiment 1: Diuron 10 uM 1.99 d 1.96 d - ~ Diuron 1 mM 2.03 d 1.99 d - - onynil 30 pMC 0.98 f 0.95 f - - onynil 100 uMC 0.76 g 0.74 g - - Buthidazole 300 pH 2.29 b 2.27 b - - Buthidazole 1 mM 2.12 bcd 2.08 cd - - Diuron 10 pM 2.25 bc - 2.28 b 2.53 a onynil 30 uMC 1.26 e - 1.24 e 1.37 e Experiment 2: Diuron 10 uM 1.31 b - 1.42 b - onynil 100 uMC 0.30 a - 0.25 d - Buthidazole 300 “M 1.28 b - 1.38 b - Tris-wash 0.31 d - 1.62 a - Heat 0.23 d - 0.77 c - aMeans within an experiment followed by the same letters are not significantly different at the 5% level by DuncanHSImJltiple range test. bAdditions are listed in the order in which they were used. Conynil decreased the variable fluorescence. However, unlike Tris- washing or heat treatment, this was partially due to an increase in F0. 65 II. However, variable fluorescence was not restored by the addition of NHZOH or DPC. This was unlike the tris-washed or heat-treated thylakoids. DPC did not restore variable fluorescence either. ‘This suggests that the site of inhibition of ioxynil is on the oxidizing side of photosystem II, but at an electron carrier which is required for electron donation by NHZOH or DPC. The herbicidal activity of buthidazole metabolites was also evaluated. .All of the buthidazole metabolites had 150 values greater than those of buthidazole (Table 5). All of the buthidazole metabo- lites, except for the amine and urea, had 150 values less than 10 0M and therefore have potential physiological significance as in vivo inhibi- tors. The amine and the urea metabolites, the proposed final products or buthidazole metabolism (Yu gt__ln,1980), have very low inhibitory activity. Metabolism of buthidazole is therefore a detoxification pro- cess. The buthidazole metabolites inhibited SiMo photoreduction less than buthidazole (Table 6). 'The slow onset of buthidazole activity has been reported previously (York gt _l,, 1981). Buthidazole metabolites showed varying degrees of delay in activity (Table 7); however, the dehydrate showed no delay in activity. Buthidazole was reported to be more abundant than any of its metabolites in pigweed (Amaranthus retroflexus LJ (Hatzios and Penner, 1982), quackgrass (AgrOpyron repens (Ls) Beauvu) (Hatzios and Penner, 1980), and corn (Z_e_a_ maxs L.) (Yu et al., 1980) over a time course of 6, 6, or 25 days, respectively. A polar metabolite presumed to be a conjugate (Hatzios and Penner, 1980), and the amine (Yu gt 1., 1980) were reported to be rapidly formed in alfalfa (Medicago sativa LJ. The amine has low herbicidal activity (Table 5) and the polar metabolite would not be expected to be active as a herbicide due to the hydr0phobic 66 Table 5.1Eg and 180 values for buthidazole and its metabolites measured by D I P pho8t00reduction. I50 I80 Metabolite (UM) (uM) Buthidazole 0.11 0.32 Methylurea 0.36 1.4 Methylol 0.95 3.7 Dehydrate 1.2 5 Dihydroxy 1.5 3.5 Desmethyl 1.7 8.5 Desmethyl-dihydroxy 9.1 30 Amine 170 - Urea 210 - 67 Table 6. Inhibition of SiMo photoreduction by buthidazole and its metabolites. All compounds were tested at 300 0M. Inhibition Metabolite (%) Buthidazole 83 a Dihydroxy 31 b Methylurea 21 bc Methylol 19 bc Dehydrate 9 cd Desmethyl-dihydroxy 1 d Desmethyl -1 d Amine -1 d Urea -3 d aMeans followed by the same letters are not significantly different at the 5% level by Duncan's multiple range test. 68 Table 7. Time for 50% inhibition, using concentrations which caused 80% inhibition at 15 min. (Table 5) measured by DCPIP photoreduction. Time for 50% Metabolite inhibition (min.) Dehydrate 0 Desmethyl 1.4 Methylurea 1.5 Methylol 3.0 Buthidazole 5.2 Desmethyl-dihydroxy 6.6 Dihydroxy 7.2 69 nature of the herbicide binding site (Hirschberg and McIntosh, 1984). The highly active metabolites reported in Table 5 were not abundant in any of the species tested (Hatzios and Penner, 1980; 1982; Yu gt al:, 1980). Therefore the in vivo toxicity of the buthidazole metabolites is presumably low in the plant species evaluated. REFERENCES / Girault, 0., and J.M. Galmiche. 1974. Restoration by silicotungstic acid of DCMU-inhibited photoreactions in Spinach chlor0plasts. Biochim. Biophys. Acta _3_§_3_:314-319. Hatzios, K.K., and 0.4 Penner. 1980. Absorption, translocation and metabolism of 1 C-buthidazole in alfalfa (Medica o sativa) and quackgrass (Agropyron repens). Weed Sci. __; - 39. Hatzios, KWK., and D. Penner, 1982. Metabolism of 14C-buthidazole in corn (lea ma 5 L.) and redroot pigweed (Amaranthus retroflexus LJ. Heed Res. __3 37-343. Hatzios, KJC” D. Penner, and D. Bell, 1980. Inhibition of photo— synthetic electron transport in isolated spinach chlor0plasts by two 1,3,4-thidiazolyl derivatives. Plant Physiol 655319-321. Hirschberg, J., and L. McIntosh. 1984. Molecular basis of herbicide resistance in Amaranthus hybridus. (Submitted) Izawa, S. 1980. .Acceptors and donors for chlorOplast electron transport. 13: Methods in Enzymol. 69(C), A. San Pietro, ed” Academic Press, N.Y., pp. 413-433. Paterson, [LR., and C.J. Arntzen, 1982. Detection of altered inhibition of photosystem II reactions in herbicide-resistant mutants. In; Methods in Chlor0plast Molecular Biology. hm Edelman, RJL Hallick, afg NJL Chua, eds. Elsevier Biomedical Press, Amsterdam, pp 109- 1 . Van Assche, CgL, and PJW. Carles, 1982. Photosystem II inhibiting chemicals: molecular interaction between inhibitors and a common target. ‘15: Biochemical Responses Induced by Herbicides, D.E. Moreland, J.B. St. John, and F.D. Hess, eds., ACS Symposium Series No.181, Washington DAL, pp.1~22. Vermaas, WJKJ., and Cu]. Arntzen. 1983. Synthetic quinones influencing herbicide binding and photosystem II electron transport. The effects of triazine resistance on quinone binding properties in thylakoid membranes. Biochim. Bi0phys. Acta 72§;483-491. 70 York, A.C., C.J. Arntzen, and F.W. Slife. 1981. Photosynthetic electron transport inhibition by buthidazole. Need Sci. 29: 59-65. Yu, C.C., Y.H. Atallah, and D.M. Whitacre, 1980. Metabolism of the herbicide buthidazole in corn seedlings and alfalfa plants. Agric Food Chem. _2_8_: 1090-1095. CHAPTER 6 Studies on the Mechanism of Paraquat Resistance in Conyza linefolia ABSTRACT A biotype of Conyza linefolia* originating in Egypt is resistant to the herbicide, paraquat (l,l'-dimethyl-4¢V—bipyridinium ion). The re- sistant and susceptible (wild) types were indistinguishable by measuring ‘in vitro photosystem I partial reactions using paraquat, diquat (6,7- dihydrodipyrido [1,2-a:2',1'-c] pyrazinediium ion), or triquat (7,8- dihydro-6H-dipyrido [1,2-o:231'-c] [1,4] diazepinediium ion) as electron acceptors. Therefore, an altered site of action is not the basis for resistance. ChlorOphyl I fluorescence measured i vivo is quenched in the susceptible biotype by leaf treatment with the bipyridinium herbicides. Resistance to quenching of_i_ vivo chlor0phyll fluorescence was observed in the resistant biotype, indicating that the herbicide was excluded from the active site. Penetration of the cuticle by 14C-paraquat was greater in the resistant biotype than the susceptible biotype; therefore resis- tance was not due to differences in uptake. 14C was localized in vascular regions of the resistant biotype when excised leaves were supplied 14C paraquat through the petiole. It is proposed that the mechanism of *Genus and species were communicated to A. Dodge by M. Parham (ICI; Jealotfls Hill, EnglandL Taxonomists have used "C. linifolt§'(not C. linefolia) in several instances to identify differéfit plant species. TAt least two such species have been reclassified under a different genus. The true identify of the species studied here must therefore be questioned, and no botanical designation can be given. 71 72 resistance to paraquat is exclusion from the prot0plast by adsorption to the extracellular matrix. However, direct evidence of selective ad- sorption to a cell wall fraction was not obtained by sucrose density gradient fractionation. INTRODUCTION Need resistance to paraquat (Figure 1) has been reported in annual bluegrass (Poa annua LJ, Philadelphia fleabane (Erigeron philadelphicus LJ and Conyza linefolia in England, Japan, and Egypt, respectively (Gressel 35; al., 1982). In every case, paraquat was applied several times per year for more than 5 years. Paraquat tolerant lines of peren- nial ryegrass [Lolium perenne L. (Causeway)] have also recently been developed (Harvey and Harper, 1982). The resistant biotype of Q; linefolia originated in the Tahrir irrigation area in Egypt. An intensive paraquat spraying program was undertaken in vine and citrus plantations in 1970 and difficulties in controlling this weed were first observed in the mid 1970's (Parham, 1982). The resistant biotype survived 2 kg/ha paraquat while the wild (susceptible) type was controlled by 0.5 kg/ha. The resistant biotype was controlled by 1 kg/ha diquat, but onlerLZ kg/ha was required for the wild type (Parham, 1983). Thus cross resistance to another bipyridinium herbicide was evident. Members of the genus Copy a_are common weeds in the U.S. and are commonly called horseweed or marestail. ‘These weeds have become a local- ly serious agronomic problem in reduced tillage systems (Anonymous, 1983), including some areas of Michigan (J. Kells, personal comnunica- tion). These weed species proliferate under conditions in which weed control often depends upon the use of paraquat. It is possible that 73 paraquat resistance may become a problem in this genus in the U.S. due to the trend toward reduced tillage and heavier reliance upon paraquat. Genetic engineering and conventional breeding techniques are being used to transfer triazine resistance into susceptible cr0p species (LeBaron, 1983). It is possible that resistance to paraquat can be exploited by the same means. The paraquat resistance trait in C; ling; fgljg_may not confer a yield loss since dry weight accumulation was equal in the two biotypes in greenhouse experiments (Parham, 1983). The mechanism of paraquat action involves the photosystem I-mediated reduction of the paraquat di-cation. This results in the formation of the mono-cation radical. The mono-cation radical reduces 02 to 02", the superoxide anion radical, resulting in the regeneration of the paraquat di-cation. Subsequently, hydrogen peroxide and the hydroxyl radical (0H0 "my be produced by a variety of reactions (Dodge, 1982; 1983). Hydroxyl radicals cause peroxidation of fatty acids. This is apparently a cause of the observed loss of membrane integrity (Harris and Dodge, 1972; Hutchison, 1979; Dodge 1983). Superoxide and hydrogen peroxide may not directly cause the paraquat-induced loss of membrane integrity (Hutchison, 1979). In addition to the formation of reactive forms of oxygen, the presence of paraquat causes the diversion of electrons which normally would reduce NADP. Cellular protective mechanisms which utilize reducing power, including carotenoids, a-toc0pherol, glutathione, and ascorbate, are assumed not to be maintained in the presence of paraquat. The action of superoxide dismutase, catalase, and peroxidase would presumably remain unaffected, however. Several hypotheses were developed which could eXplain the mechanism of paraquat resistance in C; linefolia, These were: 1) detoxification of the superoxide anion radical or other reactive forms of oxygen produced in 74 Figure 1. Structures and redox potentials of the three bipyridinium herbicides tested. 75 H,c—’N\ / /_\ LCH3 \ / /_\ N N Paraquat -446 mV Diquat -349 mV Triquat - 538 mV 76 the presence of paraquat; 2) alteration in the redox potential of the photosystem I primary electron acceptor; 3) detoxification of paraquat; and 4) altered compartmentalization of paraquat, resulting in reduced localization of the herbicide at the active site. There have been several studies on the possible mechanians) of paraquat resistance. A three-fold increase in superoxide dismutase acti- vity in the resistant C; linefolia has been reported (Youngman and Dodge, 1981). Activities of superoxide dismutase, catalase, and peroxidase were 50%, 32%, and 35% higher, respectively, in paraquat resistant lines of perennial ryegrass (Harvey and Harper, 1982). Resistance in perennial ryegrass could not be attributed to differences in uptake, translocation or metabolism of paraquat. Therefore, resistance to paraquat may in part be due to dismutation of superoxide. Resistance to paraquat but not diquat has been observed when measuring C02 fixation and chlorOphyll loss (Dodge, personal comunication). The redox potential of paraquat is more negative than that of diquat (Figure 1). This is consistent with a hypothesis that the site of action of these herbicides is modified in the resistant biotype, such that the redox potential of the electron donor to these herbicides is less negative. The objective of this study was to examine the validity of the hypotheses discussed above. METHODS AND MATERIALS Photosystem [_partial reaction.(flilor0plast thylakoid membranes of resistant and susceptible biotypes of Q; linefolia were isolated as previously described (Chapter 1). Rates of photosystem I-mediated elec- tron flow from reduced TMPD (N,NJW,N”—tetramethyl-p-phenylenediamine) to paraquat (Figure 1, Chapter 5), diquat, and triquat were monitored as oxygen uptake using an oxygen electrode. The bipyridinium herbicides 77 selected represent a wide range of redox potential (Figure 1). Rates of electron transport were measured under saturating light by continuous recording of 02 uptake using a water-jacketed Clark-type oxygen electrode maintained at 20°C. The assay buffer contained 50 ug chlor0phyll-ml‘1, 50 mM Tricine-NaOH (pH 738), 100 mM sorbitol, lmM NH4Cl, (L1 uM gramici- din, 5 mM MgCl2, 10 mM NaCl, 100 M NaN3, 10 0M diuron, 2.5 mM ascorbate, 25 pg/ml superoxide dismutase and 100 uM TMPD. Herbicide concentrations were varied as indicated in the results. Dose-response effects on excised leaves. Leaves were excised under water and dipped in solutions containing a range of herbicide concentrations and(15% surfactant [alkylaryl polyoxyethylene glycol-free fatty acid- i50pr0panol (X-77 Spreader)]. Excised leaves were supported by a sponge, in a water-filled 18 by 150 mm test tube. There were four replications of each treatment and a different plant was used for each replicate. Leaves were placed in darkness for 4 h to allow uptake of the herbicides. In vivo chlor0phyll fluorescence was monitored with a model SF-lO fluorimeter (Richard Brancker Research Lth as described previously (Ahrens 33 31:, 1981). Transients were recorded with a Nicolet Explorer digital oscillosc0pe» 'The variable fluorescence values reported repre- sent.(Fp - F0)/F0, where Fp is the peak fluorescence value (recorded at about 2 sec. in this experiment; Figure 3) and F0 is the fluorescence level at which the variable component of fluorescence begins to change (Figure 5). After recording the fluorescence transients, the leaves were moved to a chamber at 25°C with white light (450 uEPm'2~s'1) for 5 h. Leaves were then placed in darkness for 24 h to allow drying of injured tissue. Injury was evaluated by visually estimating the percent green 78 leaf area. Percent moisture was determined by measuring fresh and dry weights. Cuticular_penetration. Leaves were excised under water and supported in test tubes as described earlier. A 50 11] solution, containing 0.16 uCi 14C-paraquat (0.13 mCivmmol'l) and 0.5% X—77 surfactant, was applied uniformly to both surfaces of the leaf in 0J5 pl dr0plets. This dose of paraquat causes injury in the susceptible biotype but not the resistant biotype (data not shown). After 4 h, leaves were dipped in water for 2 min., blotted dry, dipped in chloroform 3 times and then wiped with a glass filter paper wetted with chloroform. The chloroform extract was allowed to dry. The purpose of the chloroform dips and wipes was to remove the cuticle. The amount of 14C paraquat penetrating the cuticle was estimated by subtracting the amount of 14C obtained in the washes from the amount applied. Liquid scintillation spectrometry was used to measure 14C. Leaf areas were determined on a model LI-3OO portable area meter (Lambda Instruments Coer. There were three replications for each biotype, and a different plant was used for each replicate. Autoradiograph . Uniform leaves approximately 150 mg fresh weight were excised as previously described and placed in aILB ml vial containing 50 l of a solution with 0.064 pCi 14C-paraquat (1.4 mCi'nmol‘l) in 10 mM pH 7 phosphate buffer. This dose of paraquat selectively desiccated the susceptible biotype when placed in sunlight (data not shown). In another experiment, leaves were placed in an unbuffered solution of pH 3 with the same amount of 14C-paraquat. Distilled water was added to maintain the solution level over a 4 h period. In a second experiment, leaves were transferred after 4 h from the initial vial to a second vial containing either water or 24 mg-ml‘1 nonlabelled paraquat (100x higher than the 79 concentration in the original radioactive solution). Leaves were kept in dim light and showed no signs of paraquat injury except in leaves treated at pH 3, where 10% and 15% injury was visually estimated in the resistant and susceptible biotypes, respectively. Leaves were then ly0philized overnight and mounted. X-ray film was placed in contact with the leaves for 36 hr. All experiments contained at least three replications of each treatment for each biotype and utilized a different plant for each replicate. Representative autoradiograms are shown. Sucrose density gradient. A linear sucrose density gradient, from 5 to 60% sucrose with a 1.5 ml 70% sucrose cushion was prepared in a 12 ml centrifuge tube. The gradient contained 50 mM tricine-NaOH (pH 7i”, 10 mM NaCl and 5 mM MgCl2. One leaf was excised from each of three resis- tant and three susceptible plants. 14C-paraquat was supplied to these leaves in the same manner as in the autoradiography experiments. Leaves were ground individually at 4°C in a mortar and pestle in(18 ml 5% sucrose, 50 mM Tricine-NaOH pH 7JL 10 mM NaCl and 5mM MgClz. .After grinding, 0.5 ml water was added, and 1.0 ml of the extract was loaded onto the gradient. The gradients were centrifuged at 55,000 g for 2.5 h at 2°C in a swinging bucket rotor. Fractions of(185 ml were removed from the gradient and the 1.5 ml cushion containing cellirlar debris was removed separately. The location of chlor0plasts and other subcellular fractions were monitored by spectr0photometric determinations of chloro- phyll (MacKinney, 1941) and absorbance at 280 nm (A280)- A OAS ml aliquot of each fraction was solubilized with lenn of NCS tissue solu- bilizer'(Amersham Coer at 20°C for 24 hr and then bleached with 350 pl benzoyl peroxide (100 mg/ml) at 40°C for 4 h. 14C was then determined by liquid scintillation spectrometry. 80 RESULTS AND DISCUSSION Photosystem I partial reaction. Since the bipyridinium herbicides act as electron acceptors, rates of electron transport increased as herbicide concentration increased (Figure 2). The responses of the two biotypes were indistinguishable for all three herbicides. Therefore an altered site of action does not appear to be responsible for resistance. Triquat activity (Figure 2C) was less than that of paraquat (Figure 2A) or diquat (Figure 28). This is presumably due to its highly negative redox potential. Paraquat and diquat had similar activities (Figures 2A and B). Dose-response effects on excised leaves. Resistance to the bipyridinium herbicides was observed by desiccation and by visual estimation of % green leaf area (Figure 3). Since diquat and triquat are structurally quite similar but differ greatly in their redox potentials (Figure 1), a larger degree of resistance to triquat would be anticipated if resistance is due to an altered site of action, i.e. an altered redox potential of the photosystem I primary acceptor. However, the degree of resistance to triquat was quite similar to that of diquat (Figures 3 and 4; Table I). This confirms previous discussion indicating that a modified site of action is not the basis for resistance. Bipyridinium herbicides quench chlor0phyll fluorescence by diverting electron flow from photosystem I. Quenching of i vivo chlorophyll fluorescence transients by paraquat is shown in Figure 5. Paraquat caused quenching in both biotypes, but much higher concentrations were required in the resistant biotype. .All three bipyridinium compounds caused quenching 0f.ifl vivo fluorescence (Figure 4). Higher concen- trations of all herbicides were required to cause quenching in the Figure 2. Effect of bipyridinium herbicide concentration on photosystem I-mediated electron transport in resistant (R) and susceptible (S) biotypes, using TMPD as the electron donor. hr uMoles 02 /mg Chl uMoles 021mg Chl \ hr >hr uMoles 02 /mg cm 300 I- 200 '- 82 A. Paraquat 100 L 1 n 10'5 10'5 10" 10‘3 10‘2 Paraquat Concentration (M) 300 - 200 -- B. Diquat 100 10‘6 10‘5 10-4 10'3 Unauat Concentration (M) 300 [- 20° " C. Triquat 100 I- MT 1 1 1 0‘7 10‘s 10‘s 1O_4 10-3 10-2 Triquat Concentration (M) 83 Figure 3. Response of % leaf area remaining green (Open symbols) and % moisture (closed symbols) to bipyridinium herbicide concentration in excised leaves of resistant (R) and susceptible (S) biotypes. 96 Moisture (CA) 96 Green Leaf Area (CA) 96 Moisture (0,21) 96 Green Leaf Area (OJ) 96 Moistue (O. A) 96 Green Leaf Area (CA ) 100 0| 0 100 50 84 Paraquat Concentration (mM) TR 0.01 0.1 1 Diquat Concentration (mM) l l 1 10 100 Triquat Concentration (mM) Figure 4. Response of in vivo fluorescence to bipyridinium herbicide concentration in excised leaves of resistant (R) and susceptible (S) biotypes. Variable Fluorescence (96 of Control) Variable Fluorescence (% ct Ccntrcl) Variable Fluorescence (96 of Control) 100 50 100 50 100 50 86 A. Paraquat I l I i I I .01 .1 1 10 100 Paraquat Concentration (mM) 8. Diquat I L I .01 .1 1 Diquat Concentration (mM) I I I 1 10 100 Triquat Concentration (mM) 87 Figure 5. Response of in vivo fluorescence transients to paraquat in excised TEEves of resistant and susceptible biotypes. 88 A. Resistant 0 mM 1 mM 73 ,5 100 mM 3 fr—q g |¢ 2 Sec 'é — +l :5 2' '5 C .23 i B. Susceptible 8 8 3 0 mM 8 2 u. 0.01 mM 1 mM r:0 (‘ 2 Sec >( 89 Table 1. Estimated concentrations which cause 50% injury by reduction of in vivo chlor0phyll fluorescence, % moisture or'% of leaf area remaining green. The resistance ratio was estimated by divid- ing the 150 for the resistant biotype by the 150 for the e susceptib biotype. 1,50 I50 , ReSistant Susceptible Method of Estimation Biotype Biotype Resistance Herbicide of Injury (mM) (mM) Ratio Paraquat Chlorophyll Fluorescence 2.3 0.015 150 % Moisture 62 0.62 100 % Green Leaf Area 30 0.32 94 Diquat Chlor0phyll Fluorescence 0.060 0.013 4.6 % Moisture 4.56 0.12 38 % Green Leaf Area 1.7 0.064 27 Triquat ChlorOphyll Fluorescence 7.2 0.078 9.2 % Moisture (b) 7.3 - % Green Leaf Area 130a 3.6 35 aProjected value. bNo % moisture response to triquat at the concentrations tested. Table 2. Cuticular penetration of 14C paraquat.a Average Amount penetrating leaf rea Aqueous wash Chloroform wash cuticle Biotype (c ) (% of applied) % of applied) % of appliedb) Resistant 7.8 70.0 b 0.05 a 33.0 a Susceptible 9.6 78.8 a 0.07 a 21.4 b aMeans within columns followed by the same letters are not significantly different at the 5% level according to Duncan's multiple range test. bCalculated by subtraction. 90 resistant biotype. Resistance to quenching 0f.ifl.1112 fluorescence by the bipyridinium herbicides must be attributed to exclusion of the herbicides from the active site, since the quenching of fluorescence is a result of the effects of the herbicides at their primary site of action. _This exclusion might occur by rapid detoxification or by compartment- alization. A pr0posed resistance mechanism involving detoxification of superoxide (Youngman & Dodge, 1981) or other reactive forms of oxygen must be considered to be of secondary importance in providing resistance since the primary basis for resistance involves exclusion from the active site. Resistance ratios and herbicide concentrations which caused 50% injury, as evaluated by the three techniques discussed are shown in Table 1. Resistance to paraquat and cross resistance to diquat were observed for all herbicides by all methods employed. However, the resistance ratios for paraquat were larger than those for diquat or triquat. The resistance mechanism is therefore somewhat Specific for the herbicide which provided the selective force in the field. The resistance ratios determined for paraquat by the three different methods are in reasonably close agreement (Table 1). The similarity of the ratios is consistent with the generally accepted principle that paraquat causes plant injury by its effects on the chlor0plast. However, the resistance ratios deter- mined for diquat and triquat were lower by chlorOphyll fluorescence than by the other method(s). It is possible that diquat or triquat moved from the chlor0plast subsequent to the chlorOphyll fluorescence detennina- tions, since chlorOphyll fluorescence was determined 4 h after treatment, and the other determinations were made 24 h later. Diquat was the most active of the three herbicides. The ranking of I50 values within any biotype, for any method used, yields the order: 150 diquat < I50 paraquat < 150 triquat (Table 1). Therefore, herbicidal 91 activity corresponds quite closely with the redox potential of these herbicides (Figure 1), with a less negative redox potential corresponding to a more active herbicide. High herbicidal activity in bipyridinium compounds is known to require a redox potential in the range of —350 to -450 mV (Summers, 1980). 'This explains the relatively low activity of triquat. It was previously noted that resistance was observed to para- quat but not to diquat when measuring C02 fixation and chlor0phyll loss (Dodge, personal communication). It is possible to attribute the failure to observe resistance to diquat to the use of a single herbicide concen- tration which may have been too high and therefore nonselective with respect to the two biotypes. Quenching of fluorescence, by all herbicides tested, begins at herbicide concentrations far below those that cause loss of moisture or chlor0phyll (Figures 3 and 4L Also, the 150 for fluorescence quenching was much lower than the 150 for % moisture or % green leaf area (Table 1) for each herbicide and biotype combination. It is possible that suffi- cient reducing power is still generated to maintain protective mechanisms when fluorescence is only partially quenched, and therefore when elec- trons are only partially diverted from NADP. Only highly quenched fluorescence is therefore correlated with apparent tissue injury. Cuticulargpenetration. Table 2 indicates that most of the 14C-paraquat applied was removed in the aqueous wash and that more 14C was removed in the aqueous washes of the susceptible biotype. Very little 14C was removed in the chloroform wash in either biotype. Therefore, considerab- ly more 14C-paraquat penetrated the cuticle of the resistant biotype. The mean leaf areas of the two biotypes differed; however it is difficult to explain the difference in cuticular penetration by this alone. Parham 92 previously reported by personal communication (Harvey and Harper, 1982) that paraquat adsorption on leaf tissue was greater in the resistant biotype of this species. This is consistent with our observation. Re- sistance to paraquat cannot be explained by differences in cuticular penetration. Autoradiography. 14C-Paraquat at pH 7 became uniformly distributed in the leaves of the susceptible biotype, but was localized in vascular regions especially in the lower petiole, in the resistant biotype (Figure 6). Therefore a compartmentalization mechanism may be responsible for resistance to paraquat. Compartmentalization may be due to adsorption to the extracellular matrix (cell wall). Further distribution of 14C was observed in the resistant biotype when leaves fed 14C-paraquat pH 7 were transferred to a high concentration of unlabelled paraquat (Figure 7). 14C moved out of the petiole region and became somewhat more uniformly distributed in the leaf. Redistribution of 14C was more restricted in the susceptible biotype. 'These observations are consistent with an hypothesis that 14C paraquat is primarily localized extracellirlarly in the resistant biotype and primarily intracellirlarly in the susceptible biotype. 14C was primarily localized in the peripheral parts of the leaves in both biotypes when a pH 3 solution was supplied to the excised leaves (Figure 8). It is possible that protons in this acidic solution were preventing adsorption of paraquat by adsorbtion to the cation ex- change sites in the resistant biotype. Greater cuticular penetration of 14C paraquat in the resistant biotype was previously noted. It is pos- sible that the proposed adsorption mechanism provides the driving force for this uptake. 93 Figure 6. Autoradiograms 1(41 and B) of excised leaves (C and 0) fed a solution of C paraquat pH 7. 94 Susceptible Resistant ) ' i ' - : .l .' 5‘ g L l i ' 4 r ' I 3 5‘ i J I, "'ch w‘ Figure 7. 95 Autoradiograma’(A-D) of leaves (E-H) of leaves fed a solution of C paraquat pH 7, and then transferred either to water (A,C,E,G) or to a solution of 24 mg/ml paraquat (B,D,F,H). Susceptible I p it I I; I Q U s ’ 1. ‘. '4 . O O .5 $ T . .. f... “C _. V , I)!" m' 1 . I . 3 Paraquat 96 Resistant :3 ll. VVater Paraquat Figure 8. 97 Autoradiograms A and B) of excised leaves (C and 0) fed a solution of 1 C paraquat pH 3. 98 Susceptible Resistant o M {I w . y r ,0 7 t“- , (7 I‘ fit . r m. A “a 99 The ion exchange properties of the cell wall are largely due to uronic acids, especially c11,4-linked polygalacturonic acid (pectic acid) (Hall 35 21,, 1976). (livalent cations such as Ca++ and Mg++ bind noncovalently to pectic acid. Therefore paraquat may also bind to pectic acid. Many uronic acids are nonionic due to methylation (Hall 35 21,, 1976). The degree of methylation of uronic acids may be the basis for the difference in tolerance to paraquat in the two biotypes. Paraquat is inactivated in soils by adsorption to anionic soil particles (Akhavein and Linscott, 1968). This may be similar, in principle, to the basis for bio-inactivation in paraquat resistant g; linefolia. 14C-atrazine was localized in the vascular regions of an atrazine [2-chloro-4- (ethylamino)-6-(i50pr0pylamino)1§§triazine] -tolerant line of cucumber (Cucumis sativus Ln) (Werner and Putnam, 1980). However, since atrazine is not cationic, the basis for tolerance to atrazine in cucumber is probably different from the basis for paraquat resistance in C; linefolia. Sucrose density gradient. The most dense sucrose fraction, containing cell wall materials, had similar amounts of 14C in both biotypes (Table 3). Most of the 14C was recovered in the least dense sucrose fractions (data not shown). It is possible that 14C-paraquat was readily ex- changeable under the experimental conditions since the cations present (10 mM Na+ and 5 mM MgTT) may have diSplaced paraquat from the cation adsorption sites. ACKNOWLEDGMENT I appreciate the help of Mr. Elliot Light in growing the plants. 100 Table 3. 14C-paraquat in the cushion of the sucrose density gradient.a 0pm _ (As a of total 14c (As % of total 14C recovered/chlorophyll {As % of total recovered/A280 as % as % of total Biotype C recovered) of total A280 measured) chlorophyll measured) Resistant 12 a 0.49 a 0.66 a Susceptible 14 a 0.60 a 1.03 a aMeans separated by the same letter are not significantly different at the 5% level by Duncan's multiple range test. 101 REFERENCES Ahrens, N.H., C.J. Arntzen, and E.N. Stoller. 1981. Chlor0phyll fluorescence assay for the determination of triazine resistance. Need Sci. 29:316-322. Anonymous. 1983. Southern no-till depends on new herbicides and cultivars. Agric. Age 21 (10): 20-26. 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Roberts. 1976. Plant Cell Structure and Metabolism. Longman, Hong Kong, 426 pp. Harvey, B.M.R., and 0.3. Harper. 1982. Tolerance to bipyridilium herbicides. In: Herbicide Resistance in Plants, H.M. LeBaron and J. Gressel, eds., John Wiley 8. Sons, New York, pp. 215-256. Hutchison, J.M. 1979. Hydrogen peroxide production and lipid perioxidation induced by paraquat in isolated cells and chlorOplasts of spinach (Spinocea oleracea LJ, FMLD. dissertation, University of California, 100 pp. LeBaron, H.M. 1983. Principles, problems, and potential of plant resistance. Beltsville Symposia VIII, in press. MacKinney, G. 1941. Absorption of light by chlorphyll solutions. J. Biol. Chem. 140:315-322. Parham, M. 1982. Personal communication reported by: Harvey, B.M.R., and 0.8. Harper. Tolerance to bipyridillium herbicides. In: Herbi- cide Resistance in Plants, H.M. LeBaron and J. Gressel, eds-3, John Wiley 8. Sons, New York, pp. 215-256. Parham, M. 1983. Personal communication to A. Dodge. 102 Summers, lflA. 1980. The Bipyridinium Herbicides. Acadenic Press, London;449 pp. Nerner, GAL, and Putnam, AJL 1980. Differential atrazine tolerance within cucumber (Cucumis sativus). Need Sci. 28:142-148. Youngman, RJ., and AIL Dodge. 1981. On the mechanism of paraquat resistance in Conyza Sp. In: Photosynthesis VI. Photosynthesis and Plant Productivity, Photosynthesis and Environment, G. Akoyunoglou, éflL, Balaban International Science Services, Philadelphia, pp. 537- 544.