“€st mum m nummuuz llllllfllflllljflfll “arm 1293 10063 5 Michigan Sam M This is to certify that the thesis entitled Investigations on the Mechanism and Inheritance of Atrazine Tolerance in Cucumber (Cucumis sativus L.) presented by Georgina Margaret Malloy Werner has been accepted towards fulfillment of the requirements for BIL D. degree in Horticulture Major professor Date October 9, 1979 07639 INVESTIGATIONS ON THE MECHANISM AND INHERITANCE OF ATRAZINE TOLERANCE IN CUCUMBER (CUCUMIS SATIVUS L.) By Georgina Margaret Malloy Werner A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1979 ABSTRACT INVESTIGATIONS ON THE MECHANISM AND INHERITANCE 0F ATRAZINE TOLERANCE IN CUCUMBER (CUCUMIS SATIVUS L.) By Georgina Margaret Malloy Werner Tolerance to the triazine herbicides has been reported in a number of economically important weeds that were formerly considered suscepti- ble. We hypothesized that if the capacity to devel0p tolerance to the triazines existed in susceptible weed species, it might exist in the germplasm of a susceptible crop such as cucumber. The world's cucumber collection was evaluated for atrazine tolerance, and PI 390244 was found to tolerate preemergent applicationscrfup to 0.56 kg/ha. The objectives of this research were to (i) determine if tolerance is due to differences in plant morphology or growth rates, (ii) determine if the differential response of cucumbers was based on differences in uptake, translocation or metabolism of atrazine, (iii) determine if tolerance is manifested at the chlorOplast level as it is in the tolerant weed species, (v) determine if PI 390244 is universally tolerant to the triazines and finally (vi) determine the mode of inheritance of atrazine tolerance. Results of a growth analysis conducted on P1 390244 and 'Marketmore 70' (a susceptible cultivar) revealed that the Plant Introduction had significantly more leaf area but less root biomass during the first l0 Georgina Margaret Malloy Werner days after germination than the susceptible cultivar. The relatively small root biomass of PI 390244 was first hypothesized to be a factor 14 contributing to its selectivity, but subsequent uptake studies with C- atrazine refuted this hypothesis. Isolated chloroplast preparations from both cultivars were equally 8 -7 -6 susceptible to atrazine at concentrations of 10' , lO , or 5 x -5 , l0 10 M. Although PI 390244 absorbed more total 14C-atrazine, it translocated less radioactivity from the roots. "While the concentration of radio- activity found in its roots was l0 to 30% greater than in 'Marketmore 70,’ the amount of 14C in the cotyledons. first and second true leaves of 'Marketmore 70' were 53, 34, and 55% higher, respectively, after 48 hours. A series of autoradiographs were made of the two cultivars. Roots of both cultivars were uniformly labeled with the isotope. However, differences were noted in the foliar distribution of the radioactivity. There was a distinct localization of the radioactivity in the roots and vascular system of the tolerant cultivar, where as the foliage of the susceptible cultivar was uniformly labeled. Compartmentilization of the atrazine away from the major photosynthetic regions appears to be the major mechanism for tolerance in P1 390244. A metabolism study conducted on both cultivars revealed no differ- ences in the rate of metabolism or nature of metabolites between the two cultivars. A dealkylated metabolite, 2-chloro-4-amino-6-isopropyl- amino-sftriazine was the only metabolite found and then in only very small quantities (5-l0%). The dealkylated metabolite was found only in the shoots and not in the roots of either cultivar. Georgina Margaret Malloy Werner Intraspecific difference in tolerance to several triazines was ob- served between the two cultivars. PI 390244 exhibited tolerance to several of the §7triazines and metribuzin. Tolerance in P1 390244 was restricted to the chlorine substituted sftriazines (atrazine, simazine and pr0pazine) and metribuzin. At the Castle Hayne, NC location, atra- zine, simazine and pr0pazine reduced the fresh weight of the Plant Introduction an average of 28, 6, and 23%, respectively, while 'Marketmore 70' was reduced 60, 25, and 57%. At the East Lansing, MI location where an additional treatment of metribuzin was added. the Plant Introduction was reduced 44, 26, and 32% by atrazine, simazine and metribuzin as compared to 'Marketmore 70' which was reduced 74, 52, and 77%, respec- tively. Prometryn, dipropetryn, ametryn and prometon did not signifi- cantly reduce the fresh weight of either cultivar at rates of 0.14, 0.28, and 0.56 kg/ha. The inheritance of tolerance to atrazine is most likely determined by two dominant genes with epistasis. ACKNOWLEDGMENTS I would like to especially thank Dr. Alan R. Putnam (Putt) for his guidance, understanding and patience during the course of my studies. Appreciation is given to Drs. David R. Dilley, Charles Cress, Donald Penner, and Larry R. Baker for serving on my guidance committee. Special thanks to Dr. Stanley K. Ries for his statistical guidance. Gratitude is also expressed to Dr. Homer LeBaron and the CIBA-Geigy Corporation for providing the 14C-atrazine and for financial support of this project. I would also like to gratefully acknowledge the excellent technical assistance and friendship of Ms. Sylvia Denome, Ms. Cathryn Braue, and Mr. William Chase. I would also like to acknowledge the technical assistance of Dr. Thomas Monaco and Steve Weller from North Carolina State University and Fred Cumbo from Castle Hayne Research Station, Castle Hayne. North Carolina. Finally, but most importantly, I would like to thank my husband, Denny, for his help, understanding, and friendship. ii TABLE OF CONTENTS Page LIST OF TABLES .......................... v LIST OF FIGURES ....................... . . vi INTRODUCTION . . . .................... . . . . 1 CHAPTER I: THE TRIAZINE HERBICIDES - A LITERATURE REVIEW ..... 3 1. Resistance to Herbicides in Crop and Weeds 3 11. Physical and Chemical Properties of Triazines . . . 6 7 III. Mechanism of Selectivity ............. IV. Fate of sytriazine Herbicides in Soil and and Plants .................... l0 Literature Cited ................... l5 CHAPTER 2: INTRASPECIFIC DIFFERENCES IN TRIAZINE TOLERANCE IN CUCUMBER (CUCUMIS SATIVUS) GERMPLASM ......... 21 Abstract ....................... 2l Introduction ..................... 22 Materials and Methods ................. 22 Initial Screening ................. 22 Evaluation of the Triazines ............ 23 Results and Discussion ................ 24 Literature Cited ................... 33 CHAPTER 3: MECHANISM FOR DIFFERENTIAL ATRAZINE TOLERANCE WITHIN CUCUMBER (CUCUMIS SATIVUS) .............. 34 Abstract ...................... . 34 Introduction ..................... 35 Materials and Methods ................. 36 Differential Cultivar Tolerance .......... 36 Growth Analysis .................. 37 Isolated Chloroplasts ............... 38 Uptake and Translocation ............. 39 Results and Discussion ................ 40 Differential Cultivar Tolerance .......... 40 Growth Analysis .................. 42 Isolated Chloroplasts ............... 47 Uptake and Translocation ............. 47 iii CHAPTER 4: CHAPTER 5: Literature Cited ................... 60 ATRAZINE METABOLISM IN A SUSCEPTIBLE AND RELATIVELY TOLERANT CUCUMBER CULTIVAR ........... . . . 63 Abstract ....................... 63 Introduction ............... ' ..... . 64 Materials and Methods .............. . . . 64 Results and Discussion ................ 67 Literature Cited ................... 73 INHERITANCE OF 2-CHLORO-4-ETHYLAMINO-6-(ISOPROPYLAMINO)- S-TRIAZINE (ATRAZINE) TOLERANCE IN CUCUMBER TCUCUMIS SATIVUS) ................... 74 Abstract ....................... 74 Introduction ................... . . 74 Materials and Methods ................. 75 Results and Discussion ................ 76 Literature Cited ................... 8] LITERATURE CITED . . . . . . . . ................. 84 iv CHAPTER 2 Table 1. CHAPTER 3 Table 1. CHAPTER 5 Table 1. Table 2. Table 3. LIST OF TABLES PI 390244 tolerance to 0.56 kg/ha atrazine . . . . Differential tolerance of cucumber PI 390244 and 'Marketmore 70' to atrazine in Dryden sandy loam and in nutrient culture solution ...... Segregation and goodness of fit for tolerance and susceptibility to atrazine of F1 and F2 seedlings .................... Incongruent data obtained from the F2 popula- tions of the cross PI 390244 x 'Gy 14' and 'Gy 14' x PI 390244 ............... Segregation and goodness of fit for tolerance and susceptibility to atrazine of backcross seedlings .................... Page 24 . 41 CHAPTER 2 Figure Figure Figure CHAPTER 3 Figure Figure Figure Figure Figure Figure LIST OF FIGURES The interaction of cultivar x chemical at Castle Hayne, NC location ............ The interaction of cultivar x chemical at the East Lansing, MI location .......... The interaction of triazine type x cultivar x rate . . . ._ ................. Leaf area ratios (LAR) from atrazine-tolerant PI 390244 and atrazine-susceptible 'Marketmore 70' cucumber in distilled water and half- strength Hoagland's solution two to ten days after emergence ................. Root biomass from atrazine-tolerant PI 390244 and atrazine-susceptible 'Marketmore 70' in distilled water and nutrient solution two to ten days after emergence ............ Photochemical activity of isolated chloroplasts from atrazine-tolerant PI 390244 and atrazine- susceptible 'Marketmore 70' cucumber leaves exposed to several atrazine concentrations Uptake of water and 14C-atrazine by atrazine- tolerant PI 390244 and atrazine—susceptible 'Marketmore 70' cucumber from 6 to 48 hours . . . . Accumulation of 14C in a) roots; b) cotyledons; c) first leaf; and d) second leaf of atrazine- tolerant PI 390244 and atrazine-susceptible 'Marketmore 70' cucumbers ............ Treated plants (above) and autoradiographs (below) of atrazine-susceptible 'Marketmore 70' (left) and atrazine-tolerant PI 390244 (right) 24 hours after root treatment with 14 vi C-atrazine . . Page 27 29 31 44 46 49 51 53 56 Figure 7. CHAPTER 4 Figure 1. Figure 2. Page A plant of PI 390244 that survived 0.56 kg/ha preemergent atrazine treatment. Chlorosis is localized in the vascular system of the leaf . . . 58 Interaction of cultivar and plant part on the amount of unchanged 14C-atrazine ...... . . . 69 Interaction of harvest date and plant part on the amount of unchanged 14C-atrazine ...... . 71 vii INTRODUCTION The herbicidal activity of the §:triazines was discovered by the J. R. Geigy Co. in Basle, Switzerland in 1952. The principal uses of the substituted srtriazine herbicides are the selective preemergence control of seedling grasses and broadleaf weeds in certain croplands, and with certain of these herbicides, the nonselective control of vege- tation in noncroplands. Extensive testing of the chloro-sftriazines showed the superiority of simazine, pr0pazine and atrazine for weed control in corn. Because of certain special considerations, namely the greater water solubility and post-emergence activity, atrazine was selected as the prime candi- date for use in corn. Recent figures show that in 1976, 90.3 million pounds of atrazine were applied to 75.7 million acres of corn in the United States (15). Simazine showed great promise for use in perennial crops and pr0pazine in sorghum. The sftriazine herbicides are readily absorbed by the roots and translocated upward to the leaves of plants via the transpiration stream. These herbicides are not translocated from the leaves after foliar appli- cation but do accumulate (2) and inhibit the Hill reaction in photosyn- thesis (45, 46). Recently, there has been a renewed interest in the §;triazine herbi- cide group. In the early 1970's triazine tolerance was reported in a 2 number of economically important weeds that were formerly considered susceptible (5, 6, 31, 48, 50, 51, 52, 53, 56, 68, 70, 74, 75). Because of this, it appeared that tolerance might also occur in crops that were considered susceptible. The objectives of this study were to determine if triazine tolerance existed in the usually sensitive cucumber, (Cucumis sativus L.) Additional objectives of this study were to (i) determine the mechanism for tolerance to.atrazine in cucumber, (ii) detenmine if tolerance is of a general nature for the triazine group and finally (iii) determine the mechanism for the inheritance of tolerance and the feasibility of developing tolerant cultivars. CHAPTER 1 LITERATURE REVIEW 1. RESISTANCE TO HERBICIDES IN CROP AND WEEDS Crop selectivity to the gftriazines is achieved both by physio- logical tolerance (relatively rapid detoxication of the absorbed chemi- cal) by certain plants and by herbicide placement. Corn and sorghum have been shown to possess a high physiological resistance to atrazine. while cotton and peas exhibit intermediate tolerance. Cucumbers and oats have been found to be very susceptible to injury (66). Intraspecific differences in response to herbicides exist in both weeds and crop plants. The first reported differential tolerance to a herbicide was with 2,4-D in AgrOStis stonolifera L. (1). Since several biotypes of weeds and crops have been found to be tolerant to 2,4-D (13, 28, 59, 76, 77), dalapon (7, 24, 55, 57), and siduron (58). The inability to control common groundsel (Senecio vulgaris L.) with simazine was first observed in~a nursery in Washington State in 1968 (56). Subsequent testing revealed that the resistant biotype of groundsel also exhibited tolerance to atrazine. By 1978, the resistant biotype had spread to over 200,000 ha in the state (6). Soon to follow were reports of resistance in redroot pigweed (Amaranthus retroflexus L.) (48), lambsquarters (ChenOpodium album L.) (5), common ragweed (Ambrosia artemsiifolia L.) (69), bird's rape (Brassica campestris L.) (68, 70) 3 4 and lateflowering goosefoot (Chenopodium strictum Roth var. glaucgphyllum (Aellen) Wahl.) (6). The occurrence of resistance should be of little surprise since Harper in 1954 (24) hypothesized that resistance may develop in weed populations that have been repeatedly exposed to certain persistant herbicides, as has occurred in insect and microbial popula- tions exposed to other pesticides. Triazine-resistant biotypes first appeared as scattered plants in fields wherea triazine herbicide had been used for more than six years and where no inter-row cultivation had been performed thus allowing these species to flower and produce seed (6). Without the selection pressure of the herbicide, the resistant biotypes would be in very low numbers or non-existent due to thelack of competitive ability of these biotypes in comparison to susceptible biotypes. Several studies (27, 51) have shown the susceptible biotypes to have a competitive advantage over the resistant biotypes. Hensley and Counselman (27) have reported an allelopathic inter- action between triazine resistant and susceptible biotypes of redroot pigweed. When seedlings from resistant and susceptible biotypes were grown together in a hydroponic system, only the susceptible biotype sur- vived after 4 weeks. Similar effects were seen in a soil system, but a longer time period was required. 4 The triazine herbicides are known to be potent photosynthetic in- hibitors (16, 17, 29), acting as an inhibitor of electron transport in PS II (44). Photosynthesis was measured in susceptible and resistant biotypes of lambsquarters, pigweed and common groundsel treated with atrazine and simazine. There was no inhibition observed in the resistant biotypes (50, 52, 53). The effect of atrazine on isolated chloroplasts 5 was also investigated (31, 51, 52, 53, 69, 74, 75). Similar findings were observed at this level also, regardless of species examined. Radosevich (52) hypothesized that structural or confirmational changes associated with the chloroplast membrane may account for the differen- tial inhibition of the Hill reaction. Studies on the mechanism of srtriazine tolerance in common lambs- quarters, common groundsel and redroot pigweed established no differences in absorption or metabolism between biotypes of the three species (32). Jensen et al. (33) has reported that both biotypes of common lambs- quarters detoxify ‘4 C-atrazine by hydroxylation, n-dealkylation and conjugation with glutathione. Parent atrazine accounted for greater than 83% of the 14C-activity extracted from the leaves of both biotypes. Jensen concluded that since there was no differential absorption, trans- location or metabolism between the two biotypes there must be some other mechanism responsible for resistance. Pfister et al. (49) have reported that in susceptible biotypes of common groundsel, atrazine and diuron compete for the same binding site on the chloroplast membrane. However, "chloroplasts from triazine- resistant plants showed no atrazine binding over low herbicide concen- tration ranges, in agreement with a lack of herbicide effects on electron transport." Pfister also suggested that there may be a selective altera- tion of the chloroplast membrane in the resistant biotypes thus prevent- ing the binding of the triazines. Arntzen et al. (4) has identified the component responsible for the differential binding of the chloroplast membranes. They suggest that a modification in a polypeptide component of the chloroplast membrane could be selectively modified in such a manner to alter a Specific herbicide from binding on the membrane. 6 II. PHYSICAL AND CHEMICAL PROPERTIES OF TRIAZINES R1 /\ R3'HN ‘\\ N NH'RZ Gysin and Knusli (19) demonstrated that if the two side chains are bisalkylated (equal), substituents at R1 may be ranked in the following order of increasing inhibitory effectiveness: C > CH(CH > CH > 2“5 3’2 3 C1 > OCH3 > Br > SCH3. There is no inhibitory effect on the Hill reac- tion if H or OH is substituted at R]. All economically important s7 triazines may be placed in one of three subgroups based on whether the substitution at the R1 position of the ring structure is a chlorine (Cl) atom, a methoxy (O-CH3) group, or a methylthio (S-CH3) group. Two imido hydrogens are required for maximum inhibitory effective- ness, this places strict limitations on the substitutions at R2 and R3 (30). Mixed short chain alkyl or symmetrical substitutions favoring lipid solubility increase the inhibitory effectiveness, such that, isopropyl > ethyl > n-prOpyl > methyl (30). The following are the common and chemical names of some members of the grtriazine herbicide family. Subgroup R1 R2 R3 Common Name 2-Cl CH(CH3)2 CH(CH3)2 Simazine 2-Cl CHZ-CH3 CHz-CH3 pr0pazine 2-0-CH3 CH(CH3)2 CH(CH3)2 prometon 2-S-CH3 CHZCH3 CH(CH3)2 prometryn 2-S-CH3-CH3 CH(CH3)2 CH(CH3)2 dipropetryn Atrazine, the most economically important grtriazine, is synthesized by reacting cyanuric chloride, with one equivalent of ethylamine and one equivalent of isopropylamine in the presence of an acid acceptor. Atrazine has low water solubility at 33 ppmw, however, it is much more soluble than simazine (5 ppmw) (3). III. MECHANISMS OF SELECTIVITY There are three major metabolic pathways (hydroxylation, dealkyla- tion and conjugation with glutathione) for the detoxication of atrazine and simazine (61, 66). Corn and sorghum have been shown to be extremely tolerant to atra- zine with peas and cotton exhibiting intermediate tolerance and wheat as being susceptible (12, 20, 63, 66). Shimabukuro et al. (66) have demonstrated that in corn, all three detoxication pathways are utilized. Hydroxy atrazine and hydroxy Simazine were the first degradation products reported in higher plants (9, 19, 43). Hydroxylation has been shown to occur only with the chlorine substitution at R1 (18). 8 Hydroxylation of the chloro-§;triazines occurs non-enzymatically by the cyclic hydroxamate, benzoxazinone (2,4-dihydroxy-3-deto-7-methoxy-l,4- Benzoxazincne) and/or its 2-glucoside. It is suggested (10) that the catalyst participates in the nucleophilic attack of the chlorine ion. The rate of conversion is proportional to the endogenous levels of benzoxazinone and its 2-glucoside (20, 21, 47, 61). Roots of certain species have been found to contain higher quantities of the compound than do shoots (64). Dealkylation of atrazine and simazine occurs to some extent in all higher plants (61, 65). Dealkylation involves the loss of either an n-ethyl or n-isopropyl sidechain. It is enzymatically catalyzed (61) and results in only partially detoxified metabolites (60)., Glutathione conjugation is another means by which plants may detoxify atrazine (41, 66). An enzyme, glutathione S-transferase, has been iso- lated from corn leaves and identified. Glutathione S-transferase has also been found in large quantities in leaf extracts of sorghum and sugar cane (40). No detectable levels of the enzyme were found in peas, oats, wheat or barley. The conjugated metabolites which were isolated and identified were S-(4-ethy1amino-6-isopr0pyl-s-triaziny1-2)-glutathione (GS-atrazine) and S-A-L-glutamyl-(4-ethylamino-6-i50propylamino-s- triazinyl-2)-L-cysteine (40). Reaction studies have shown that this enzyme has a specific requirement for a chlorine at the 2 position. This enzyme is strongly inhibited if the substitution at the 2 position is a methylthio group. The rate of atrazine metabolism and detoxication appears to deter- mine the basis for resistance in corn and sorghum (61). The rate of metabolism must be rapid enough to prevent the accumulation of a lethal 9 level of atrazine in the plant, and the pathway of metabolism must re- sult in the formation of nonphytoxotic metabolites (61). Studies con- ducted on corn have Shown that very little parent atrazine remained after 48 hours (61). Atrazine metabolism was monitored in corn leaf discs by Shimabukuro et al. (66). They reported that at the end of a 5 hr incubation period where the discs were incubated in 55 uM 14C- atrazine, only 23% of the parent atrazine was extracted. The remaining 77% was in the form of glutathione conjugated atrazine. In sorghum and pea, dealkylation occurred readily (61, 63, 68). Shimabukuro et al. (66) summarized the results obtained on corn, sorghum, 14 peas and wheat that were treated-with C-atrazine either in the root media or feliarly. When corn and sorghum plants were treated foliarly, the major mechanism of detoxication was the conjugation of atrazine with 14C-atrazine nutrient culture treatments 14 glutathione. Conversely, with on corn and sorghum, corn metabolized the C-atrazine non-enzymatically with benzoxazinone to the nonphytotoxic hydroxyatrazine. Sorghum was unable to detoxify the atrazine to any large extent. A small amount of dealkylation occurred in both roots and shoots of corn and sorghum. The major metabolite of atrazine in both roots and shoots of young peas (which exhibit intermediate tolerance to atrazine) was 2-chloro- 4-amino-6-isopropylamino-sftriazine, a dealkylated metabolite (60). No hydroxyatrazine was found, and only a small quantity of glutathione con- jugated atrazine was present. The dealkylated metabolite in this form is only partially detoxified and must be further dealkylated to a non- phytotoxic form (60). This may explain the intermediate degree of toler- ance exhibited in peas. Very small quantities of hydroxyatrazine. con- jugated atrazine and dealkylated metabolites were found in wheat, a 10 susceptible species (66). These were present in such minute quantities that their presence did not alter the susceptibility of the crop (66). Thompson (72) has reported that in six Setaria species and three Panicium species treated with atrazine, no differences were found in absorption or translocation between the nine species. Glutathione con- jugation was the only major metabolite found. Small quantities of hydroxyatrazine were also found. The rate of atrazine and pr0pazine metabolism by Setaria and Panicium species correlates highly with their tolerance to these herbi- cides. The metabolism of atrazine and simazine by wild cane resulted in the detoxication of atrazine by hydroxy derivatives and glutathione conjugation. Only 30% of the simazine absorbed by the wild cane was detoxified (73). Jensen (32) has reported that in 53 grass Species, dealkylation, hydroxylation and glutathione conjugation occurred in all species al- though rates of these metabolic pathways varied among the species tested. The recovery of net C02 exchange was correlated with fermation of gluta- thione atrazine conjugates. Conjugation was the major detoxication pathway in species exhibiting tolerance to atrazine. IV. FATE OF SrTRIAZINE HERBICIDES IN SOIL AND PLANTS The fate of the sftriazines in soil is characterized by enormous complexity in both systems. Several factors are known to influence the fate and behavior of herbicides in soil systems: 1) photochemical decomposition, 2) chemical ll degradation, 3) microbial degradation, 4) volatilization, 5) movement, 6) adsorption, 7) plant or organism uptake (2). There Is evidence for non-biological detoxication of the grtriazine herbicides via photodecomposition, volatilization, hydroxylation and dealkylation (25, 26, 34, 38). Adsorption appears to be one of the major factors involved in the fate of the sytriazine compounds in soil. Many excellent reviews have been written on this subject (18). Talbert and Fletchall (71) have reported that in the case of a Marshall silty clay loam with an organic matter content of 4.2%, adsorption of five §:triazine herbicides de- creased in the order: prometryn > prometone > simazine > atrazine > pr0pazine. Prometryn and prometon were bound significantly more than the others. The effect of chlorosubstitution (atrazine, simazine and pr0pazine) appears to lessen the attractive forces between the herbicide and soil particles (25). The fact that adsorption has a great influence on the leaching and movement of the s-triazine compounds can be observed. It is felt that adsorption, and not solubility regulates the extent of movement (25). Detoxication of the chloro1§-triazine compounds may occur chemically in the soil through hydrolysis to form nonphytotoxic hydroxyanalogs (26). Harris (26) has reported that within a few weeks of treatment, hydroxy derivatives of atrazine, simazine and pr0pazine were extracted from soil samples. Recovery at 8 weeks accounted for almost 50% of the radioactivity. Microbial degradation is also an important component in the detoxi- cation process in soil. Numerous soil microorganisms have been identi- fied as having the capacity to degrade the sftriazines (18). Soil fungi appear to be the major participants in the degradation of the §;triazine compounds (8, 14, 35, 36, 37, 54). 12 McCormick and Hilbold (42) have reported that the decomposition of triazine herbicides was directly related to the breakdown of soil organic matter. They believe the triazines are passively degraded, incidental to the metabolism of soil-derived substances and are non-stimulatory to the growth or enzymatic capability of the microflora. They also believe that possibly the only reason microorganisms would actively degrade the §:triazines would be as a source of energy. Many methods have been developed to study the degradation capacity of microorganisms: 1) isolates of microorganisms in nutrient cultures where the §:triazines were used as the sole source of carbon or nitrogen (35, 43), 2) evolution of 14C02 increased oxygen consumption, 3) the use of bioassays to follow oxygen consumption, 4) the use of bioassays to fbllow the dissipation of the compounds from microbial culture solutions (35). Various reports can be cited in the literature as to the ability of soil fungi to utilize the sftriazines as a source of nitrogen or carbon, and it can be concluded that soil microorganisms can indeed utilize these compounds as sources of energy (37). There are three methods that have been documented by which micro- organisms in the soil may detoxify the grtriazine compounds: dealkyla- tion, hydroxylation or cleavage of the ring. Dealkylation is the major mechanism involved in the microbial degradation of the chloro-§:triazines (35, 36, 37, 39). Simazine detoxi- cation has been reported to occur almost immediately after soil micro- organisms were exposed to the compound (37). The most effective soil fungi reported by Kearney et a1. (37) was Aspergillus fumigatus which could almost completely detoxify simazine in 12 days. The major l3 metabolite identified was 2-chloro-4-amino-6-ethylamino-grtriazine (39). Hydroxylation is also a common mechanism by which microorganisms may detoxify herbicides. It is frequently the initial reaction step in the degradation of halogenated pesticides (18). Ring cleavage occurs only after the chloro-§:triazines are first converted to the hydroxyanalogs (43). During the first 91 hours after 14 exposure, the rate of C-ring labeled simazine decomposition by corn, 14 cucumbers and soil microorganisms to CO2 was high, after which the rate decreased dramatically. 14 Kearney et al. (39) found . CO2 being evolved from organisms that have been exposed only to chain-labeled simazine but not from ring- labeled simazine. These were the same soil fungi which could dealky- late simazine very readily. Evolution of low levels of 14 14 C02 from microbial systems treated with C-ring labeled srtriazines have been reported (11). Small quanti- ties (less than 5%) of the 14 14 C-labeled prometryn, atrazine and simazine were evolved as C02 (42). They hypothesized that the §:triazine ring is somewhat resistant to microbial attack. I The fate of the §:triazine herbicides in plants has been fairly well documented. The detoxication processes associated with higher plants have been previously discussed. In summary, the sftriazine herbi- cides may remain in plants as unchanged material, partially or totally nonphytotoxic forms as hydroxyanalogs, dealkylated compounds or conju- gated with glutathione. Many factors determine the ultimate fate of the srtriazine com- pounds. It is the interaction of many components which lead to the l4 degradation of these herbicides into the nonphytotoxic compounds which are found in plant residues and soil. 10. 11. 12. LITERATURE CITED Albrecht, H. R. 1947. Strain differences in tolerance to 2,4-D in creeping bent grasses. J. Am. Soc. Agron. 39:163-165. Anderson, W. P. 1977. Weed Science: principles. West Publishing Co., St. Paul, MN. 598 pp. Anonymous. 1979. Herbicide Handbook of the Weed Science Society of America. Fourth Edition. Champaign, IL. 479 pp. Arntzen, C. J., K. Pfister and C. L. Ditto. 1979. Alterations in the Photosystem II complex in chloroplasts from herbicide- resistant weed biotypes. Weed Sci. Soc. Am. Abstr. #238. Bandeen, J. D. and R. D. McLaren. 1976. Resistance of Chenopodium album L. to triazines. Can. J. Plant Sci. 56:411-412. Bandeen, J. 0., J. V. Parochetti, G. F. Ryan, 8. Maltais and D. V. Peabody. 1979. Discovery and distribution of triazine re- sistant.weeds in North America. Weed Sci. Soc. Am. Abstr. #229. Bucholtz, K. P. 1958. Variations in the sensitivity of clones of quackgrass to dalapon. Proc. 15th North Cent. Weed Contr. Conf. 18-19. Burnside, 0. C., E. L. Schmidt and R. Behrens. 1961. Dissipation of simazine from the soil. Weeds. 9:477-484. Castlefranco, P., C. L. Foy and D. B. Deutsch. 1961. Nonenzymatic detoxification of 2-chloro-4,6bis(ethylamino)-§-triazine (simazine) by extracts of gee mays. Weeds. 9:580-591. Castlefranco, P. and M. S. Brown. 1962. Purification and proper- ties of the simazine-resistant factor of geg_mays. Weeds. 10:131-136. Couch, R. W., J. V. Gramlich, D. E. Davis and H. H. Funderburk Jr. 1965. The metabolism of atrazine and simazine by soil fungi. Proc. 5. Weed Conf. 18:623. Davis, D. E., J. V. Gramlich and H. H. Funderburk Jr. 1964. Atra- zine absorption and degradation by corn, cotton, and soybeans. Weeds. 12:252-255. 15 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 16 Devine, T. E., R. E. Seaney, D. L. Linscott, R. D. Hagin and N. Brace. 1975. Results of breeding for tolerance to 2,4-D in Birdsfoot Trefoil. Crop Sci. 15:721-724. Duke, H. B. 1964. The decomposition of 2-chloro-4-ethylamino-6- isopropylamino-s-triazine (atrazine and related s-triazine herbicides) by soil microorganisms. M.S. Thesis. Ore. State Univ. Eichers, T. R., P. A. Andrilenas, T. W. Anderson. 1976. The farmers use of pesticides. Agric. Econ. Report. 418. 58 pp. Good, N. E. 1961. Inhibitors of photosynthesis. Plant Physiol. 34:584-607. Good, N. E. and S. Izawa. 1964. Selective inhibitors of photo- synthesis. Rec. Chem. Prog. 4:225-237. Gunther, F. A. ed. 1970. The triazine herbicides: Residue Re- views. Springer-Verlag, NY. 413 pp. Gysin, H. and E. Knusli. 1960. Chemistry and herbicidal proper- ties of triazine derivatives. Adv. Pest Contr. Res. 3:289- 358. ' Hamilton, R. H. 1964. A corn mutant deficient in 2,4-dihydroxy- 7-methoxy-l.4-benzoxazinone with an altered tolerance to atrazine. Weeds. 12:27-31. Hamilton, R. H. 1964. Tolerance of several grass species to 2- chloro-s-triazine herbicides in relation to degradation and content of benzoxazinone derivatives. J. Agr. Food Chem. 12:14-17. Hamilton, R. H. and D. E. Moreland. 1963. Fate of ipazine in cotton plants. Weeds. 11:213-217. Hamilton, K. C. and H. Tucker. 1964. Response of selected and random plantings of Johnsongrass to dalapon. Weeds. 15: 220-222. Harper, J. L. 1956. The evolution of weeds in relation to re- sistance to herbicides. Proc. 3rd Br. Weed Contr. Conf. 179-188. Harris, 0. I. 1965. Monuron and g-triazines in soil. Weeds. 13:6'90 Harris, C. I. 1967. Fate of 2-chloro-s-triazine herbicides in soil. J. Agr. Food Chem. 15:157-T62. Hensley, J. R. and C. J. Counselman. 1979. Allelopathic inter- actions between triazine resistant and susceptible strains of 28. 29. 30. 3]. 32. 33. 34. 35. 36. 37. 39. 40. 17 redroot pigweed (Amaranthus retroflexus L.). Weed Sci. Soc. Am. Abstr. #232. Hodgson, J. M. 1970. The response of Canada thistle ecotypes to 2,4-0, amitrole, and intensive cultivation. Weed Sci. 18: 253-255. Izawa, S. and N. E. Good. 1965. The number of sites sensitive to 3-(3,4-dichloropheny1)-l,l-dimethy1urea, 3-(4-chlorophenyl) -l,l-dimethylurea and 2-chloro-4-(2-propylamino)-6-ethy1amino- s-triazine. Biochem. Biophys. Acta. 102:20-38. Jensen, K. I. N. 1975. Atrazine detoxification in three gramineae subfamilies. Ph.D. Thesis. Univ. of Guelph. Ontario. Jensen, K. I. N., J. D. Bandeen and V. Souza Machado. 1977. Studies on the differential tolerance of two lambsquarters selections to triazine herbicides. Can. J. Plant Sci. 57: 1169-1177. Jensen, K. I. N., G. R. Stephenson and L. A. Hunt. 1977. Detoxi- fication of atrazine in three gramineae subfamilies. Weed SCI. 25:212-220. Jensen, K. I. N., J. D. Bandeen and V. Souza Machado. 1979. Role of triazine herbicide uptake, translocation, accumulation and metabolism in plant selectivity. Weed Sci. Soc. Am. #234. Jordan, L. 5., B. E. Day and W. A. Clerx. 1964. Photodecomposi- tion of triazines. Weeds. 12:5-7. Kaufman, D. 0., C. Kearney and T. J. Sheets. 1963. Simazine: Degradation by soil microorganisms. Science. 142:405- Kaufman, D. D., C. Kearney and T. J. Sheets. 1964. Degradation of simazine by soil microorganisms. Weed Sci. Soc. Am. Abstr. p 12. Kaufman, D. 0., C. Kearney and T. J. Sheets. 1965C Microbialde- gradation of simazine. J. Agr. Food Chem. 13:238-242. Kearney, C., T. J. Sheets and J. W. Smith. 1964. Volatility of seven s-triazines. Weeds. 12:83-87. Kearney, C., D. D. Kaufman and T. J. Sheets. 1965. Meabolites of simazine by Aspergillus fumigatus. J. Agr. Food Chem. 13: 369-372. Lamoureux, G. L., R. H. Shimabukuro, H. R. Swanson and D. S. Frear. 1970. Metabolism of 2-chloro-4-ethy1amino-6-isopropylamino- s-triazine (atrazine) in excised sorghum leaf sections. J. fibre FOOd Chemo 18:81-86. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 18 Lamoureux, G. L., L. E. Stafford, R. H. Shimabukuro and R. C. Zaylskie. 1973. Atrazine metabolism in sorghum: Catabolism of the glutathione conjugate of atrazine. J. Agr. Food Chem. 21:1020-1030. McCormick, L. L., A. E. Hiltbold. 1966. Microbial decomposition of atrazine and diuron in soil. Weeds. 14:77-82. Montgomery, M. L. and V. H. Freed. 1961. The uptake, transloca- tion and metabolism of simazine and atrazine by corn plants. Weeds. 9:231-237. Moreland. D. E. 1967. Mechanism of action of herbicides. Ann. Rev. Plant Physiol. 18:365-386. Moreland. D. E., W. A. Gentner, J. L. Hilton and K. L. Hill. 1959. Studies on the mechanism of herbicidal action of 2- chloro-4,6bis(ethylamino)§7triazine. Plant Physiol. 34: 432-435 0 Moreland. D. E. and K. L. Hill. 1962. Interference of herbicides with the Hill reaction of isolated chloroplasts. Weeds. 10: 229-236. Palmer, R. D. and C. 0. Grogan. 1965. Tolerance of corn lines to atrazine in relation to content of benzoxazinone 2-glucoside. Peabody, D. 1973. Aatrex tolerant pigweed found in Washington. Weeds Today. 4:17. Pfister, K., S. R. Radosevich and C. J. Arntzen. 1979. Modifica- tion of herbicide binding to the chlor0p1ast membranes of weed biotypes showing differential herbicide susceptibility. Weed Sci. Soc. Am. Abstr. #237. Radosevich, S. R. 1977. Mechanism of atrazine resistance in lambs- quarters and pigweed. Weed Sci. 25:316-318. Radosevich, S. R. 1979. Physiological responses to triazine herbicides in susceptible and resistant weed biotypes. Weed Sci. Soc. Am. Abstr. #235. Radosevich, S. R. and A. P. Appleby. 1973. Studies on the mech- anism of resistance to simazine in common groundsel. Weed Sci. 21:497-500. Radosevich, S. R. and 0. T. Devilliers. 1976. Studies on the mechanism of s-triazine resistance in common groundsel. Weed Sci. 24:229-232. Ragab, M. I. H. and J. P. McCollum. 1961. Degradation of 14C- labelled simazine by plants and soil microorganisms. Weeds. 9:72-84. 55. 56. 57. 59. 60. 61. 62. 63. 65. 66. 67. 68. 19 Roche, B. F. and T. M. Muzik. 1964. Ecological and physiological study of Echinochloa crus alli L. Beauv. and response of its biotypes to sodium 2,2-dichloropropionate (dalapon). Agron. J. 56:155-160. Ryan, G. I. 1970. Resistance of common groundsel to simazine and atrazine. Weed Sci. 18:614-616. Santlemann, P. W. and J. A. Meade. 1961. Variation in morpholOgi- ‘cal characteristics and dalapon susceptibility within the species Seteria lutescens and S, faberii. Weeds. 9:406-410. Schooler, A. B., A. R. Bell and J. D. Nalewaja. 1972. Inheritance of siduron tolerance in foxtail barley. Weed Sci. 20:167- 169. Sexsmith, J. J. 1964. Morphological and herbicide susceptibility differences among strains of Hoary Cress. Weeds. 15:19-22. Shimabukuro, R. H. 1967. Significance of atrazine dealkylation in root and shoot of pea plants. J. Agr. Food Chem. 15: 557-562. Shimabukuro, R. H. 1967. Atrazine metabolism and herbicidal selectivity. Plant Physiol. 42:1269-1276. Shimabukuro, R. H. N., R. E. Kadunce and D. S. Frear. 1966. De- alkylation of atrazine in mature pea plants. J. Agr. Food Chem. 14:392-395. Shimabukuro, R. H. and H. R. Swanson. 1969. Atrazine metabolism in cotton as a basis for intermediate tolerance. Weed Sci. 18:231-234. Shimabukuro, R. H. and H. R. Swanson. 1969. Metabolism of root applied vs. foliarly applied atrazine in corn. Weed Sci. Soc. Am. Abstr. p. 197. Shimabukuro, R. H. and H. R. Swanson. 1969. Atrazine metabolism, selectivity and mode of action. J. Agr. Food Chem. 17:199- 205. Shimabukuro, R. H., H. R. Swanson and W. C. Walsh. 1970. Gluta- thione conjugation; atrazine detoxification mechanism in corn. Plant Physiol. 46:103-107. Shimabukuro, R. H., W. C. Walsh, G. L. Lamoureux and L. E. Stafford. 1973. Atrazine metabolism in sorghum: chloroform-soluble intermediates in the n-dealkylation and glutathione conjuga- tion pathways. J. Agr. Food Chem. 21:1031-1036. Souza Machado. V. J. D. Bandeen, W. D. Taylor and P. Lavigne. 1977. Atrazine resistant biotypes of common ragweed and bird's rape. Can. Weed Report East. Sec. p. 306. 69. 70. 71. 72. 73. 74. 75. 76. 77. 20 Souza Machado. V. J. D. Bandeen, G. R. Stephenson and K. I. N. Jensen. 1977. Differential atrazine interference with the Hill reaction of isolated chloroplasts from Chenopodium album L. biotypes. Weed Res. 17:407-413. Souza Machado. N. J. D. Bandeen and P. C. Bhowick. 1978. Triazine tolerance of bird's rape biotypes. Can. Weed Report East. Sec. p. 338. Talbert, R. E., 0. H. Fletchall. 1965. The adsorption of some g-triazines in soils. Weeds. 13:46-51. Thompson, L. Jr. 1972. Metabolism of triazine herbicides. Weed Sci. 20:584-587. Thompson, L. Jr. 1972. Metabolism of simazine and atrazine by wild cane. Weed Sci. 20:153-155. Thompson, L. Jr., R. W. Schumacher and C. J. Rieck. 1974. An atrazine resistant strain of redroot pigweed. Weed Sci. Soc. Am. Abstr. #196. West, L. D., T. J. Muzik and R. I. Witters. 1976. Differential gas exchange of two biotypes of redroot pigweed to atrazine. Weed Sci. 24:68-72. Whitehead, C. W. and C. M. Switzer. 1963. The differential re- sponse of strains of wild carrot to 2,4-D and related herbi- cides. Can. J. Plant Sci. 43:255-262. Whiteworth, J. W. and T. J. Muzik. 1967. Differential response of selected clones of bindweed to 2,4-D. Weeds. 15:275-280. CHAPTER 2 INTRASPECIFIC DIFFERENCES IN TRIAZINE TOLERANCE WITHIN CUCUMBER (CUCUMIS SATIVUS) GERMPLASM ABSTRACT Tolerance to several s-triazine herbicides was determined on two cultivars of cucumber, PI 390244 and 'Marketmore 70' (a susceptible cultivar). PI 390244 has been previously reported tolerant to atrazine at 0.56 kg/ha preemergence. These studies indicated that tolerance in P1 390244 was restricted to the chlorine substituted s-triazines (atrazine, simazine, and pr0pazine) and metribuzin. At the Castle Hayne, NC location, weights of the PI were reduced only 28, 6. and 23% by atrazine, simazine and pr0pazine, respectively, as compared to 63. 24, and 57% for 'Marketmore 70'. Weights of the PI at the East Lansing, MI location were reduced 44, 26, and 32% by atrazine, simazine and metri- buzin while 'Marketmore 70' was reduced 74, 52, and 77%, respectively. Prometryn, diprOpetryn. prometon, and ametryn did not significantly reduce the fresh weight of either cultivar at rates of 0.14, 0.28, and 0.56 kg/ha. 21 22 INTRODUCTION Intraspecific differences in herbicidal response exist in both weed and crop plants. Biotypes tolerant to the g-triazines have been reported in common groundsel (Senecio vulgaris L.) (5), lambsquarters (Chenopodium album L.) (l, 2), common ragweed (Ambrosia artemisiifolia L.) (7), redroot pigweed (Amaranthus retroflexus L.) (3), bird's rape (Brassica campestris L.) (6, 7) and cucumber (Cucumis sativus L.) (9). The response of two biotypes of common groundsel to six s-triazines has been reported by Radosevich and Appleby (4). The sensitive biotype of common groundsel was found significantly more susceptible to all triazines except terbutryn. Universal s-triazine tolerance has also been demonstrated in biotypes of bird's rape (6). The atrazine tolerant biotype did not Show phytotoxic symptoms with the methoxy, chloro and methylthio-g-triazines. Similar results were also observed in atrazine tolerant biotypes of lambsquarters (1). This study was conducted to determine if a cucumber cultivar rela- tively tolerant to atrazine was also tolerant to other s-triazines and metribuzin. MATERIALS AND METHODS Initial Screenigg A collection of 424 accessions of the world's cucumber germplasm was obtained from the Regional Plant Introduction Station at Ames, Iowa and 41 selected cultivars were obtained from Dr. L. R. Baker, Michigan State University, East Lansing, MI and Asgrow Seed Company, Kalamazoo, MI. 23 The seeds were sown on July 8, 1977 at East Lansing, M1 on a Miami silt loam with an organic matter content of 2.1%. The number of seeds planted per accession varied from 10-200 seeds. Seeds were sown 1.9 cm deep using a V-belt Planter Jr. Atrazine was applied preemergence at 0.56 kg/ha in a spray volume of 333 1/ha over the entire planting. Evaluation of the Triazines Cucumber seed of both cultivars, relatively tolerant PI 390244 (The survivors of the initial screening were selfed and maintained individually for three generations - The most tolerant and most homo- zygous line was selected for this study.) and susceptible 'Marketmore 70' (obtained from the Asgrow Seed Company, Kalamazoo, M1) were sown 1.9 cm deep in individual rows 6.2 m long spaced 30.4 cm apart using a V-belt Planter Jr. Each plot contained 90 seeds of each cultivar. Seven s-triazines: 2-chloro-4,6-bis(isopropylamino)-§-triazine (pr0pazine), 2,4-bis(isoprooylamino)-6-(methy1thio)-§-triazine(prometryn), 2-(ethylamino)-4,6-bis(isopropy1amino)-§-triazine(dipropetryn), 2,4-bis (isopropylamino)-6-methoxy-§-triazine(prometon), 2-(ethylamino)-4- (isopropylamino)-6-(methylthio)-§-triazine(ametryn), 2-chloro-4,6-bis (ethylamino)-§-triazine(simazine). and 2-chloro-4-(ethy1amino-6- (isopropylamino)-§-triazine(atrazine) were applied preemergence at rates of 0, 0.14, 0.28, and 0.56 kg/ha. The first experiment was conducted in Castle Hayne, NC on April 19, 1979 on a Portsmith fine sandy loam with an organic matter content of 3.2 to 4.4%. The experiment was blocked to minimize variation through the organic matter gradient. The second experiment was initiated in East Lansing, MI on June 16, 1979 on Miami silt loam with an organic matter content of 2.1%. The second 24 study included 4-amino-6-t_e_r_i_:_-buty1-3-(methylthio— _s_ -triazin-5(4H)-one (metribuzin) as a chemical treatment. Three replicates were used in each experiment. _Plants were harvested at the second leaf stage. Data obtained were number of surviving plants and fresh weight per plot. Analysis of variance were determined on all data and means were compared using the LS0 test at the 5% level (8). RESULTS AND DISCUSSION The initial study conducted on the world's cucumber collection and selected cultivars for atrazine tolerance resulted in several survivors from the various Plant Introductions. At 17 and 36 days after emergence, plants from 28 accessions had survived the 0.56 kg/ha of atrazine. How- ever, most of these accessions contained either one or two plants and were considered escapes. One accession, PI 390244 exhibited the best tolerance to atrazine (Table 1). Of the 12 seeds initially planted, 9 seeds germinated and 7 plants survived the treatment. The survivors of the Plant Introduction were selfed and the seed obtained was used for all subsequent studies. Table 1. PI 390244 tolerance to 0.56 kg/ha atrazine. Number of seed planted 12 Number of seed emerged 9 Number of surviving seedlings 7 78% survival 25 Applications of the s-triazines on both cultivars yielded similar results at both locations (Figures 1 and 2). There was a two-fold dif- ference in soil organic matter content between sites which may explain the slight increase in activity obtained at East Lansing. It was fbund that several of the s-triazines did not injure either cultivar at the rates we selected. Prometryn. dipropetryn, prometon and ametryn did not significantly reduce the fresh weight of either cultivar at either location. Higher rates of application may have pro- duced differences. However, the chloro-substituted triazines, atrazine simazine and pr0pazine at the three rates tested greatly reduced the fresh weight of 'Marketmore 70.'.the susceptible cultivar, in comparison to the Plant Introduction at both locations (Figure 3). At Castle Hayne, PI 390244 had been reduced 28. 6, and 23%. respectively, while 'Marketmore 70' was reduced by 63, 24, and 57% (Figure 1). The chloro-triazines and metribuzin were also much more toxic to 'Marketmore 70' at the East Lansing location. Atrazine, simazine and pr0pazine decreased the fresh weight of 'Marketmore 70' by 74. 52, and 50%, respectively, as compared to the Plant Introduction which was reduced 44, 26, and 26%. Metribuzin, which is an asymmetrical triazine was extremely toxic UJ'Marketmore 70.' Reductions in fresh weight of 'Marketmore 70' were 77% while PI 390244 was reduced only by 32% (Figure 2). Although both cultivars manifested some injury symptoms, the sus- ceptible cultivar showed extensive chlorosis, necrosis, and high mor- tality. PI 390244 had a low mortality rate and showed only limited chlorosis. When summarizing all the data over two locations (excluding metri- buzin), the interaction of cultivar x rate x chloro-triazines versus 26 Figure l. The interaction of cultivar x chemical at the Castle Hayne, NC location. Data are averages for the three rates of appli- cation. * indicates that means are significantly different at the 5% probability level. 27 «fin—9:0 I0 gin-:3 I “— o:_uoao._n_I m 5:380 In 4! M 206895 I U 5:23.91 I m 553an I< HNm am.— ou 9959.62 - $88 _a N ._.zoE0ID \ . \ \ - \ x \ a .36an U \E \ \ 9.3.0.5052... I m \ m “ 5:3an I. W on em 8. 8. ON 9.0.9.3102 I ON— 388 a a (Iowm IO %) 'IM Hsaaa Figure 3. The interaction of triazine type x cultivar x rate. Data are averages of both locations. Metribuzin data has been omitted. ** indicates that means are significantly different at the 1% probability level. 31 Aofms: 20.535 end and 3.0 H»: mm.— ou - E -II .77 wvuoom E ..I. 3050/ O O O O 0 co 0 V N 0°me I0 %) 'lM mm o 2 ON— 32 others was significant at the 1% level (Figure 3). Although both culti- vars were affected by the chloro-triazines,'Marketmore 70' was much more susceptible at the higher rates of 0.28 and 0.56 kg/ha. Since only atrazine, simazine, pr0pazine (all chlorine substituted s-triazines), and metribuzin were the only triazines which affected the growth of both cultivars, it may have been that we erroneously selected poor rates of the others. Differences might have been noted with the other s-triazines had they been applied at higher rates. Due to a shortage of PI 390244 seed, this has not been accomplished. Resistant biotypes of lambsquarters, bird's rape, and common groundsel have been reported very tolerant to all of the triazines. The resistance in these particular weeds occurs at the chloroplast level. The tolerance to the triazines exhibited among cucumbers is of a different nature. A previous study (10) has shown that the mechanism of tolerance in P1 390244 may be related to its ability to sequester the herbicide in its vascular system thus preventing the movement of the compound into the leaves and the chlor0p1asts. This is the most likely mechanism responsible for the relatively good tolerance manifested by P1 390244. 10. LITERATURE CITED Bandeen, J. D. and R. D. McLaren. 1976. Resistance of Chenopodium album L. to triazines. Can. J. Plant Sci. 56:411-412. Jensen, K. I. N., J. D. Bandeen and V. Souza Machado. 1977. Studies on the differential tolerance of two lambsquarters selections to s-triazine herbicides. Can. J. Plant Sci. 57:1169-1177. Peabody, D. 1973. Aatrex tolerant pigweed found in Washington. Weeds Today:l7. Radosevich, s. R. and A. P. Appleby. 1973. Studies on the mechanism of resistance to Simazine in common groundsel. Weed Sci. 21:497-500. A Ryan, 6. I. 1970. Resistance of common groundsel to simazine and atrazine. Weed Sci. 18:614-616. Souza Machado. V., J. D. Bandeen and P. C. Bhowick. 1978. Triazine tolerance of bird's rape biotypes. Can. Weed Comm. East Sec. Rep. p. 338. Souza Machado. V., J. D. Bandeen, W. D. Taylor and P. Lavigne. 1977. Atrazine resistant biotypes of common ragweed and bird's rape. Can. Weed Comm. East Sec. Rep. p. 306. Steel. R. G. D. and J. H. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., NY, 481 pp. Werner, G. M. and A. R. Putnam., 1977. Triazine tolerance in Cucumis sativus L. Proc. North Cent. Weed Contr. Conf. 32 p. 26. Werner, G. M. and A. R. Putnam. 1979. Atrazine metabolism in a susceptible and relatively tolerant cucumber cultivar. Weed Sci. (in preparation). 33 CHAPTER 3 MECHANISM FOR DIFFERENTIAL ATRAZINE TOLERANCE WITHIN CUCUMBER (CUCUMIS SATIVUS) ABSTRACT The cucumber (Cucumis sativus L.) accession PI 390244 tolerated up to 0.56 kg/ha atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)- s-triazine] whereas most cultivars and accessions were killed by 0.14 kg/ha. The possible mechanisms fer tolerance were investigated by com- paring growth rates, effects on photosystem II in isolated chloroplasts, and differences in uptake and translocation of 14C-atrazine between tolerant PI 390244 and the susceptible cultivar 'Marketmore 70.' PI 390244 had significantly more leaf area than 'Marketmore 70.' but less root biomass. Isolated chlor0p1ast preparations from both cultivars were equally susceptible to the herbicide. Although PI 390244 absorbed 14 more total C-atrazine, it translocated less radioactivity from the 14C found in the PI 390244 roots was roots. While the concentration of 10 to 30% greater than in 'Marketmore 70.' the amount of radioactivity in the cotyledons, first and second true leaves of 'Marketmore 70' were 53, 34, and 55% higher, respectively, after 48 hours. 34 35 INTRODUCTION Intraspecific differences in response to herbicides exist in both weeds and crop plants. The first reported differential tolerance to an herbicide was with 2,4-D (2,4-dichlorophenoxy)acetic acid in creeping bentgrass (Agggstis stolonifera L.) (1). Since then, several biotypes of weeds and crops have been found to be tolerant to 2,4-D (10, 22, 30, 31), dalapon (2,2-dichloropropionic acid) (4, 7, 18, 20), and siduron [l-(2-methyl-cyclohexyl)-3-phenylurea] (21). Tolerance to the triazines has been reported in a number of important weeds that were formerly con- sidered susceptible. Biotypes tolerant to the 2-chloro-s-triazines were first reported in 1970 by Ryan (19) in conmon groundsel (53393319 vulgaris L.). Other broadleaf weed species with triazine-tolerant bio- types are common lambsquarters (Chenopodium album L.) (3), common rag- weed (Ambrosia artemisiifolia L.) (23), redroot pigweed (Amaranthus retroflexus L.) (14), and bird's rape (Brassica campestris L.) (23, 25). Studies on the mechanism of s-triazine tolerance in common lambs- quarters, common groundsel and redroot pigweed established no differences in absorption or metabolism between biotypes of the three Species. Iso- lated chloroplasts of the resistant biotypes were not affected by the triazines (12, 15, 16, 17, 24, 26, 29). The development of tolerance to herbicides is not surprising. Harper (8) hypothesized that resistant biotypes may be selected in weed populations that have been exposed repeatedly to certain persistant herbi- cides as has occurred in insect and microbial populations. We hypothesized that if the capacity to develop tolerance to tria- zines existed in susceptible weed species. it might also exist in 36 germplasm of susceptible crops. Previous research (27) has revealed tolerance to s-triazines in the usually sensitive cucumber. The world's cucumber collection, obtained from the Regional Plant Introduction Station, Ames, Iowa, was evaluated in the summer of 1977 and Plant Introduction (PI) 390244 was found to tolerate 0.56 kg/ha atrazine, whereas most other PIS were killed at one fifth that rate. Studies described in this paper were conducted to (1) determine whether the differential response of atrazine tolerant cucumbers was based on differences in uptake and translocation of atrazine, (ii) determine if tolerance was due to differences in plant morphology or growth rates, (iii) determine if tolerance is at the chloroplast level as reported by Souza Machado et al. (24) in common lambsquarters. MATERIALS AND METHODS Differential Cultivar Tolerance Initial evaluations in the field and greenhouse demonstrated atra- zine tolerance in cucumber PI 390244 (27). A more definitive test was established by planting five cucumber seeds per pot in Dryden sandy loam soil and preemergence application of atrazine at O, 0.14, 0.28, 0.56, 0.84, and 1.12 kg/ha in 926 l/ha. Five replications of PI 390244 and the susceptible cultivar 'Marketmore 70' were assigned to a randomized complete block design and maintained under a 16 hour photoperiod with 2 sec-1) supplementing natural sunlight metal halide lights (842 uE m' in the greenhouse. To study cultivar tolerance in nutrient culture, 2 day-old seedlings were rinsed with distilled water and transferred directly into 220 ml 37 cups wrapped in aluminum foil containing 180 ml of O, 0.1. 0.2, or 0.3 uM atrazine in half-strength Hoagland's solution (9). Three seedlings were suspended in the solution by a sponge rubber disc. The nutrient solution was replenished at 2-day intervals. Plants were grown in a growth chamber with a 16 hour photoperiod maintained with cool-white 2 sec") and day and fluorescent and 25 W incandescent lamps (225 uE m' night temperatures of 31 and 21 : 2°C, respectively. A randomized com- plete block design with five replications was used. The fresh weights of the shoots were recorded after 16 days. The experiment was repeated twice. Growth Analysis A grOwth analysis was conducted to compare PI 390244 and 'Marketmore 70.' Plant growth was monitored for 10 days after seedling emergence. Plants were grown individually in 10 cm styrofoam pots containing vermi- culite. A randomized complete block design (five replications) was used with five harvest dates and two nutrient regimes, distilled water and half-strength Hoagland's containing 12 mM N03 (pH 6.5). The pots were initially watered with 200 ml of either distilled water or half-strength Hoagland's solution. At 2-day intervals, 100 ml of the nutrient treat- ments were surface applied to the pots. The pots were placed in a growth chamber under the conditions described previously. Plants were harvested at 2-day intervals after the seedlings had emerged from the growth medium. Leaf area and shoot and root dry weights were recorded. Plant parts were oven-dried for 2 days at 45°C. Leaf area ratio (LAR), net assimilation rate (NAR), and relative growth rate (RGR) were calculated (6). The dry weight data obtained for the shoots 38 and roots were analyzed using planned comparisons with the LSD test (5% level). Each growth analysis was repeated two or more times. Isolated Chloroplasts Chloroplasts were isolated from mature leaves of 3 Week-old cucum- ber (PI 390244 and 'Marketmore 70') plants that had been grown in coarse grade vermiculite, and were watered daily with half-strength Hoagland's solution. Plants had been grown for 3 weeks in a growth chamber’with a 16 hour photoperiod using cool white fluorescent light (300 E III"2 sec-I) with day and night temperatures of 30 and 20 :_2°C, respectively. ChlorOplastS were isolated according to Izawa and Good (11) with the following modifications: plants were harvested at the end of the dark period, but before exposure to light. Fifty grams of deveined leaf tissue were ground for 5 sec in a Waring blender with 75 ml 0.3 mM NaCl, 0.03 M tricine (N-tris[Hydroxymethy1]methy1 glycine), 3 mM MgC12, 0.5 mM EDTA (pH 7.8). The homogenate was squeezed through 16 layers of cheese-cloth and then centrifuged at 2500 x g fbr 90 seconds. The sediment was resuspended in 10 ml of a medium consisting of 0.2 M sorbitol, 0.005 M HEPES (N-2-hydroxyethyl piperazine-Nl-Zethanesulfbnic acid), 2 mM MgCl2 and bovine serum albumin (BSA) (0.5 g/100 ml) (pH 7.4) and centrifuged at 2000 x g for 45 seconds. The supernatant was then filtered through tissue paper. The filtered supernatant solution was centrifuged at 2500 x g for 3 minutes. The supernatant solution was de- canted, the pellet resuspended in a final volume of 1.5 ml of suspension medium, and the chlor0p1ast content was determined (2). All extractions 8 7 were made in a cold room at 0°C. Treatments consisted of 0, 10' , 10' , -6 5 10 , or 5 x 10' M atrazine. 39 The Hill reaction assay was carried out in a darkened room. The reaction mixture was made up in a 13 by 100 mm cuvette. The reaction mixture (2 ml final volume) consisted of 15 ug/ul chloroplast suspension, 50 IIM HEPES, 2 IIM MgClZ. 0.5 II" ferricyanide, and 0.2 M sorbitol, with the various concentrations of atrazine. The reaction was measured on a modified Bausch and Lamb Spectronic 505 Spetrophotometer at 420 nm. The experiment was repeated twice for all concentrations. Uptake and Translocation Cucumber seeds (PI 390244 and 'Marketmore 70') were germinated in 77 ml plastic cups containing coarse grade vermiculite and watered with half-strength Hoagland's solution. Two days after emergence, the seed- lings were rinsed carefully with distilled water to remove the vermicu- lite. The seedlings were suspended by sponge rubber discs in aluminum foil-wrapped cups that contained 180 ml nutrient solution containing 12 mM N03 (pH 6.5). The nutrient solution was changed every two days. Seedlings were placed in a growth chamber under conditions described previously fbr the chloroplast study. At 16 days, the seedlings were 14C) placed in 170 m1 of nutrient solution to which 0.2 uCi of (ring - fortified atrazine with a specific activity of 27.2 uCi/4.63 M had been added. Corrections for evaporation were made following each ex- posure time using a control treatment that was identical to the others except that it contained no plants. All tests were initiated at the beginning of the light period. A randomized complete block design con- taining four replications (three seedlings/replication) was used. Plants were harvested at 6. 12, 24, and 48 hours after treatment and the roots were rinsed three times with distilled water. Solution volume was 40 recorded at harvest after the removal of the plants, and absorption cal- culated by difference, after correcting for evaporation. Harvested plants were divided into roots, stem plus cotyledons, first leaf and second leaf, and the parts individually frozen in vials with dry ice and acetone and 1yophi1ized. Dry weights were recorded and samples were combusted in a Harvey Biological Oxidizer. The 14CD2 released by com- bustion was captured in 15 ml of Carbon 14 Cocktail1 and quantitated by liquid scintillation. Data from two experiments were averaged and analyzed as a three-way factorial. Planned comparisons were made using the LS0 test (5% level). Plants of PI 390244 and 'Marketmore 70,‘ grown as previously described, were suspended in 14 C-atrazine (0.2 uCi/40 ml) solution for 24 hours. Following exposure, the roots were rinsed three times with distilled water, blotted, pressed, frozen, lyophilized, and mounted. The plants were then exposed for one week to Kodak no-screen x-ray film. The treatments were replicated four times. RESULTS AND DISCUSSION Differential Cultivar Tolerance 'Marketmore 70' cucumber was injured more extensively than PI 390244 by an atrazine treatment of 0.14 kg/ha (Table 1). Several PI 390244 plants survived treatment with 0.56 kg/ha, but there was considerable variability among siblings of this cultivar. Plants of the two cultivars grown for 16 days in half-strength Hoagland's solution containing 0.1, 1R. J. Harvey Instrument Company; Hillsdale, NJ 07642. 41 Table 1. Differential tolerance of cucumber PI 390244 and 'Marketmore 70' to atrazine in Dryden sandy loam and in nutrient culture solution. Soil Treatment Atrazine rate Plant survivala (kg/ha) 'Marketmore 70' PI 390244 ---------------- (%)--------------- 0.14 12 75 0.28 0 50 0.56 0 16 0.84 0 0 1.12 0 0 aF value f0r cultivar is Significantly different at the 1% level. Nutrient Culture Atrazine Fresh wt/plant (uM) 'Marketmore 70' PI 390244 ---------------- (g)--------------- 0 10.5 14.0 0.1 11.0 13.3 0.2 5.1 11.6 0.3 0.9 6.8 L.S.D. at .01 1.82 1.82 42 0.2, or 0.3 M atrazine responded quite different (Table 1). None of the susceptible cultivar 'Marketmore 70' survived treatment with 0.3 uM atrazine, but PI 390244 was reduced in fresh weight to only 48% of the control. At the lowest and intermediate concentrations of atrazine, decreases noted on P1 390244 were 95% and 83% of control compared to 104% and 48% exhibited by 'Marketmore 70.' Growth Analysis The net assimilation rates of the two cultivars were not signifi- cantly different regardless of nutrient treatment. Highly significant differences in the relative growth rates were observed between different nutrient regimes. 1 PI 390244 had approximately 15% more leaf area than 'Marketmore 70' after 8 to 10 days, when averaged over both nutrient regimes (Figure 1). Both cultivars produced greater leaf areas with nutrient solution than with distilled water. 'Marketmore 70' had Significantly more root biomass than PI 390244 at 6 days and thereafter (Figure 2). The nutrient solution increased root biomass of both cultivars. The relatively small root biomass of PI 390244 at 6 to 10 days after seedling emergence was first hypothesized to be a faCtor contri- buting t0 the selectivity of this cultivar to atrazine by restricting absorption and dilution of the quantity absorbed throughout the larger leaf area. Later uptake studies with 14C-atrazine refuted this hypo- thesis. Figure l. 43 Leaf area ratios (LAR) from atrazine-tolerant PI 390244 and atrazine-susceptible 'Marketmore 70' cucumber in distilled water and half-strength Hoagland's solution two to ten days after emergence. Asterisks indicate values that are Signi- ficantly different at the 5% level. lAll (cm2 loaf/III: dry wt) 44 340 320 ‘ 300 . Hoagland ’s 280 ”5 .....oocooooooooooooooo 'Qouufl”” * 26° ' — P1390244 I coco. M-70 240 '. 220 200 DAYS 45 Figure 2. Root biomass from atrazine-tolerant PI 390244 and atrazine- susceptible 'Marketmore 70' in distilled water and nutrient solution two to ten days after emergence. Asterisks indi- cate values that are Significantly different at the 5% level. 46 O— ONIE no... Vfiuoon _n— I 0— ON on 0% On 00 ON (Wild/3'”) II All! 47 Isolated Chloroplasts The g-triazines are known to be potent inhibitors of the Hill reac- tion (12). There were no differences in response between the two culti- vars (Figure 3). Therefore, unlike the results reported with common lambsquarters (16, 23), the differential response is not manifested at the chloroplast level. Uptake and Translocation Atrazine was readily absorbed from the nutrient solution by the roots and translocated ace0petally to the leaves of both cultivars. PI 390244 consistently absorbed more water and more 14C-atrazine than 'Marketmore 70' (Figure 4), even though PI 390244 had less root biomass at the time of treatment. Perhaps, greater transpiration losses from larger leaf areas caused an increase in root uptake. PI 390244 contained 10 to 13% more 14C/plant than 'Marketmore 70' through the duration of the experiment (Figure 4). There were no significant differences between cultivars in distri- bution of radioactivity (dpm/mg) within a plant part 6 or 12 hours after 14 treatment. However, at 24 and 48 hours, PI 390244 accumulated more C 14 than 'Marketmore 70' in the roots (Figure 5a). Although no C-activity accumulated in the roots after 24 hours in either cultivar, ‘4 C-activity continued to accumulate in both the cotyledons and leaves. 'Marketmore 70' had 32% more dpm/mg in the cotyledons after 48 hours than PI 390244 (Figure 5b). The 14C concentration in the first and second leaves was not Significantly different between cultivars at 6 and 12 hours after treatment, but after 24 and 48 hours, 'Marketmore 70' had consistently more dpm/mg in these two areas (Figures 5c and 5d). 48 Figure 3. Photochemical activity of isolated chloroplasts from atrazine- tolerant PI 390244 and atrazine-susceptible 'Marketmore 70' cucumber leaves exposed to several atrazine concentrations. 49 — PI 390244 00000 M-7o 2.3 0:. £\..o no.057v 29.22.50“. «EH .3 ZO_._.ODDmm mo_z<>0_mmmu_ 10" 5X1O'5 10" ATRAZINE CONCENTRATION (molar) 10" 50 Figure 4. Uptake of water and 14C-atrazine by atrazine-tolerant PI 390244 and atrazine-susceptible 'Marketmore 70' cucumber from 6 to 48 hours. In N 51 .01 x .lNVld/WdCl 1v101 1- z u, 5 x: a: <01 \N 580 580 300T on? n:— _ was 210.2 5‘15” 31 I—d In F O O O O m 0 1' N (Iw) axvmn alarm 48 36 24 12 TIME (hours) 52 14C in a) roots; b) cotyledons; C) fIVSt 193T; Figure 5. Accumulation of and d) second leaf of atrazine-tolerant PI 390244 and atrazine- susceptible 'Marketmore 70' cucumbers. 53 4's 140 601 .I 120 50. COTYLEDONS .°' 0... 100- 40 ‘ 3 up on 0'. E E .o \ \ 0. . 8° ' I 30 ' .0. H. I. a B °°‘ — 91390244 2°' .-°' — M 390244 ooooo M-70 00... M-70 40- 10 4 [.51). l.S.ll. 5x In 0 V V f ‘I c I V I fl 0 6 12 124 48 0 6 12 ‘24 48 TIME (hours) TIIE (hours) 70 - 0* :' :' 604 :0 90 ‘ :* 50' FIRST LEAF _: 75. SECOND lEAF ,o' :' :' - 403 ,0: n 60‘ .0: E .’ E : \ .' \ .’ a : a _: g 30- :1: 2; 451 .- .i 20 - 30 10 - 15 ' o — P1390244 soooo M-7o so... M‘70 o _ _ a o . . - I) 6 12 24 48 0 6 12 214 TIME (hours) TIIE (hours) 'TIME DISPLAYED on LOG SCALE 54 Although PI 390244 contained more total radioactivity than the 'Marketmore 70.' there were pronounced differences in the distribution of the isotope. Differences in atrazine tolerance by the two cultivars does not seem to be explained by differential uptake, but may be related to differential translocation. The differential tolerance of plants to the s-triazines has not previously been associated with differences in uptake and translocation. 14 14 Studies conducted by Thompson et al. (26) with C-atrazine and C- simazine 0n resistant and susceptible strains of redroot pigweed did not reveal differences in uptake or subsequent translocation. Jensen et a1. (12) found no difference between two selections of common lambsquarters in foliar or root uptake of 14C-atrazine or in translocation and accumu- 14C-atrazine within the plants following root uptake. 14 lation of Radosevich and Appleby (15) found no difference in uptake of C-simazine in two biotypes of common groundsel. ‘ A series of autoradiographs were made of the two cultivars to further elucidate translocation differences. Roots of both cultivars were uniformly labeled with the isotope (Figure 6). However, differences were noted in the foliar distribution of the 14 C. There appeared to be a high accumulation of radioactivity distributed uniformly throughout the leaves of the susceptible cultivar while the tolerant cultivar exhibited localization of radioactivity only in the vascular regions of the plant. Similar symptomology was seen in a greenhouse experiment using f0rmulated atrazine (Figure 7). Differential atrazine tolerance in cucumber may be based on several factors: (i) differences in plant morphology between the two cultivars with P1 390244 possessing less root biomass and more leaf area than the 55 Figure 6. Treated plants (above) and autoradiographs (below) of atrazine- susceptible 'Marketmore 70' (left) and atrazine-tolerant PI 14 390244 (right) 24 hours after root treatment with C-atrazine. 56 57 Figure 7. A plant of PI 390244 that survived 0.56 kg/ha preemergent atrazine treatment. Chlorosis is localized in the vascular system of the leaf. 58 59 14 more susceptible cultivar; (ii) differences in translocation of C- atrazine; and lastly (iii) evidence from autoradiographs suggests that 14C-activity in PI 390244 was more localized in the roots and xylem of the plant. Possibly, there is some type of binding of the herbicide in the xylem. Further research is needed to determine if cucumber tolerance to the triazine group is of a general nature and to determine if metabolism may also contribute to the tolerance in P1 390244. A genetic study has ascertained the inheritance of tolerance to atrazine is most likely determined by two dominant genes with epistasis (28). 10. 11. 12. LITERATURE CITED Albrecht, H. R. 1947. Strain differences in tolerance to 2,4-D in creeping bentgrasses. J. Am. Soc. Agron. 39:163-165. Arnon, D. I. 1949. Copper enzyme in isolated chloroplasts, Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24:1-15. Bandeen, J. D. and R. D. McLaren. 1976. Resistance of Chenopodium album L. to triazines. Can. J. Plant Sci. 56:411-412. Bucholtz, K. P. 1958. Variations in the sensitivity of clones of quackgrass to dalapon. Proc. 15th North Cent. Weed Contr. Conf. 18-19. Devine, T. E., R. E. Seaney, D. L. Linscott, R. D. Hagin, and N. Brace. 1975. Results of breeding f0r tolerance to 2,4-D in birdsfoot trefoil. Crop Sci. 15:721-724. Evans, B. C. 1972. The quantitative analysis of plant growth. Univ. of California Press, Berkeley, Los Angeles. 734 pp. Hamilton, K. C. and H. Tucker. 1964. Response of selected and random plantings of johnsongrass to dalapon. Weeds 12:220-222. Harper, J. L. 1956. The evolution of weeds in relation to resist- ance to herbicides. Proc. 3rd Br Weed Contr. Conf. 179-188. Hoagland, D. R. and D. I. Arnon. 1938. The water culture method for growing plants without soil. Univ. Calif. Agric. Exp. Stn. Circ. 347, 32 pp. Hodgson, J. M. 1970. The response of Canada thistle ecotypes to 2.4-0, amitrole, and intensive cultivation. Weed Sci. 18: 253-255. Izawa, S. and N. E. Good. 1965. The number of Sites sensitive to 3-(3,4-dichlor0phenyl)-l,1-dimethylurea,3-(4-chloropheny1)-l, 1-dimethylurea and 2-ch1oro-4-(2-pr0pylamino)-6-ethy1amino-s- triazine. Biochim. Biophys. Acta. 102:20-38. Jensen, K. I. N., J. D. Bandeen and V. Souza Machado. 1977. Studies on the differential tolerance of two lambsquarters selections to triazine herbicides. Can. J. Plant Sci. 57:1169-1177. 6O 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 61 Moreland. D. E. and K. L. Hill. 1962. Interference of herbicides with the Hill reaction of isolated chloroplasts. Weeds 10: 229-236. Peabody, D. 1973. Aatrex tolerant pigweed found in Washington. Weeds Today 4:17. Radosevich, S. R. and A. P. Appleby. 1973. Studies on the mechanism of resistance to simazine in common groundsel. Weed Sci. 21:497-500. Radosevich, S. R. and 0. T. Devilliers. 1976. Studies on the mechanism of s-triazine resistance in common groundsel. Weed Sci. 24:229-232. Radosevich, S. R. 1977. Mechanism of atrazine resistance in lambsquarters and pigweed. Weed Sci. 25:316-318. Roche, B. F. and T. J. Muzik. 1964. Ecological and physiological study of‘ Echinochloa crusgalli L. Beauv. and response of its biotypes to sodium 2,2-dichloropr0pionate (dalapon). Agron. J. 56:155-160. Ryan, 0. I. 1970. Resistance of common groundsel to simazine and atrazine. Weed Sci. 18:614-616. Santlemann, P. W. and J. A. Meade. 1961. Variation in morphologi- cal characteristics and dalapon susceptibility within the species Setaria lutescens and S, faberii. Weeds 9:406-410. Schooler, A. B., A. R. Bell and J. D. Nalewaja. 1972. Inheritance of Siduron tolerance in foxtail barley. Weed Sci. 20:167-169. Sexsmith, J. J. 1964. Morphological and herbicide susceptibility differences among strains of hoary cress. Weeds 12:19-22. Souza Machado, V., J. D. Bandeen, W. D. Taylor and P. Lavigne. 1977. Atrazine resistant biotypes of common ragweed and bird's rape. Can. Weed Comm. East. Sect. Rep. p. 306. Souza Machado, V., J. D. Bandeen, G. R. Stephenson and K. I. N. Jensen. 1977. Differential atrazine interference with the Hill reaction of isolated chloroplasts from Chenopodium album L. biotypes. Weed Res. 17:407-413. Souza Machado, V., J. D. Bandeen and P. C. Bhowmik. 1978. Tria- zine tolerance of bird's rape biotypes. Can. Weed Comm. East. Sect. Rep., p. 338. Thompson, L., Jr., R. W. Schumacker and C. J. Rieck. 1974. An atrazine resistant strain of redroot pigweed. Weed Sci. Soc. Am. Abstr. #196z85. 27. .28. 29. 30. 31. 62 Werner, G. M. and A. R. Putnam. 1977. Triazine tolerance in Cucumis sativus L. Proc. North Cent. Weed Contr. Conf. 32 p. 26. Werner, G. M. and A. R. Putnam. 1979. Inheritance of 2-chlor0- 4-(ethylamino)-6-(isopropylamino)-§-triazine. (In preparation). West, L. 0., T. J. Muzik and R. I. Witters. 1976. Differential gas exchange responses of two biotypes of redroot pigweed to atrazine. Weed Sci. 24:68-72. Whitehead, C. W. and C. M. Switzer. 1963. The differential response of strainSIrfwild carrot to 2,4-D and related herbi- cides. Can. J. Plant Sci. 43:255-262. Whiteworth, J. W. and T. J. Muzik. 1967. Differential response of selected clones of bindweed to 2,4-D. Weeds 15:275-280. CHAPTER 4 ATRAZINE METABOLISM IN A SUSCEPTIBLE AND RELATIVELY TOLERANT CUCUMBER CULTIVAR ABSTRACT Metabolism of the herbicide 2-chloro-4-ethylamino-6-isopropylamino- s-triazine (atrazine) was investigated in two cultivars of cucumber (Cucumis sativus L.). PI 390244 which has been identified previously as exhibiting tolerance to the herbicide and 'Marketmore 70.' a susceptible cultivar. This study revealed no differences in the rate of metabolism or nature of metabolites between the two cultivars. The only metabolite found was 2-chloro-4-amin0-6-isopropylamino-s-triazine and then in only very small quantities (5-10%). N0 hydroxy-atrazine 0r glutathione-conju- 1 gated atrazine was found. The dealkylated metabolite was found only in the Shoots and not in the roots of either cultivar during the length of the experiment. The slight metabolism and minimal differences found between the two cultivars cannot explain the differences in tolerance between the two cultivars. 63 64 INTRODUCTION Dealkylation of atrazine occurs to some extent in all higher plants investigated (2, 4). The rate of atrazine metabolism and detoxication appears to determine the basis for resistance in corn, sorghum and peas (2). The rate of metabolism must be rapid enough to prevent the accumu- lation of a lethal level of atrazine in the plant, and the pathway of metabolism must result in the f0rmation of non-phytotoxic metabolites (2, 5). ‘ The major metabolite of atrazine in both roots and shoots of young peas, which exhibit intermediate tolerance to atrazine, was 2-chloro- 4-amino-6-i50prOpylamino-s-triazine, a dealkylated metabolite (4). No hydroxyatrazine or glutathione canjugated atrazine was present. The dealkylated metabolite in this form is only partially detoxified and must be further dealkylated to a non-phytotoxic form (4). Shimabukuro (3) suggests this may be the basis for the intermediate tolerance ex- hibited by peas. Atrazine tolerance in cucumber has recently been reported in P1 390244 obtained from the Regional Plant Introduction Station at Ames, Iowa (7). It was hypothesized that the rate of metabolism and detoxica- tion of atrazine might be responsible for the differential response ob- served in cucumber. MATERIALS AND METHODS Cucumber seeds (PI 390244 and 'Marketmore 70.' a susceptible culti- var) were germinated in 77 ml plastic cups containing coarse grade 65 vermiculite and watered with half-strength Hoagland's solution (1). Two days after emergence, the seedlings were rinsed carefully with distilled water to remove the vermiculite. The seedlings were transplanted to 220 ml cups wrapped in aluminum foil which contained 180 ml nutrient solu- tion containing 12 mM nitrate nitrogen (pH 6.5). A sponge rubber disc was used to suspend seedlings in the nutrient solution, which was changed every two days. Seedlings were placed in a growth chamber with a 16 hour 2 sec-1) with day photoperiod from cool white fluorescent light (300 uE m' and night temperatures of 31° and 21°C :_1°C, respectively. After 11 days, the cucumber seedlings were placed in 150 ml of nutrient solution that had 0.2 uCi of uniformally ring-labeled 14C-atrazine with a specific activity of 27.2 uCi/4.63 M added. All tests were initiated at the beginning of the light period. The experimental design was a split-split plot containing four replications (two seedlings/replication). Plants were harvested at 1, 3 and 5 days after treatment. At harvest, the roots were rinsed three times with distilled water. Water uptake was recorded at the time of harvest after the removal of the plants by measuring the solution remaining and subtracting the water lost by evaporation. Har- vested plants were then divided into four parts: roots, stem plus coty- ledons, first leaf and second leaf. These plant parts were then frozen in a dry ice and acetone bath and lyophilized. Samples were kept frozen at -25°C until analyzed. The two plant replication were combined prior to storage. The extraction procedure followed was one furnished by the CIBA-Geigy Corporation of Greensboro, NC and is as follows. Individual plant parts were homogenized with 15 ml chloroform for 5 minutes in a Sorvall Omni-mixer. The homogenate was filtered through 66 glass wool and 20 g anhydrous sodium sulfate. The filtrate was evapor- ated to dryness in a 50 m1 round bottom flask on a rotary evaporator at 40°C. The flask was rinsed twice with 50 ml of hexane. The hexane was then decanted into a 125 m1 separatory funnel. The flask was also rinsed with 25 ml of acetonitrile and was thus combined with the hexane and swirled f0r 1 minute. The acetonitrile was decanted into a second 125 m1 separatory funnel. The hexane fraction was rinsed with 25 ml acetonitrile for 1 minute and the acetonitrile fractions then were com- bined. To the separatory funnel containing the acetonitrile, 50 ml V hexane was added. The acetonitrile fraction was decanted into a 250 ml round bottom flask. The filtrate was evaporated to dryness. To flasks containing root extractions, 2 aliquots of 10 ml methanol were added. Two aliquots of 10 ml carbon tetrachloride were added to flasks contain- ing chlorophyll. The methanol and carbon tetrachloride extracts were then decanted into vials f0r storage purposes until analyzed. All sam- ples were stored at -15°C. Before assaying, all samples were evaporated to dryness under a stream of nitrogen and brought up to volume of 1 ml with methanol. A 50 1.11 sample was streaked ona0.250 nm Silica Gel G thin-layer chroma- tography plate. The plates were developed 15 cm in benzene:acetic acid (50:4 v/v) (Solvent A). The silica gel was scraped at 1 cm intervals and assayed fer radioactivity using 15 ml of a 4 g PPO (2,5-diphenyloxa- zole) plus 50 mg POPOP (l,4-bis[2-(4-methyl~5-phenyloxazoly)]-benzene in 1 liter toluene. A second 50 01 was streaked on a similar plate and developed in benzene:acetic acid:water (50:50:3 v/v) (Solvent B). Stan- 14 dards<3f C-atrazine and the dealkylated metabolite 2-chloro-4-amino- 6-isopropylamino-s-triazine were also streaked on each plate. The Rf 67 values of atrazine and the dealkylated metabolite were 0.40 and 0.26, respectively for Solvent A and 0.98 and 0.96 for Solvent B. Rf values of the unknown spots were compared with the standards. The atrazine and the nonlabeled metabolite were detected by viewing the chromatogram under ultraviolet light. Rf values of the unknown spots were compared with the standards. Data were converted to percent (%) unchanged atrazine for direct comparisons. The experiment was conducted twice and significance of the means was determined using the LS0 test at the 1% level (6). RESULTS.AND DISCUSSION Thin-layer chromatographic analysis of chloroform-soluble compounds in the plant extracts showed that only dealkylation occurred in cucumbers. There were no differences in the rate of metabolism or dealkylated meta- bolites between the two cultivars. The only metabolite found was 2-chloro- 4-amino-6-isopropylamino-s-triazine. and then in only very small quanti- ties (5-10%). No hydroxyatrazine or glutathione conjugated atrazine were found. There was a significant difference (1% level) in the amount of un- changed atrazine only in the first leaf between the two cultivars. In general, 'Marketmore 70' appeared to metabolize more ‘4C-atrazine al- though the differences were not significant (Figure l). The dealkylated metabolite was not found in the roots of either cultivar during the length of the experiment. The metabolite was f0und in the other plant parts of both cultivars (Figure 2). 68 Figure 1. Interaction of cultivar and plant part on the amount of un- changed 14C-atrazine. 69 .52.. p215 .0215qu *8. .3: 612.53 \ \ .\ k HN— am.— 2 $0250.22 I 388 a n m ‘00.— 00 no #0 00 mo 00— 3NIZV81V OBQNVHDNH % 70 Figure 2. Interaction of harvest date and plant part on the amount of unchanged 14C-atrazine. 71 has. ._.Z<.E 00 E33 .00. .2: V II II: m 9.000.500 \ L\\'\\‘\\\\\\\\\ .00. .\ oo No #0 00 mo 00— 3NIZV2|1V GSONVHDNH % 72 There was very little metabolism of the atrazine by either cultivar during the 5 day period. At five days after treatment the amount of un- changed atruzine was 90%. The slight metabolism and minimal differences found between the two cultivars cannot explain differences in tolerance between the two cultivars. Shimabukuro et a1. (2, 4) have stated that slight metabolism occurs in all higher plants investigated, even plants that are considered susceptible to the s-triazines. Another mechanism is more likely responsible for the differential tolerance exhibited by PI 390244. Autoradiograms of both Cultivars (treated with 14 C-atrazine) revealed that PI 390244 appeared to restrict the movement of atrazine to the vascular system of the plant, thus preventing the movement of the herbicide into the leaves of the plant while 'Marketmore 70.' the sus- ceptible cultivar, had a uniform diStribution of the radioactive material throughout the leaves and root system of the plant (8). The basis f0r differential tolerance in cucumber is not due to dif- ferences in the rate of metabolism or differences in metabolites formed. Slight metabolism did occur at 3 days (95% unchanged atrazine) and con- tinued at 5 days with only 10% of the total as the dealkylated metabolite. LITERATURE CITED Hoagland, D. R. and D. I. Arnon. 1938. The water-culture method for growing plants without soil. Univ. Calif. Agric. Exp. Stn. Circ. 347, 32 pp. Shimabukuro, R. H. 1967. Atrazine metabolism and herbicidal selectivity. Plant Physiol. 42:1269-1276. Shimabukuro, R. H. 1967. Significance of atrazine dealkylation in root and shoot of pea plants. J. Agr. Food Chem. 15:557- 563. Shimabukuro, R. H. and H. R. Swanson. 1969. Atrazine metabolism, selectivity and mode of action. J. Agr. Food Chem. 17:199- 205. ~ Shimabukuro, R. H., R. E. Kadunce and D. S. Frear. 1966. Dealky- lation of atrazine in mature pea plants. J. Agr. Food Chem. 14:392-395. Steel, R. G. D. and J. H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hill Book Co., Inc., NY,_481 pp. Werner, G. M. and A. R. Putnam. 1977. Triazine tolerance in Cucumis sativus L. Proc. North Cent. Weed Contr. Conf. 32, p. 26. Werner, G. M. and A. R. Putnam. 1979. Differential atrazine tolerance within cucumber. Weed Sci. (In press). 73 CHAPTER 5 INHERITANCE OF 2-CHLORO-4-ETHYLAMINO-6-ISOPROPYLAMINO-Sy TRIAZINE (ATRAZINE) TOLERANCE IN CUCUMBER (CUCUMIS SATIVUS) ABSTRACT Genetic control of atrazine tolerance appears to be determined by two genes. Analysis of the F2 population from the cross of PI 390244 (tolerant) and 'Gynoecious 14' (susceptible) suggested that two dominant epistatic genes conditioned atrazine tolerance in cucumber. Variability existed in some of the F2, BC}, and BCz data. A possible explanation could be that the Plant Introduction was not completely homozygous for the trait or that there may be different levels of gene penetrance or a combination of the two. Penetrance was incomplete in some cases and variable from cross to cross. INTRODUCTION Many crop Species normally regarded as susceptible to the s- triazines may contain Significant intraspecific variability f0r toler- ance to these compounds. Genetic variability for s-triazine tolerance has been observed recently in soybeans (Glycine max Merr. L.) (1), flax ‘(Linum usitatissimum L.) (2), wheat (Triticum aestivum L.) (3), rape (BrasSica napus L.) (5), mustard (Sinapis alba L.) (3) and cucumber 74 75 (Cucumis sativus L.) (6, 7) which are normally considered extremely sensitive. The genetic investigation of triazine resistance in bird's rape conducted by Souza Machado et a1. (5) indicates that resistance is inheri- ted uniparentally, through the female parent. Triazine tolerance in flax was reported to be quantitatively inherited (2). The purpose of this investigation was to determine the genetic in- heritance of atrazine tolerance in cucumber. MATERIALS AND METHODS The mode of inheritance of the triazine tolerant character was examined by making crosses with cucumbers that were previously selfed for three generations and screened for homozygosity fer atrazine toler- ance. PI 390244, the tolerant parent, was crossed with the susceptible 'Gynoecious 14.I Throughout the course of this study the Plant Intro- duction was maintained by vegetative pr0pagation. Appropriate F1 hybrids were selfed to obtain F2 progeny, and also backcrossed as pollen parents to 'Gynoecious 14.‘ Additional Fl's were used as maternal parents with PI 390244 as the pollen parent. A11 seeds were sown May 21, 1979 at Castle Hayne, NC on a Portsmith sandy loam with an organic matter content of 3.2 to 4.4%. A randomized complete block design with six replicates was used. Atrazine was applied preemergence at 0.56 kg/ ha in a spray volume of 333 l/ha over the entire planting. Parental. F1, F2 and backcross seedlings were evaluated at three weeks after germi- nation. The seedlings were classified in two phenotypic classes: alive or dead. Family data have been derived from individual F1 plants which have been selfed. Individual crosses were maintained separately. 76 RESULTS AND DISCUSSION Segregation ratios obtained fer the individual F2 populations are found in Table 1. F2 families of the reciprocal crosses which manifested 9:7 ratios did not deviate significantly from the ratio tested thus suggesting that two dominant genes with epistasis conditioned this factor. Segregation occurred in both F1 and F2 seedlings for atrazine toler- ance. The results are shown in Table l. Segregation ratios obtained for atrazine tolerance in the F1 population did not deviate significantly from a 1:1 ratio suggesting that possibly one parent was not completely homozygous. When the Plant Introduction was first identified as being atrazine tolerant, the percent survival to 0.56 kg/ha atrazine was 78% (8). The survivors were selfed and maintained individually for three , generations and repeatedly tested for atrazine tolerance. The most tolerant and most homozygous line was selected for this study. After the third generation some variability still existed. Some incongruent data was obtained from the F2 populations which is presented in Table 2. The cross of PI 390244 x 'Gynoecious 14' for three of the F2 families tested had a higher total number of survivors at three weeks. They did not fit a 9:7 ratio as did the other crosses but more closely fit a 3:1 ratio. Reciprocal differences were also ob- served. These F2 families most closely fit 7:9 ratios, since increased mortality reversed the ratio of the phenotypic classes. The backcross data is presented in Table 3. The data obtained was also variable but for the most part fit a 1:1 ratio with the exception of one cross of ('Gynoecious 14' x PI 390244) x PI 390244 which fir a 3:1 ratio. A possible explanation for the variability of the data for this 77 om.o-oe.o wn.o wmumm .m om.o-ou.o PN.o aoupm .N mum o~.o-ow.o mo.o eoumw .P N b .25., open a M .ep ac. x eemomm Ha ow.o-om.o , No.o umumm .m om.o-ow.o mo.o mmumw .N mum oo.o-on.o NF.o mmuwm .P N APPEmm name a eemomm H; x .ep ac. _”_ om.o-oe.o oN._ emuom .e_ >5. x eemomm Ha _"_ om.o-oe.o mm.o mN mm «emoam Ha x .ep aw. umumoh nwumm a Nx owpem mmocu .mm mom N cwpu a one _m we ocw~mcam cu xpwpvnvuamomam wen mocagmpo» co; a?» mo mmocuoom new cowummmcmmm .P anah 78 ov.o:om.o mm.o qumm .N oo.o on o , an“ - . ¢_.o qumm .F s_csac Samoan Ha x .e_ so. oe.o-om.o om.o 3e woe .m om.o-om.o mo.o am «_F .N Pam om.o-om.o mo.~ om oo_ ._ Apogee .ep ea. x eemomm Ha ambush sebum a Nx among amoeu .eemoom Ha x .e_ am. new .e_ aw. x ee~omm Ha omega may co meowpapsaoa we as» eoec cacwaono moan beasemcoueo .N mpnae 79 05.0-00.0 00.0 . 00HN0 .0 00.0-00.0 00.0 00n00 .N _u_ 00.0-00.0 00.0 00 00 ._ 2.0, 00. x 00~000 000 x .00 >0. 0”_ 00.0-00.0 _ F0.0 00.20 .N _"m 00.0-0A.0 00.0 AN 00 .F 00~000 00 x 2000000 00 x .0F 00.0 00.0-00.0 00.0 emumm .N 0._ 00.0-00.0 00._ 00HN0 .0 2000000 00 x .0, 00.0 x .0? x0. 000000 00000 a 0x 00000 00000 .mmcwpuomm mmocoxuan mo ocw~0cua 00 apwpwnwpamomsm 000 00000000» cow “0* 00 00000000 0:0 cowpmmmcmom .m m_0me 80 experiment may be due to different levels of gene penetrance. Penetrance, in this instance was incomplete and variable from cross to cross. Another factor that may have confounded the experiment and the results might have been the variation in organic matter content of the soil used for this study. In an evaluation of PI 390244 versus 'Marketmore 70,‘ to determine the level of atrazine tolerance in nutrient culture, the Plant Introduc- tion's response was very uniform and no differences were noted among the plants utilized for the study (7). The presence of varying organic matter content, even though no replication differences were noted, nay have con- tributed to the variability expressed. By selection for the proper back- ground genotypes, completely penetrant lines could be developed. Since the atrazine tolerant trait appears to be dominant, it should not be difficult to incorporate this trait into existing cucumber culti- vars. The development of cultivars that possess limited tolerance to the s-triazines would be advantageous in allowing use of low rates for chemical weed control, or the planting of cucumbers in crop rotation systems where a carry-over problem might exist due to the persistance of these herbicides. Cucumbers have normally been considered so sensitive to the s: triazines that they are used as indicators in bioassays to determine soil persistance. This study demonstrates the importance of selecting culti- vars that have been evaluated for triazine susceptibility prior to their use in bioassays. LITERATURE CITED Andersen, R. N. 1970. Influence of soybean seed size on response to atrazine. Weed Sci. 18:162-164. Comstock, V. E. and R. N. Andersen. 1968. An inheritance study of tolerance to atrazine in a cross of flax (Linum usitatissimum L.). Crop Sci. 8:508-509. Karim, A. and A. D. Bradshaw. 1968. Genetic variation in simazine resistance in wheat, rape and mustard. Weed Res. 8:283-291. Snedecor, G. W. and W. C. Cochran. 1967. Statistical methods. Iowa State University Press, 593 pp. Souza Machado, V., J. D. Bandeen, G. R. Stepheson and P. Lavigne. 1978. Uniparental inheritance of chloroplast atrazine tolerance in Brassica campestris. Can. J. Plant Sci. 58: 977-981. Werner, G. M. and A. R. Putnam. 1977. Triazine tolerance in Cucumis sativus L. Proc. North Cent. Weed Contr. Conf. 32, p. 26. Werner, G. M. and A. R. Putnam. 1979. Differential atrazine tolerance within cucumber. Weed Sci. (In press). Werner, G. M. and A. R. Putnam. 1979. Intraspecific differences in triazine tolerance in cucumber (Cucumis sativus) germplasm. (In preparation). 81 LITERATURE CITED LITERATURE CITED Albrecht, H. R. 1947.. Strain differences in tolerance to 2,4-D in creeping bentgrasses. J. Am. Soc. Agron. 39:163-165. Andersen, R. N. 1970. Influence of soybean seed size on response to atrazine. Weed Sci. 18:162-164. Anderson, W. P. 1977. Weed Science: principles. West Publishing Co., St. Paul, MN, 598 pp. Anonymous. 1979. Herbicide Handbook of the Weed Science Society of America. Fourth Edition. Champaign, IL, 479 pp. Arnon, D. I. 1949. Copper enzyme in isolated chloroplasts, Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24:1-15. Arntzen, C. J., K. Pfister and C. L. Ditto. 1979. Alterations in the Photosystem II complex in chloroplasts from herbicide- resistant weed biotypes. Weed Sci. Soc. Am. Abstr. #238. Bandeen, J. D. and R. D. McLaren. 1976. Resistance of Chenopodium album L. to triazines. Can. J. Plant Sci. 56:411-412. Bandeen, J. 0., J. V. Parochetti, G. F. Ryan, B. Maltais and D. V. Peabody. 1979. Discovery and distribution of triazine resistant weeds in North America. Weed Sci. Soc. Am. Abstr. #229. Bucholtz, K. P. 1958. Variations in the sensitivity of clones of quackgrass to dalapon. Proc. 15th North Cent. Weed Contr. Conf. 18-19. Burnside, 0. C., E. L. Schmidt and R. Behrens. 1961. Dissipation of simazine from the soil. Weeds 9:477-484. Castlefranco, P., C. L. Foy and D. B. Deutsch. 1961. Nonenzymatic detoxification of 2-chloro-4,6-bis(ethylamino)-§-triazine (simazine) by extracts of Zea mays. Weeds 9:580-591. Castlefranco, P. and M. S. Brown. 1962. Purification and pr0perties of the simazine-resistant factor of Zga_mays. Weeds 10:131-136. Comstock, V. E. and R. N. Andersen. 1968. An inheritance study of tolerance to atrazine in a cross of flax (Linum usitatissimum L.). Crop Sci. 8:508-509. 82 83' Couch, R. W., J. V. Gramlich, D. E. Davis and H. H. Funderburk, Jr. 1965. The metabolism of atrazine and Simazine by soil fungi. Proc. 5. Weed Conf. 18:623. Davis, D. E., J. V. Gramlich and H. H. Funderburk, Jr. 1964. Atrazine absorption and degradation by corn, cotton, and soybeans. Weeds 12:252-255. Devine, T. E., R. E. Seaney, D. L. Linscott, R. D. Hagin and N. Brace. 1975. Results of breeding f0r tolerance to 2.4-0 in Birdsfoot Trefoil. Crop Sci. 15:721-724. Eichers, T. R., P. A. Andrilenas, T. W. Anderson. 1976. The farmers use of pesticides. Agric. Econ. Report 418, 58 pp. Evans. B. C. 1972. The quantitative analysis of plant growth. Univ. of California Press, Berkeley, Los Angeles. 734 pp. Good, N. E. 1961. Inhibitors of photosynthesis. Plant Physiol. 34: 584-607. Good, N. E. and S. Izawa. 1964.1 Selective inhibitors of photosynthesis. Rec. Chem. Prog. 4:225-237. Gunther, F. A. ed. 1970. The triazine herbicides: Residue Reviews. Springer-Verlag, NY, 413 pp. . Gysin, H. and E. Knfisli. 1960. Chemistry and herbicidal properties of triazine derivatives. Adv. Pest. Contr. Res. 3:289-358. Hamilton, R. H. 1964. A corn mutant deficient in 2,4-dihydroxy-7- methoxy-l,4-benzoxazinone with an altered tolerance to atrazine. Weeds 12:27-31. Hamilton, R. H. 1964. Tolerance of several grass species to 2-chloro- §ftriazine herbicides in relation to degradation and content of benzoxazinone derivatives. J. Agr. Food Chem. 12:14-17. Hamilton, R. H. and D. E. Moreland. 1963. Fate of ipazine in cotton plants. Weeds 11:213-217. Hamilton, K. C. and H. Tucker. 1964. Response of selected and random plantings of Johnsongrass to dalapon. Weeds 15:220-222. Harper, J. L. 1956. The evolution of weeds in relation to resistance to herbicides. Proc. 3rd Br. Weed Contr. Conf. 179-188. Harris. 0. I. 1965. Monuron and g-triazines in soil. Weeds 13:6-9. Harris, 0. I. 1967. Fate of 2-chloro-s-triazine herbicides in soil. J. Agr. Food Chem. 15:157-162. 84 Hensley, J. R. and C. J. Counselman. 1979. Allelopathic interactions between triazine resistant and susceptible strains of redroot pigweed (Amaranthus retroflexus L.). Weed Sci. Soc. Am. Abstr. #232. Hoagland, D. R. and D. I. Arnon. 1938. The water culture method for growing plants without soil. Univ. Calif. Agric. Exp. Stn. Circ. 347. 32 pp. Hodgson, J. M. 1970. The response of Canada thistle ecotypes to 2,4-0, amitrole, and intensive cultivation. Weed Sci. 18:253-255. Izawa, S. and N. E. Good. 1965. The number of sites sensitive to 3-(3, 4- dichlorophenyl)- 1, l -dimethylurea, 3- (4-chlorophenyl)- l, l- dimethylurea and 2- chloro- 4- (2- propylamino)- 6- ethylamino-S- triazine. Biochem. Biophys. Acta. 102: 20- 38. Jensen, K. I. N. 1975. Atrazine detoxification in three gramineae subfamilies. Ph.D. Thesis, Univ. of Guelph, Ontario. Jensen, K. I. N., J. D. Bandeen and V. Souza Machado. 1977. Studies on the differential tolerance of two lambsquarters selections to triazine herbicides. Can. J. Plant Sci. 57:1169-1177. Jensen, K. I. N., G. R. Stephenson and L. A. Hunt. 1977. Detoxifica- tion of atrazine in three gramineae subfamilies. Weed Sci. 25: 212-220. Jensen, K. I. N., J. D. Bandeen and V. Souza Machado. 1979. Role of triazine herbicide uptake, translocation, accumulation and meta- bolism in plant selectivity. Weed Sci. Soc. Am. #234. Jordan, L. S., B. E. Day and W. A. Clerx. 1964. Photodecomposition of triazines. Weeds 12:5-7. Karim, A. and A. D. Bradshaw. 1968. Genetic variation in Simazine resistance in wheat, rape and mustard. Weed Res. 8:283-291. Kaufman, D. D., C. Kearney and T. J. Sheets. 1963. Simazine: Degradation by soil microorganisms. Science 142:405- Kaufman, D. D., C. Kearney and T. J. Sheets. 1964. Degradation of Simazine by soil microorganisms. Weed Sci. Soc. Am. Abstr. p. 12. Kaufman, D. D., C. Kearney and T. J. Sheets. 1965. Microbial degrada- tion of simazine. J. Agr. Food Chem. 13:238-242. Kearney, C., T. J. Sheets and J. W. Smith. 1964. Volatility of seven g-triazines. Weeds 12:83-87. Kearney, D., D. D. Kaufman and T. J. Sheets. 1965. Metabolites of simazine by Aspergillus fumigatus. J. Agr. Food Chem. 13:369-372. 85 Lamoureux, G. L., R. H. Shimabukuro, H. R. Swanson and D. S. Frear. 1970. Metabolism of 2- chloro- 4- ethylamino-6- -isopropylamino-s- triazine (atrazine) in excised sorghum leaf sections. J. Agr. Food Chem. 18: 81- 86. Lamoureux, G. L., L. E. Stafford, R. H. Shimabukuro and R. C. Zaylskie. 1973. Atrazine metabolism in sorghum: Catabolism of the gluta- thione conjugate of atrazine. J. Agr. Food Chem. 21:1020-1030. McCormick, L. L., A. E. Hiltbold. 1966. Microbial decomposition of atrazine and diuron in soil. Weeds 14:77-82. Montgomery, M. L. and V. H. Freed. 1961. The uptake, translocation and metabolism of simazine and atrazine by corn plants. Weeds 9:231-237. Moreland, D. E. 1967. Mechanism of action of herbicides. Ann. Rev. Plant Physiol. 18:365-386. Moreland, D. E., W. A. Gentner, J. L. Hilton and K. L. Hill. 1959. Studies on the mechanism of herbicidal action of 2-chloro-4.6- bis(ethylamino)-§-triazine.. Plant Physiol. 34:432-435. Palmer, R. D. and C. 0. Grogan. 1965. Tolerance of corn lines to atrazine in relation to content of benzoxazinone 2-glucoside. Weeds 13:219-222. Peabody, D. 1973. Aatrex tolerant pigweed found in Washington. Weeds Today 4:17. Pfister, K., S. R. Radosevich and C. J. Arntzen. 1979. Modification of herbicide binding to the chloroplast membranes of weed biotypes showing differential herbicide susceptibility. Weed Sci. Soc. Am. Abstr. #237. Radosevich, S. R. 1977. Mechanism of atrazine resistance in lambsquarters and pigweed. Weed Sci. 25:316-318. Radosevich, S. R. 1979. Physiological responses to triazine herbicides in susceptible and resistant weed biotypes. Weed Sci. Soc. Am. Abstr. #235. Radosevich, S. R. and A. P. Appleby. 1973. Studies on the mechanism of resistance to Simazine in common groundsel. Weed Sci. 21: 497-500. Radosevich, S. R. and 0. T. Devilliers. 1976. Studies on the mechanism of s-triazine resistance in common groundsel. Weed Sci. 24:229- 232. Ragab, M. I. H. and J. P. McCollum. 1961. Degradation of 14C-labeled simazine by plants and soil microorganisms. Weeds 9:72-84. 86 Roche, B. F. and T. M. Muzik. 1964. Ecological and physiological study of Echinochloa crusgalli L. Beauv. and response of its bio- types to sodium 2, 2- -dichloropr0pionate (dalapon). Agron. J. 56: 155— 160. Ryan, G. I. 1970. Resistance of common groundsel to simazine and atra- zine. Weed Sci. 18: 614-616. . Santlemann, P. W. and J. A. Meade. 1961. Variation in morphological characteristics and dalapon susceptibility within the species Seteria lutescens and S, faberii. Weeds 9:406-410. Schooler, A. B., A. R. Bell and J. D. Nalewaja. 1972. Inheritance of siduron tolerance tn foxtail barley. Weed Sci. 20:167-169. Sexsmith, J. J. 1964. Morphological and herbicide susceptibility dif- ferences among strains of Hoary Cress. Weeds 15:19-22. Shimabukuro, R. H. 1967. Significance of atrazine dealkylation in root and shoot of pea plants. J. Agr. Food Chem. 15:557-562. Shimabukuro, R. H. 1967. Atrazine metabolism and herbicidal selecti- vity. Plant Physiol. 42:1269-1276. Shimabukuro, R. H. N., R. E. Kadunce and D. S. Frear. 1966. Dealkyla- tion of atrazine in mature pea plants; J. Agr. 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Differential atrazine tolerance within cucumber. Weed Sci. (In press). Werner, G. M. and A. R. Putnam. 1979. 'Intraspecific differences in triazine tolerance in cucumber (Cucumis sativus) germplasm. (In preparation). Werner, G. M. and A. R. Putnam. 1979. Atrazine metabolism in a sus- ceptible and relatively tolerant cucumber cultivar. Weed Sci. (In preparation). West, L. 0., T. J. Muzik and R. I. Witters. 1976. Differential gas exchange of two biotypes of redroot pigweed to atrazine. Weed Sci. 24:68-72. Whitehead, C. W. and C. M. Switzer. 1963. The differential response of strains of wild carrot to 2,4-D and related herbicides. Can. J. Plant Sci. 43:255-262. Whiteworth, J. W. and T. J. Muzik. 1967. Differential response of selected clones of bindweed to 2,4-D. Weeds 15:275-280. "I11111111111111“