Reciprocal Antagonism between the Herbicides, Diclofop-Methyl and 2,4-D, in Corn and Soybean Tissue Culture

The antagonistic interaction between the grass herbicide, diclofopmethyl (methyl 2-[4(2′,4′-dichlorophenoxy)phenoxy]propanoate) (DM), and 2,4-dichlorophenoxyacetic acid (2,4-D), was demonstrated in DM-resistant soybean (Glycine max [L.] Merr.) and DM-susceptible corn (Zea mays L.). 2,4-D caused root shortening and thickening, and induced callus growth in soybean and corn root tissue cultures at 1 and 10 micromolar. Normal soybean root growth was unaffected by 10 micromolar DM whereas corn root growth was inhibited completely by 1 to 10 micromolar DM. DM at 10 micromolar reversed completely the induction of callus growth by 1 micromolar 2,4-D in soybean roots. In corn, 10 micromolar 2,4-D reversed the growth inhibiting activity of 1 micromolar DM and induced callus growth. The antagonistic interaction between DM and 2,4-D was reciprocal and the activity of either compound depended upon the relative concentration of the other. 2,4-D did not antagonize or decrease the activity of DM by decreasing its uptake by root tissues or increasing the rate of its detoxication. The antagonistic interaction between DM and 2,4-D probably involves involves cellular activity associated with actively growing and proliferating cells and requires the presence of both compounds at the sensitive site.     

In vitro selection for 2,4-D tolerance in red clover

Summary In vitro, selection is a viable method of selecting herbicide-tolerant crops. This research was to evaluate in vitro selection techniques for enhancing 2,4-D [(2,4-dichlorophenoxy) acetic acid] tolerance in red clover (Trifolium pratense L.). In vivo and in vitro responses to 2,4-D of eight diverse red clover populations were correlated (r=0.77), justifying in vitro selection for 2,4-D tolerance. Suspension cultures of a red clover genotype capable of regeneration were plated onto agar-based nutrient media supplemented with 0.18 mM 2,4-D for selection experiments. After two cycles of selection, 16 2,4-D tolerant callus lines were identified based on visual growth assessment. These lines were evaluated for 2,4-D tolerance (based on 2,4-D content), using a 2,4-D bioassay procedure which consisted of placing selected callus tissue pieces on top of oat (Avena sativa L.) coleoptile or internode sections. The relative amount of 2,4-D in the callus tissue was estimated by the amount of oat section elongation after 24 h. Two of the more tolerant callus lines had 61% and 83% less 2,4-D in their tissues than the susceptible control tissue. These studies indicated that in vitro selection can enhance the levels of 2,4-D tolerance in red clover callus tissue.Florida Agricultural Experiment Station Journal Series No. 8943     

Aryl hydroxylation: A selective mechanism for the herbicides, diclofop-methyl and clofop-isohutyl, in gramineous species

Esters of substituted phenoxy-phenoxy propionic acid constitute a new class of herbicides that are effective against gramineous weed and crop species. Slight changes in chemical structure alter drastically the spectrum of weeds controlled by this class of herbicides. Wheat (Triticum aestivum L.) is resistant to diclofop-methyl (methyl 2-[4-(2′,4′-dichlorophenoxy)phenoxy] propanoate) (DM) and clofop-isobutyl (iso-butyl 2-[4-(4′-phenoxy)phenoxy] propanoate) (CI), oat (Avena sativa L.) and wild oat (Avena fatua L.) are susceptible to DM but resistant to CI, and corn (Zea mays L.) is susceptible to both compounds. The antagonism of IAA-induced elongation in the coleoptile straight growth test was determined to measure biological activity of the herbicides. The basis for the differential responses by gramineous species was related to the metabolism and deioxication of the herbicides in coleoptiles. Growth of wheat coleaptiles was relatively unaffected by both compounds, oat coleoptile growth was inhibited by DM but not by CI. but corn coleoptile growth was inhibited equally by both compounds. Coleoptiles and excised shoots of the three species rapidly hydrolyzed both compounds to their respective acids (diclofop, clofop). The acids were conjugated to a water-soluble ester conjugate or they were hydroxylated in the chlorine-substituted phenyl ring and conjugated as a phenolic conjugate. Aryl hydroxylation is a detoxication mechanism in resistant plants. Plants resistant to DM or CI contained low concentrations of the parent ester and the free or bound (ester conjugate) acid and a high concentration of free or bound (phenolic conjugate) aryl hydroxylated acid in coleoptile and shoot tissues, Differential responses by the three gramineous species to DM and CI axe due apparently to differences in their detoxication mechanism. The enzyme for aryl hydroxylation in oat appears to have a higher affinity for the 4-chloro- than for the 2,4-dichloro-substituted moiety. Therefore, oat hydroxylated clofop rapidly and was tolerant to CI but the limited ability of oat to hydroxylate diclofop resulted in oat being extremely susceptible to DM.     

Influence of nutrient supply and plant growth regulators on phytotoxicity of imazamethabenz in wild oat (Avena fatua L.)

The influences of nutrient supply and plant growth regulators on the phytotoxicity of imazamethabenz in wild oat (Avena fatua L.) were evaluated in the greenhouse. Wild oat plants supplied with half-strength rather than one-eighth-strength Hoagland solution were more susceptible to imazamethabenz, showing greater growth reduction in main shoot and tillers. The improved herbicide efficacy at higher nutrient levels appeared related to increased herbicide interception by the greater leaf surface available. Leaves developing at either nutrient level did not differ significantly in epicuticular wax, so differential absorption appeared unlikely. Wild oat plants supplemented with nutrient, switching from low to high levels at the time of herbicide application, were as susceptible to imazamethabenz or even more so than plants growing with a constant high level of nutrition. The wild oat pure-line Montana 73, a strongly tillering line, was more susceptible to imazamethabenz than the limited-tillering line, Crop Science 40. Both 2,4-D and GA₃ reduced imazamethabenz-induced tillering. Imazamethabenz efficacy was increased by GA₃ but not by 2,4-D. These results support the hypothesis that lowering apical dominance of wild oat increases imazamethabenz activity in tillers, and that increased tillering following sublethal doses of imazamethabenz treatment is associated with the release of apical dominance.     

Effect of diclofop-methyl and 2,4-D on transmembrane proton gradient: a mechanism for their antagonistic interaction

The site of action of the postemergence graminicide, diclofop-methyl (DM), in susceptible plants is possibly the plasmalemma. Indole-acetic acid (IAA)- and fusicoccin (FC)-induced net proton excretion in Avena coleoptiles was inhibited by the free acid, diclofop. However, net proton excretion recovered within 2 h when 2,4-dichlorophenoxy acid (2,4-D) was added simultaneously with diclofop. Diclofop depolarized the membrane potential (Em) within 12 min but the Em recovered within 30 min when diclofop was removed and replaced with either IAA or 2,4-D. The inhibition of IAA-induced coleoptile growth by DM and the membrane effects of its acid, diclofop, were partially reversed by 2,4-D if it was added shortly after treatment of the tissue. These results are consistent with the reversal of DM injury in whole plants with 2,4-D.     

The effect of light on the production of ethylene from 1-aminocyclopropane-1-carboxylic acid by leaves

White light inhibits the conversion of 1-amino-cyclopropane-1-carboxylic acid (ACC) in discs of green leaves of tobacco (Nicotiana tabacum L.) and segments of oat (Avena sativa L.) leaves by from 60 to 90%. Etiolated oat leaves do not show this effect. The general nature of the effect is shown by its presence in both a mono- and a dicotyledon. Since the leaves have been grown and pre-incubated in light, yet can produce from 2 to 9 times as much ethylene in the dark as in the light, it follows that the light inhibition is fully reversible. The inhibition by light is about equal to that exerted in the dark by CoCl₂; it can be partly reversed by dithiothreitol and completely by mercaptoethanol. Thus the light is probably acting, via the photosynthetic system, on the SH group(s) of the enzyme system converting ACC to ethylene.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

Analytical pyrolysis-pattern recognition for the characterisation of leafy spurge (Euphorbia esula L.) biotypes

Curie-point pyrolysis-gas chromatography-pattern recognition was used to elucidate chemical variations within leafy spurge (Euphorbia spp.). Hierarchical cluster analysis (HCA) readily identified two major clusters corresponding to E. esula and E. cyparissias. The E. esula cluster further separated into three distinct subclusters. Results from principal components analysis (PCA) and fuzzy c-varieties (FCVPC) pattern recognition were similar, verifying the presence of three biotypes among the E. esula samples studied. It is suggested that analytical pyrolysis in combination with pattern recognition may predict the behaviour of biocontrol agents introduced into fields to control leafy spurge.     

2,4-Dichlorophenoxy acetic acid and indoleacetic acid partially counteract inhibition of organogenesis by difluoromethylornithine

Difluoromethylornithine (DFMO) is a well known inhibitor of putrescine biosynthesis that has been reported to interact in varying ways with auxins such as indoleacetic acid (IAA) and 2,4-dichlorophenoxy acetic acid (2,4-D). In the present report DFMO is shown to inhibit root formation in isolated hypocotyl segments of Euphorbia esula L. (leafy spurge) grown in the dark on solidified nutrient media in Petri dishes. Shoot formation was only slightly inhibited by DFMO and only on media with salts and vitamins diluted 10-fold. 2,4-D inhibited both root and shoot formation in full strength or diluted media. Simultaneous application of both compounds resulted in partial reversal of root inhibition, but only at 450 n M 2,4-D, the highest concentration used. In both media IAA also partially reversed DFMO effects on root formation. The effects of DFMO, 2,4-D or IAA on root (or shoot) formation do not appear to be closely related to endogenous content of the polyamines determined by high performance liquid chromatography.     

Effect of herbicide diclofop-methyl on proton extrusion from Lolium multiflorum seedlings differing in resistance and in fungal endophyte (Neotyphodium sp.) infection

Lolium multiflorum (Italian ryegrass), an annual weed invading wheat and barley cropping systems, has evolved resistance to diclofop-methyl (DM) herbicide. Earlier studies on the mode of action of DM in susceptible L. multiflorum and L. rigidum populations have shown that herbicide promotes oxidative stress leading to senescence, a process reversible through the action of auxins. The disruption of cell membrane potential ( E _m ) appears to be correlated with DM phytotoxicity in susceptible populations, suggesting that the continuous H⁺ extrusion from plasmalemma to extracellular space is inhibited. L. multiflorum usually establishes a symbiotic relationship with fungal endophytes of the Neotyphodium genus. This fungus confers to host plants higher survival at sublethal dosages of DM, probably due to the production of auxinic compounds. Our goal was to characterize DM-resistant and DM-susceptible L. multiflorum populations infected (E+) and non-infected (E−) with endophytes, by studying the capacity of H⁺ bumping of plasmalemma of intact roots under DM selection. We correlated the effects of DM on H⁺ disruption with plant survival. DM inhibited acidification markedly more in susceptible than in resistant populations. Continued extrusion of H⁺ by DM-resistant cell membranes was positively related to plant survival and growth. There was no detectable difference in the capacity of bumping H⁺ between DM-susceptible E+ and E− seedlings, even though survival was higher in E+ plants. The basis for the differential response in H⁺ extrusion between resistant and susceptible populations of L. multiflorum is discussed.     

Biology and host specificity of Spurgia capitigena (Bremi) (Dipt., Cecidomyiidae), for the biological control of Euphorbia esula L. in North America

Abstract: Spurgia capitigena (Bremi) was evaluated as a potential biological control agent of leafy spurge, Euphorbia esula L. (species complex) in North America. To ascertain the host specificity of this gall midge, tests were conducted in the field at Cavaillon, France, and at laboratories located in Rome, Italy and Bozeman, Montana, USA, in 1994 and 1995. Twenty-nine plant species, including eight native North American euphorbias and biotypes of E . esula , were evaluated. In the laboratory, the gall-midge was able to induce galls on seven species of spurges (all belonging to the subgenus Esula) and the three accessions of leafy spurge. In field tests, S . capitigena infested only E . esula . The biology and host specificity of S . capitigena from France appears to be similar to that observed for Spurgia esulae Gagné from Italy (= Bayeria capitigena Bremi). In France, this gall-midge was commonly observed from early May until October in habitats of very heavy and moist soils located along water channels, roadsides and in fruit orchards. Galls were induced on meristematic tissues, and thereby prevented flowering.     

Differential response of young and adult leaves to herbicide 2,4-dichlorophenoxyacetic acid in pea plants: role of reactive oxygen species

In this work the differential response of adult and young leaves from pea (Pisum sativum L.) plants to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) (23 mm) applied by foliar spraying was investigated. The concentration of 2,4-D (23 mm) and the time of treatment (72 h) were previously optimized in order to visualize its toxic effects on pea plants. Under these conditions, the herbicide induced severe disturbances in mesophyll cells structure and proliferation of vascular tissue in young leaves and increased acyl-CoA oxidase (ACX), xanthine oxidase (XOD) and lipoxygenase (LOX) activities in young leaves, and only ACX and LOX in adult leaves. This situation produced reactive oxygen species (ROS) over-accumulation favoured by the absence of significant changes in the enzymatic antioxidants, giving rise to oxidative damages to proteins and membrane lipids. An increase of ethylene took place in both young and adult leaves and the induction of genes encoding the stress proteins, PRP4A and HSP 71,2, was observed mainly in young leaves. These results suggest that ROS overproduction is a key factor in the effect of high concentrations of 2,4-D, and ROS can trigger a differential response in young and adult leaves, either epinasty development in young leaves or senescence processes in adult tissues.     

Ethylene production in habituated and auxin-requiring tobacco callus cultures. Does ethylene play a role in the habituation?

Ethylene production of habituated and auxin-requiring tobacco ( Nicotiana tabacum L. cv. Xanthi) callus cultures were compared. More ethylene was produced by auxinrequiring i.e. auxin-heterotrophic cultures than by habituated ones. Treatment with 2,4-dichlorophenoxyacetic acid increased the ethylene evolution of habituated cultures over the range 10⁻⁷ to 10⁻⁴ M , which suggests that the higher ethylene production of auxin-dependent callus is caused by the 2,4-D in the medium. The IAA levels depended on the age of both types of callus cultures.     

Identification, Pathogenicity and Comparative Virulence of Fusarium spp. Associated with Diseased Euphorbia spp. in Europe

Fusarium spp. isolated from diseased Euphorbia spp. in Europe were assessed for pathogenicity to North American accessions of leafy spurge ( Euphorbia esula/virgata ). Of the nine strains of Fusarium spp. isolated from diseased E. stepposa or E. virgata in the Caucasus region of Russia and E. esula/virgata in southern France, all were pathogenic to leafy spurge. There were significant differences in virulence among strains. Four strains, including the two that were most virulent, were identified as F. oxysporum . Four of the five other strains were identified as F. solani and one was identified as F. proliferatum . Three of the four most virulent strains to leafy spurge were isolated from E. stepposa . The most virulent strain was associated with root damage caused by insect biological control agents, as found earlier with domestic strains of Fusarium spp. pathogenic to leafy spurge. Two strains identified as F. solani were vegetatively compatible. It was concluded that further screening of a larger set of strains of foreign Fusarium spp. under quarantine conditions in the US or in limited overseas facilities would be justified, and could yield promising biological control agents for leafy spurge.     

Tocotrienols, the unsaturated forms of vitamin E, can function as antioxidants and lipid protectors in tobacco leaves

Vitamin E is a generic term for a group of lipid-soluble antioxidant compounds, the tocopherols and tocotrienols. While tocotrienols are considered as important vitamin E components in humans, with functions in health and disease, the protective functions of tocotrienols have never been investigated in plants, contrary to tocopherols. We took advantage of the strong accumulation of tocotrienols in leaves of double transgenic tobacco (Nicotiana tabacum) plants that coexpressed the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene (PDH) and the Arabidopsis (Arabidopsis thaliana) hydroxyphenylpyruvate dioxygenase gene (HPPD) to study the antioxidant function of those compounds in vivo. In young leaves of wild-type and transgenic tobacco plants, the majority of vitamin E was stored in thylakoid membranes, while plastoglobules contained mainly delta-tocopherol, a very minor component of vitamin E in tobacco. However, the vitamin E composition of plastoglobules was observed to change substantially during leaf aging, with alpha-tocopherol becoming the major form. Tocotrienol accumulation in young transgenic HPPD-PDH leaves occurred without any significant perturbation of photosynthetic electron transport. Tocotrienols noticeably reinforced the tolerance of HPPD-PDH leaves to high light stress at chilling temperature, with photosystem II photoinhibition and lipid peroxidation being maintained at low levels relative to wild-type leaves. Very young leaves of wild-type tobacco plants turned yellow during chilling stress, because of the strongly reduced levels of chlorophylls and carotenoids, and this phenomenon was attenuated in transgenic HPPD-PDH plants. While sugars accumulated similarly in young wild-type and HPPD-PDH leaves exposed to chilling stress in high light, a substantial decrease in tocotrienols was observed in the transgenic leaves only, suggesting vitamin E consumption during oxygen radical scavenging. Our results demonstrate that tocotrienols can function in vivo as efficient antioxidants protecting membrane lipids from peroxidation.     

2,4-Dichlorophenoxyacetic acid and kinetin in anther culture of cultivated and wild oats and their interspecific crosses: plant regeneration from A. sativa L.

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin was studied in anther culture of oat Avena sativa L., wild oat A. sterilis L. and progeny of crosses between them. A high 2,4-D concentration (5–6 mg l^–1) increased embryo production in genotypes of both species and promoted plant regeneration in anther cultures of A. sterilis and A. sativa×A. sterilis progeny, while kinetin caused severe browning. However, a low concentration of kinetin was essential for initiation of regenerable embryos from anther culture of A. sativa cv. Kolbu: one green and one albino plant were produced. In addition, medium containing W₁₄ salts gave higher regenerant recovery compared with medium containing Murashge and Skoog salts, when cross progeny were tested. Received: 6 March 1998 / Revised: 30 April 1998 / Accepted: 16 November 1998     

Selectivity of diclofop-methyl between wheat and wild oat: growth and herbicide metabolism

Abstract Growth of the second leaf of susceptible wild oat (Avena fatua L.) was inhibited within 2 days after treatment with the herbicide, diclofop-methyl, in the 1-1/2 leaf stage. Leaf growth of resistant wheat (Triticum aestivum L.) was unaffected by diclofop-methyl. In wild oat. chlorosis developed 1 day after leaf growth was inhibited. Foliar absorption of diclofop-methyl was similar between wild oat and wheat with 67 and 61% of the recovered radioactivity from [¹⁴C]diclofop-methyl being absorbed by wild oat and wheat, respectively, after 4 days. Wild oal was equally sensitive to the methyl ester and acid forms of the herbicide when the compounds were injected into the stem. Wheat was unaffected by both forms when treated similarly. Very little diclofop-methyl and diclofop (combined total of 10 to 12% in wild oat and 5 to 7% in wheat) remained in plant tissues 2 days after leaf treatment in both susceptible and resistant plants. Therefore, the active form of the herbicide must inhibit growth of susceptible plants very rapidly and at relatively low concentrations. Diclofop-methyl was rapidly hydrolyzed to diclofop by wild oat and wheat. Wild oat predominantly conjugated diclofop to an ester conjugate but wheat hydroxylated the 2,4-dichlorophenyl ring and formed a phenolic conjugate. The formation of the different conjugates between wild oat and wheat was the most significant difference in metabolism between the two species. Nearly 60 and 70% of the methanol-soluble radioactivity was present as water-soluble conjugates in wild oat and wheat, respectively, 4 days after treatment.     

Effect of Leafy Spurge (Euphorbia esula) Genotype on Feeding Damage and Reproduction of Aphthona spp.: Implications for Biological Weed Control

North American leafy spurge (Euphorbia esula L.) is genetically diverse and composed of multiple genotypes introduced from several areas of Europe and Asia. Five species of leafy spurge flea beetle (Aphthona spp.) have been introduced as biological control agents for leafy spurge, but were collected in a relatively small region of Europe. Greenhouse and field experiments were conducted to determine if observed variation in feeding preference and reproduction of Aphthona spp. on North American leafy spurge may be due in part to leafy spurge genotype. Leafy spurge genotypes were collected from Austria; Manitoba in Canada; and the states of Montana, Nebraska, North Dakota, South Dakota, and Wyoming in the United States. Leafy spurge genotype affected feeding but not egg laying by Aphthona spp. adults. Aphthona czwalinae/lacertosa fed slightly less in a free-choice test on a genotype from Manitoba (7%) compared to genotypes from Nebraska and North Dakota (14%). Aphthona flava tended to feed less on the Nebraska genotype than any other genotype evaluated. Reproduction of Aphthona spp. was greatly affected by leafy spurge genotype. For instance, A. czwalinae/lacertosa produced 72 adults per plant from a Nebraska genotype compared to 11 to 32 adults per plant from all other genotypes evaluated. Thus, some observed variation in establishment and reproduction of Aphthona spp. can be attributed to leafy spurge genotype.     

Modeling Responses of Leafy Spurge Dispersal to Control Strategies

Leafy spurge （Euphorbia esula L.） has substantial negative effects on grassland biodiversity, productivity, and economic benefit in North America. To predict these negative impacts, we need an appropriate plant-spread model which can simulate the response of an invading population to different control strategies. In this study, using a stochastic map lattice approach we generated a spatially explicitly stochastic process-based model to simulate dispersal trajectories of leafy spurge under various control scenarios. The model integrated dispersal curve, propagule pressure, and population growth of leafy spurge at local and short-temporal scales to capture spread features of leafy spurge at large spatial and long-temporal scales. Our results suggested that narrow-, medium-, and fat-tailed kernels did not differ in their ability to predict spread, in contrast to previous works. For all kernels, Allee effects were significantly present and could explain the lag phase （three decades） before leafy spurge spread accelerated. When simulating from the initial stage of introduction, Allee effects were critical in predicting spread rate of leafy spurge, because the prediction could be seriously affected by the low density period of leafy spurge community. No Allee effects models were not able to simulate spread rate well in this circumstance. When applying control strategies to the current distribution, Allee effects could stop the spread of leafy spurge; no Allee effects models, however, were able to slow but not stop the spread. The presence of Allee effects had significant ramifications on the efficiencies of control strategies. For both Allee and no Allee effects models, the later that control strategies were implemented, the more effort had to be input to achieve similar control results.     

Attack behavior and host utilization ofAphthona chinchihi (Col.: Chrysomelidae), a potential biological control agent ofEuphorbia esula (Euphorbiaceae, leafy spurge) in North America

Aphthona chinchihi Chen was collected in China feeding on leafy spurge (Euphorbia esula L.). Studies were conducted on its host specificity in the laboratory, using field collected adults and their progeny.Aphthona chinchihi can effectively complement the impact of the other natural enemies of leafy spurge established from Europe in the U.S.A. and Canada. The adults feed on leaves and shoots and the larvae, which cause the main damage to the plant, feed on the hypogeous portion of the plant, seriously stressing the plant and preventing its vegetative spread. The host range ofA. chinchihi was studied with tests on adult feeding and oviposition, larval survival and host suitability, using 40 plant species or varieties distributed in 12 families. The experiments demonstrated that it has a high level of specificity. This flea beetle completed its life cycle only on leafy spurge. Also, because of its ecological valence,A. chinchihi has a very good potential as a biocontrol agent in North America.