Influence du piclorame sur l'activité phénylalanine-ammoniac lyase de Nicotiana tabacum induite par la réaction d'hypersensibilité au virus de la mosaïque du tabac

In Nicotiana tabacum var. Xanthi n.c. phenylalanine ammonia lyase (PAL) activity, which increases significantly during the hypersensitivity reaction to tobacco mosaic virus (TMV), is only partially affected when plants are treated with piclorame, which is known to be an inhibitor of PAL in vivo. Using piclorame together with TMV, it has been possible to distinguish between that increase in PAL activity due to the virus and that dependent on the photoperiod.     

Atrazine metabolism and herbicidal selectivity

Metabolism of the herbicide 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine) was investigated in resistant corn (Zea mays L.) and sorghum (Sorghum vulgare Pers.), intermediately susceptible pea (Pisum sativum L.), and highly susceptible wheat (Triticum vulgare Vill.) and soybean (Glycine max Merril.). This study revealed that 2 possible pathways for atrazine metabolism exist in higher plants. All species studied were able to metabolize atrazine initially by N-dealkylation of either of the 2 substituted alkylamine groups. Corn and wheat, which contain benzoxazinone, also metabolized atrazine initially by hydrolysis in the 2-position of the s-triazine ring to form hydroxyatrazine. Subsequent metabolism by both pathways resulted in the conversion of the parent atrazine to more polar compounds and eventually into methanol-insoluble plant residue. No evidence for s-triazine ring cleavage was obtained.

Both pathways for atrazine metabolism appear to detoxify atrazine. The hydroxylation pathway results in a direct conversion of a highly phytotoxic compound to a completely non-phytotoxic derivative. The dealkylation pathway leads to detoxication through one or more partially detoxified, stable intermediates. Therefore, the rate and pathways of atrazine metabolism are important in determining the tolerance of plants to the herbicide. Both quantitative and qualitative differences in atrazine metabolism were detected between resistant, intermediately susceptible, and susceptible species. The ability of plants to metabolize atrazine by N-dealkylation and the influence of this pathway in determining tolerance of plants to atrazine are discussed.

     

A plant selectable marker gene based on the detoxification of the herbicide dalapon

Summary A gene from Pseudomonas putida coding for a dehalogenase capable of degrading 2,2 dichloropropionic acid (2,2DCPA), the active ingredient of the herbicide dalapon, has been isolated and characterised. In plant transformation experiments the gene was shown to confer resistance to 2,2DCPA at a tissue culture level where 2,2DCPA could be used to select for transformants. At the whole plant level, transformed plants showed resistance to 2,2DCPA at concentrations up to 5 times the recommended dose rate of dalapon when it was sprayed on their leaves. At lower concentrations, the herbicide caused a non-lethal yellowing of sensitive plants which clearly distinguished them from resistant plants. The mode of action of chlorinated aliphatic acids is not known but they probably affect many enzyme pathways. The results described here are the first example of engineering a plant resistant to a herbicide that does not have one specific enzyme as its target site. This gene has several advantages as a marker in plant breeding and genetic studies. For example, the herbicide is readily available and has low toxicity, transformants can be selected at both the tissue culture and the whole plant level, a large number of transformed plants can easily be screened even in the field, and there is a very low probability of selecting spontaneous mutants.Abbreviations 2MCPA 2, monochloropropionic acid - 2,2DCPA 2,2 dichloropropionic acid - TCA trichloroacetic acid     

Comparing Metabolic Functionalities,Community Structures,and Dynamics of Herbicide-Degrading Communities Cultivated with Different Substrate Concentrations

Two 4-chloro-2-methylphenoxyacetic acid (MCPA)-degrading enrichment cultures selected from an aquifer on low (0.1 mg liter⁻¹) or high (25 mg liter⁻¹) MCPA concentrations were compared in terms of metabolic activity, community composition, population growth, and single cell physiology. Different community compositions and major shifts in community structure following exposure to different MCPA concentrations were observed using both 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and pyrosequencing. The communities also differed in their MCPA-mineralizing activities. The enrichments selected on low concentrations mineralized MCPA with shorter lag phases than those selected on high concentrations. Flow cytometry measurements revealed that mineralization led to cell growth. The presence of low-nucleic acid-content bacteria (LNA bacteria) was correlated with mineralization activity in cultures selected on low herbicide concentrations. This suggests that LNA bacteria may play a role in degradation of low herbicide concentrations in aquifers impacted by agriculture. This study shows that subpopulations of herbicide-degrading bacteria that are adapted to different pesticide concentrations can coexist in the same environment and that using a low herbicide concentration enables enrichment of apparently oligotrophic subpopulations.     

Alterations in peroxidase activity and phenylpropanoid metabolism induced by Nacobbus aberrans Thorne and Allen, 1944 in chilli (Capsicum annuum L.) CM334 resistant to Phytophthora capsici Leo.

Chilli CM334 (Capsicum annuum L.) is resistant to Phytophthora capsici Leonian (Pc), but Nacobbus aberrans Thorne and Allen, 1944 (Na) broke down its resistance in plants previously infected by the nematode. Peroxidase (POD) and L-phenylalanine ammonia-lyase (PAL) activity, total soluble phenols (TSP) and chlorogenic acid concentration in CM334 plants inoculated with either or both pathogens (Na-Pc) were compared; also, the toxic effect of some phenolic acids on Na was tested in vitro. The highest POD activity (5.3 μM tetraguaiacol mg^?1 protein min^?1) was registered in plants inoculated only with Pc, while those inoculated only with Na showed the lowest (3.3 μM) (P?≤?0.05). PAL activity was 39.9 nM trans-cinnamic acid μg^?1 protein min^?1 in plants inoculated only with Pc, and it was lower (19.3 nM) and similar in non-inoculated plants or those with Na and with Na-Pc (P?≤?0.05). Usually, plants inoculated with Pc alone had higher contents of TSP (P?≤?0.05) (1.9 mg tannic acid g^?1 dry matter) and plants inoculated with Na or Na-Pc had lower levels (0.8 and 0.9 mg) than those non-inoculated (1.3 mg). CM334 plants inoculated with Na showed a significant reduction (10–37% and 12–17%, in roots and leaves) in the concentration of chlorogenic acid as compared to the non-inoculated. Vanillic, trans-cinnamic, p-coumaric and syringic acids had greater nematicidal effects (P?≤?0.05) than chlorogenic acid in vitro. Apparently Na modified the defence responses in CM334 plants as POD and PAL activities and TSP and chlorogenic acid concentrations were reduced.     

The influence of form of deposit on the phytotoxicity of MCPA, paraquat and glyphosate applied as individual drops

MCPA, paraquat and glyphosate were applied as individual drops (200–400 μm) to pot-grown plants of radish (Raphanus sativus) or wild oat (Avena fatua), using concentrations appropriate to very low volume applications of these herbicides. For a given dose per plant, herbicide activity was unaffected by concentration of MCPA or paraquat but was enhanced as the concentration of glyphosate was increased. The activity of all three herbicides on both species was affected by variation of the site of application but not by drop size. On radish the greatest activity resulted when paraquat was applied to the cotyledons, glyphosate to the interveinal areas of true leaves and MCPA to the veins of true leaves. This is discussed in relation to herbicide mobility and local toxicity following applications at high concentration.     

Potassium influx and efflux of 2,4-D- and MCPA-treated rice plants

Summary A study was made of the effects of the herbicides 2,4-D (2,4-dichlorophenoxyacetic acid) and MCPA (4-chloro-2-methyl-phenoxyacetic acid) on ion uptake, leakage and growth of rice seedlings. Using isotopically-labelled solutions containing different concentrations of 2,4-D or MCPA, it was established that 10^–4 M 2,4-D or MCPA effectively inhibited potassium ion uptake, while K-ion leakage from the roots occurred only at 10^–3 M. The growth of the rice seedlings was markedly retarded even at low (10^–6 M) concentrations, and the roots and shoots showed different tolerances to the herbicide. At 10^–8 M herbicide, the effects were not injurious, but rather favourable. Reduction in root length by herbicides was not in accordance with dry-matter production.     

Atrazine Resistance in a Velvetleaf (Abutilon theophrasti) Biotype Due to Enhanced Glutathione S-Transferase Activity

We previously reported that a velvetleaf (Abutilon theophrasti Medic) biotype found in Maryland was resistant to atrazine because of an enhanced capacity to detoxify the herbicide via glutathione conjugation (JW Gronwald, Andersen RN, Yee C [1989] Pestic Biochem Physiol 34: 149-163). The biochemical basis for the enhanced atrazine conjugation capacity in this biotype was examined. Glutathione levels and glutathione S-transferase activity were determined in extracts from the atrazine-resistant biotype and an atrazine-susceptible or “wild-type” velvetleaf biotype. In both biotypes, the highest concentration of glutathione (approximately 500 nanomoles per gram fresh weight) was found in leaf tissue. However, no significant differences were found in glutathione levels in roots, stems, or leaves of either biotype. In both biotypes, the highest concentration of glutathione S-transferase activity measured with 1-chloro-2,4-dinitrobenzene or atrazine as substrate was in leaf tissue. Glutathione S-transferase measured with 1-chloro-2,4-dinitrobenzene as substrate was 40 and 25% greater in leaf and stem tissue, respectively, of the susceptible biotype compared to the resistant biotype. In contrast, glutathione S-transferase activity measured with atrazine as substrate was 4.4- and 3.6-fold greater in leaf and stem tissue, respectively, of the resistant biotype. Kinetic analyses of glutathione S-transferase activity in leaf extracts from the resistant and susceptible biotypes were performed with the substrates glutathione, 1-chloro-2,4-dinitrobenzene, and atrazine. There was little or no change in apparent K_m values for glutathione, atrazine, or 1-chloro-2,4-dinitrobenzene. However, the V_max for glutathione and atrazine were approximately 3-fold higher in the resistant biotype than in the susceptible biotype. In contrast, the V_max for 1-chloro-2,4-dinitrobenzene was 30% lower in the resistant biotype. Leaf glutathione S-transferase isozymes that exhibit activity with atrazine and 1-chloro-2,4-dinitrobenzene were separated by fast protein liquid (anion-exchange) chromatography. The susceptible biotype had three peaks exhibiting activity with atrazine and the resistant biotype had two. The two peaks of glutathione S-transferase activity with atrazine from the resistant biotype coeluted with two of the peaks from the susceptible biotype, but peak height was three- to fourfold greater in the resistant biotype. In both biotypes, two of the peaks that exhibit glutathione S-transferase activity with atrazine also exhibited activity with 1-chloro-2,4-dinitrobenzene, with the peak height being greater in the susceptible biotype. The results indicate that atrazine resistance in the velvetleaf biotype from Maryland is due to enhanced glutathione S-transferase activity for atrazine in leaf and stem tissue which results in an enhanced capacity to detoxify the herbicide via glutathione conjugation.     

Glutathione conjugation: atrazine detoxication mechanism in corn

Glutathione conjugation (GS-atrazine) of the herbicide, 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine) is another major detoxication mechanism in leaf tissue of corn (Zea mays, L.). The identification of GS-atrazine is the first example of glutathione conjugation as a biotransformation mechanism of a pesticide in plants. Recovery of atrazine-inhibited photosynthesis was accompanied by a rapid conversion of atrazine to GS-atrazine when the herbicide was introduced directly into leaf tissue. N-De-alkylation pathway is relatively inactive in both roots and shoots. The nonenzymatic detoxication of atrazine to hydroxyatrazine is negligible in leaf tissue. The hydroxylation pathway contributed significantly to the total detoxication of atrazine only when the herbicide was introduced into the plant through the roots. The metabolism of atrazine to GS-atrazine may be the primary factor in the resistance of corn to atrazine.     

Effects of atrazine on the assimilation of inorganic nitrogen in cereals

The inclusion of sub-lethal amounts ofthe herbicide atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] in the nutrient solution supplied to maize and barley increased the growth of the root and shoot and the uptake of nitrate. The activities of nitrate and nitrite reductases, glutamine synthetase and glutamate synthase were enhanced and the amino acid and nitrate contents of the xylem sap increased. All these effects of atrazine were found only in plants grown with nitrate as the nitrogen source. The uptake of ¹⁵NO₃^? and its incorporation into protein in the root and shoot of maize and barley seedlings was significantly greater in the atrazine treated plants. However, a stimulation in the incorporation of leucine-[¹⁴C] into TCA-precipitable protein of detached leaves from 7-day-old barley seedlings was obtained only in the absence of a supply of combined nitrogen either in the culture medium or in the in vitro incubation mixture containing the labelled amino acid.     

A new concept for reduction of diffuse contamination by simultaneous application of pesticide and pesticide-degrading microorganisms

Pesticide residues and their transformation products are frequently found in groundwater and surface waters. This study examined whether adding pesticide-degrading microorganisms simultaneously with the pesticide at application could significantly reduce diffuse contamination from pesticide use. Degradation of the phenoxyacetic acid herbicides MCPA (4-chloro-2-methylphenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) was studied in soil microcosm experiments after simultaneous spraying of herbicide and herbicide-degrading bacteria on an agricultural soil and on a sand with low degradation potential. The latter represented pesticide use on non-agricultural soils poor in microbial activity. Degradation and possible loss of herbicidal effect were also tested in a system with plants and the amounts of bacteria needed to give satisfactory MCPA-degradation rate and the survival of degrading bacteria in formulated MCPA were determined. The results showed >80–99% degradation of 2,4-D and MCPA in soil within 1 day and >99% within 3 days after inoculation with 10⁵–10⁷ herbicide-degrading bacteria g⁻¹ dry weight of soil. Enhanced degradation of MCPA was also obtained in the presence of winter wheat and white mustard without loss of the intended herbicidal effect on white mustard. The survival of an isolated MCPA-degrading Sphingomonas sp. in three realistic concentrations of formulated MCPA was very poor, showing that in practical applications direct contact between the microorganisms and the pesticide formulation must be precluded. The applicability and economic feasibility of the method and the information needed to obtain a useable product for field use are discussed.     

Effects of metribuzin herbicide on nitrogen,pigments, protease and nitrate reductase activity of normal and NaCl-stressed castor bean and maize plants

Addition of 0.5 and 2.5 gm^?3 of metribuzin into Hoagland nutrient media, either alone or in combination with NaCl, induced significant decreases in nitrate-, amino-, ammonia-, and total soluble-N contents, whereas significant increases in these nitrogen fractions were apparent in maize and castor bean seedlings and plants treated with high concentrations (5 and 10 g m^?3) of the herbicide, again either alone or in combination with NaCl. Protein- and total-N contents increased and decreased at low and high concentrations of the herbicide, respectively. The contents of chlorophyllsa andb, as well as carotenoids of both castor bean and maize seedlings and plants treated with low concentration of herbicide, either alone or supplemented with NaCl, were unaffected, whereas at high concentrations of the herbicide a significant decrease in chloroplast pigments was found. Nitrate reductase activity (NRA) was increased significantly at low concentrations of the herbicide alone and decreased significantly at high levels. Inclusion of NaCl into the herbicide media induced significant decreases in NRA of both castor bean and maize seedlings and plants. Unlike NRA changes, protease activity was increased significantly with high concentrations (5 and 10 g m^?3) of metribuzin and decreased significantly with its low (0.5 and 2.5 g m^?3) concentrations.     

Selection of atrazine-resistant plants by <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

The effects of atrazine on cotyledon cultures of Capsicum annuum (L.) cv. G₄ were investigated with a view of establishing a system for in vitro selection of resistant mutants. At low levels of herbicide produced little growth inhibition, some chlorophyll loss occurred associated with the production of albino shoots. At 20 mg l⁻¹ atrazine bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenized cotyledon explants resulted in production of herbicide-resistant plants on medium containing selective levels of sucrose (0.5%) and atrazine (20 mg l⁻¹). Differential morphogenetic responses were observed when the levels of sucrose (0.5–5%) were altered. Shoot regeneration was maximum in 2 sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3%–5%). However, lowering of sucrose concentration from 2 to 0.5% caused complete bleaching of explants and permitted the selection of herbicide-resistant plants. Complete atrazine-resistant plantlets were obtained after rooting of regenerated green shoots on rooting medium containing 10 mg l⁻¹atrazine, 1.0 mg l⁻¹IAA and 0.5% sucrose. Leaf-segment assay of differentiated plants revealed that all regenerants were resistant to the atrazine. Reciprocal crosses between atrazine-resistant and -sensitive plants showed a non-Mendelian transmission of resistance trait.     

Effect of herbicide residues in a sandy loam on the growth,nodulation and nitrogenase activity (C2H2/C2H4) of Trifolium subterraneum

The herbicides 2,4-D, amitrole, atrazine, diclofop-methyl, diquat, paraquat and trifiluralin were applied at rates of 0, 2, 5 and 10 μg ai. g⁻¹ to a sandy loam soil and allowed to degrade for 120 days. After this period, subterranean clover seedlings were transplanted into treated soil and the effect of herbicide residues on plant growth, number of nodules formed and nitrogenase activity was investigated. At all rates of atrazine and chlorsulfuron, and at all rates of amitrole in excess of 2 mg ai g⁻¹ of soil, sufficient herbicide remained to be lethal to the seedlings. When amitrole was applied at the rate of 2 mg ai g⁻¹ of soil, plant growth, nodulation and nitrogenase activity of plants were reduced. Residues of diquat reduced all plant parameters studied while, residues of 2,4-D reduced plant growth and nodule formation, but plant nitrogenase activity was unaffected. Residues of trifluralin had no effect on plant growth parameters but the number of nodules formed per plant was reduced. Residues of paraquat and diclofop-methyl had no effect on any of the plant parameters studied.     

Modification of Herbicide Binding to Photosystem II in Two Biotypes of Senecio vulgaris L

The present study compares the binding and inhibitory activity of two photosystem II inhibitors: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron [DCMU]) and 2-chloro-4-(ethylamine)-6-(isopropyl amine)-S-triazene (atrazine). Chloroplasts isolated from naturally occurring triazine-susceptible and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.) showed the following characteristics. (a) Diuron strongly inhibited photosynthetic electron transport from H₂O to 2,6-dichlorophenolindophenol in both biotypes. Strong inhibition by atrazine was observed only with the susceptible chloroplasts. (b) Hill plots of electron transport inhibition data indicate a noncooperative binding of one inhibitor molecule at the site of action for both diuron and atrazine. (c) Susceptible chloroplasts show a strong diuron and atrazine binding (¹⁴C-radiolabel assays) with binding constants (K) of 1.4 × 10⁻⁸ molar and 4 × 10⁻⁸ molar, respectively. In the resistant chloroplasts the diuron binding was slightly decreased (K = 5 × 10⁻⁸ molar), whereas no specific atrazine binding was detected. (d) In susceptible chloroplasts, competitive binding between radioactively labeled diuron and non-labeled atrazine was observed. This competition was absent in the resistant chloroplasts.     

Genetic mapping of non-target-site resistance to a sulfonylurea herbicide (Envoke®) in Upland cotton (Gossypium hirsutum L.)

Acetolactate synthase (ALS) is responsible for a rate-limiting step in the synthesis of essential branched-chain amino acids. Resistance to ALS-inhibiting herbicides, such as trifloxysulfuron sodium (Envoke^®), can be due to mutations in the target gene itself. Alternatively, plants may exhibit herbicide tolerance through reduced uptake and translocation or increased metabolism of the herbicide. The diverse family of cytochrome P450 proteins has been suggested to be a source of novel herbicide metabolism in both weed and crop plants. In this study we generated a mapping population between resistant and susceptible cotton (Gossypium hirsutum L.) cultivars. We found that both cultivars possess identical and sensitive ALS sequences; however, the segregation of resistance in the F2 progeny was consistent with a single dominant gene. Here we report the closely linked genetic markers and approximate physical location on chromosome 20 of the source of Envoke herbicide susceptibility in the cotton cultivar Paymaster HS26. There are no P450 proteins in the corresponding region of the G. raimondii Ulbr. genome, suggesting that an uncharacterized molecular mechanism is responsible for Envoke herbicide tolerance in G. hirsutum. Identification of this genetic mechanism will provide new opportunities for exploiting sulfonylurea herbicides for management of both weeds and crop plants.     

Effect of plant stage, <Emphasis Type="Italic">Colletotrichum truncatum</Emphasis> dose,and use of herbicide on control of <Emphasis Type="Italic">Matricaria perforata</Emphasis>

Colletotrichum truncatum (Ct) was examined in a tank mix with the herbicide 2,4-D, clopyralid plus MCPA (Caurtail M^®), or metribuzin (Sencor^®) for control of scentless chamomile at 8- (younger) and 11-leaf stages (older) under controlled conditions. In initial trials, Ct at 7 × 10⁶ spores/ml (200 l/ha) reduced the fresh weight of scentless chamomile only slightly. However, its combinations with herbicides improved the efficacy variably depending on the herbicide used and stage of the weeds. Ct plus 2,4-D reduced the fresh weight by about 50% at both leaf stages of scentless chamomile when compared to untreated controls but no plants were killed. The fungus plus Curtail M consistently killed younger but not older plants, and the efficacy was substantially greater than that of the herbicide alone. The herbicide Sencor was highly effective on younger plants, and adding Ct did not achieve additional benefits. On older plants, however, Ct plus Sencor was substantially more effective than the herbicide alone, causing 76% fresh-weight reduction when compared to controls and killing 9 out of 16 older plants in four trials. Sencor applied alone reduced the fresh weight of older plants by 65%, but no plants were killed. Tested at doses ranging from 2 × 10⁶ to 20 × 10⁶ spores/ml, Ct plus Curtail M was most effective at the highest fungal inoculum dose, consistently killing younger but not older plants. In comparison, Ct at a medium dose (7 × 10⁶ spores/ml) plus Sencor killed the majority of older chamomile plants (7 out of 12), whereas the herbicide alone did not cause plant mortality. Further increasing fungal inoculum dose from this medium level did not enhance the weed control by Ct plus Sencor.     

Characterization of Naturally Occurring Atrazine-Resistant Isolates of the Purple Non-Sulfur Bacteria

Six isolates of the purple non-sulfur bacteria, which upon primary isolation were naturally resistant to the herbicide atrazine, were characterized with respect to their taxonomic identity and the mechanism of their resistance. On the basis of electron microscopy, photopigment analysis, and other criteria, they were identified as strains of Rhodopseudomonas acidophila, Rhodopseudomonas palustris, or Rhodocyclus gelatinosus. These isolates exhibited degrees of atrazine resistance which ranged from 1.5 to about 4 times greater than that of cognate reference strains (American Type Culture Collection) tested. Furthermore, all of the reference strains tested were more intrinsically resistant to atrazine than was Rhodobacter sphaeroides. No unique plasmids which might encode for herbicide degradation or inactivation were found in these isolates. Resistance to the herbicide in these isolates was not the result of diminished binding of the herbicide to the L subunit of the bacterial reaction center. Differences in herbicide resistance among the various species of this group may be the result of compositional and chemical differences in the individual reaction centers. However, the increase in atrazine resistance for the isolates characterized in this study probably occurs by undefined mechanisms and not necessarily by changes in the binding of the herbicide to the L subunit of the photosynthetic reaction center.