Detached Leaf Culture

Abstract

To study the viability of detached leaf culture technique, studies were carried out with detached leaves from cotton apex (true trilobed leaves). The prepared leaves were sprayed with 2,4-D amine and ester, at rates of 10, 30, 70, and 100% of the recommended doses. Detached leaves without herbicide spray were used as controls. Simultaneously, a greenhouse experiment was conducted with the same treatments as used for the detached leaves experiment. Toxicity was measured through a 0-to-5 grading according to the percentage of affected leaf area in the detached leaves experiment or examining the affected rate of whole plant as indicated in the greenhouse. Results showed that the ester form of the herbicide induced earlier and more severe toxicity symptoms in detached leaves and greenhouse grown plants. Positive and significant correlations (p < 0.001) were found between toxicity results obtained at 7 and 14 days after application in detached leaves and greenhouse plants (r = 0.97 and 0.92, respectively). Negative, significant correlations (p < 0.005) were found between the toxicity levels found at 7 and 14 days after application in detached leaves and dry matter of cotton plants grown in the greenhouse (r = ?0.92 and ?0.92, respectively).     

Biological responses of wheat (Triticum aestivum) plants to the herbicide chlorotoluron in soils

Chlorotoluron is a phenylurea herbicide that is widely used for controlling grass weeds in the land of cereal, cotton and fruit production. However, extensive use of this herbicide may lead to its accumulation in ecosystems, thus inducing the toxicity to crops and vegetables. To assess chlorotoluron-induced toxicity in plants, we performed the experiment focusing on the metabolic adaptation of wheat plants (Triticum aestivum) to the chlorotoluron-induced oxidative stress. The wheat plants were cultured in the soils with chlorotoluron at concentrations of 0-25mg/kg. Chlorotoluron accumulation in plants was positively correlated with the external chlorotoluron concentrations, but negatively with the plant growth. Treatment with chlorotoluron induced the accumulation of O(2)(-) and H(2)O(2) in leaves and resulted in the peroxidation of plasma membrane lipids in the plant. We measured the endogenous proline level and found that it accumulated significantly in chlorotoluron-exposed roots and leaves. To understand the biochemical responses to the herbicide, activities of the antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were assayed. Analysis of SOD activity by non-denaturing polyacrylamide gel electrophoresis (PAGE) revealed that there were three isoforms in the roots and leaves, but the isoforms in the tissues showed different patterns. Also, using the native PAGE, 6 isoforms of root POD and 10 in leaves were detected. The total activity of POD in roots was significantly enhanced. Activities of APX in roots and leaves showed a similar pattern. The CAT activities were generally suppressed under the chlorotoluron exposure.     

Transient effect of the herbicide flazasulfuron on carbohydrate physiology in Vitis vinifera L

In the Champagne vineyard, most of the areas treated in early 2000 with the newly approved herbicide flazasulfuron had vines with altered growth and yellow leaves throughout the growing season. In order to clarify the physiological perturbations caused on the non-target grapevine and their potential consequences, C nutrition of grape plants grown in vineyards treated or not with flazasulfuron in 2000 was characterized during the following season. Vines from treated areas exhibited yellow leaves and an alteration of photosynthetic activity, characterized by declines in leaf gas exchanges (by 85%) and photosynthetic pigment concentrations (by 88%), and a marked disorganization of the leaf plastids. The herbicide also caused a decrease in leaf starch and soluble carbohydrate levels (-74% and -90%, respectively). Surprisingly, some vines re-greened after bloom, then exhibiting similar carbohydrate physiology to those grown in a non-treated area. Thus, recovery of CO(2) fixation rates, plastid ultra-structure, pigment concentrations and carbohydrate levels was found in re-greening leaves. Unlike the informations available in the literature, our results showed that flazasulfuron may be phytotoxic for grapevine. However, this toxicity was overcome the following year, indicating that vines have the potential to recover from this herbicide stress after one season.     

Absorption and translocation of 2,4-D in plants of Memora peregrina

The objective of this work was to evaluate absorption and translocation of the herbicide 2,4-D in plants of Memora peregrina. The herbicide 2,4-D was used alone with the formulation DMA 806 BR and associated with the herbicide picloram in the commercial product Padron. Levels of radioactivity on the treated leaves were determined in sample obtained after washing them with methanol and chloroform at different times after the application of the radiolabelled formulation (1, 2, 4, 8, 24, and 48 h). Translocation was evaluated by cutting plants between stem and root. The parts obtained were: root, stem, leaf treated, leaves above the leaf treated, leaves below the leaf treated, and leaf opposite of the leaf treated. These parts were weighted, dried, ground, burnt, and radioactivity in the samples was determined. The results suggest that the translocation of the radioactive herbicide 2,4-D was insignificant in plants of M. peregrina in the two treatments evaluated. Absorption of 14C 2,4-D in the treatment with DMA 806 BR and the mixture of DMA 806 BR plus Padron had the same behavior. These observations explain the inefficient control obtained with this herbicide in plant species under study.     

Boron toxicity in the rare serpentine plant, Streptanthus morrisonii

The release of boron-laden mist from the cooling towers of some geothermal power stations in northern California potentially threatens nearby populations of the rare serpentine plant, Streptanthus morrisonii F. W. Hoffm. To assess the tolerance of S. morrisonii to high levels of boron, the effect of boron on leaf condition, life history, germination rate, growth rate, allocation and photosynthesis was measured on plants grown in a greenhouse. Relative to other species, S. morrisonii was tolerant of excess boron. On serpentine soil, mild to moderate toxicity symptoms (older leaves exhibiting chlorosis and necrosis, but few leaves killed) were apparent when the boron concentration in applied nutrient solutions was 240-650 microm. Severe toxicity symptoms (significant leaf loss, young leaves with toxicity symptoms) were apparent when the applied solution was over 1000 microm boron. Above 1000 microm boron, S. morrisonii appeared unable to complete its life cycle. On a tissue basis, boron toxicity was first observed when leaf boron content was 40-90 micromol g(-1) dry weight. In leaves with severe boron toxicity (> 35% injury), the boron content was generally above 130 micromol g(-1) dry weight. These levels were an order of magnitude above the tissue boron content of plants in the field. Prior to the onset of pronounced boron toxicity symptoms, growth rate, allocation patterns, and photosynthesis were unaffected by high boron. These results indicate that inhibition of growth and photosynthesis occurred because of a loss of viable tissue due to boron injury, rather than a progressive decline as leaf boron levels increased.     

Selectivity of the herbicide flazasulfuron applied after postemergence in sugarcane (Saccharum spp. L.) crop

The selectivity of the herbicide flazasulfuron was evaluated when applied at two rates (50 and 100 g/ha) with and without surfactants (Aterbane or Agral at 0.2% v/v). The treatment was applied at early (three leaves) and late (five to six leaves) stages of the postemergence of sugarcane plants (var. RB845257) grown in two soils (sandy and clay) with good moisture status before and after application. Despite the toxicity symptoms, especially at the late stage with the higher rate of application in the sandy soil, the herbicide did not affect the growth nor the stalk yield. The presence of the surfactants had no effect on the toxicity symptoms.     

Fate of imidacloprid in soil and plant after application to cotton seeds

This study aimed to investigate the persistence of imidacloprid in soil after application to cotton seeds and to obtain a complete picture on the mass balance of this compound in soil and cotton plants. The study was carried out as a pot culture experiment under laboratory conditions using a Gaucho formulation containing ¹⁴C-labeled imidacloprid. Three treatments of cotton seeds were made in sandy loamy soil: live seeds grown in autoclaved soil, dead seeds put in live soil and live seeds grown in live soil. Results showed that total ¹⁴C recoveries decreased by time ranging 93.8–96.2, 77.1–88.4 and 53.5–62.4% of the applied radioactivity at 7, 14, and 21 d after application, respectively. The reduction in the extracted ¹⁴C from soil coincided with the increase of non-extracted ones. Levels of bound ¹⁴C was always less in autoclaved soil than in live ones. Results revealed also that only 1.8–6.8% of the applied ¹⁴C was taken up by the plants and fluctuated within the test period. ¹⁴C levels were higher in plants grown in autoclaved soil than those in live ones and the radioactivity tended to accumulate on the edges of cotton leaves. Most of the radioactivity in the soil extracts was identified as unchanged ¹⁴C-imidacloprid.     

Phytotoxicity to and uptake of RDX by rice

Phytoremediation is an emerging strategy to remediate soils contaminated with pollutants like explosives in which plants will uptake, degrade and/or accumulate pollutants. To implement this technology on a site contaminated with RDX, we chose rice, which is able to grow in lagoons, and we tested its ability to grow in soils with high levels of RDX and to decrease RDX concentrations in soil. Rice was grown for 40 days in soil contaminated with increasing [14C]RDX concentrations. Emergence and growth were not affected by RDX. Total chlorophyll content decreased with RDX concentrations of over 500 mg kg(-1). Amounts of chlorophyll were correlated with the appearance of necrosis in leaf extremities. After 40 days, rice translocated 89% of uptaken radioactivity to leaves with 90% in leaf extremities. Analyzes of leaf extracts showed that 95% of radioactivity was RDX in its parent form. Necrosis appears to be a phytotoxic symptom of RDX accumulation.     

Vapour of the free acid of the herbicide 2,4-D is toxic to tomato and lettuce plants

Tomato and lettuce plants were exposed to vapour of the free acid of [14C-phenyl] 2,4-D at concentrations in the range 1-600 pg litre(-1) for periods of 6, 24 or 72 h. The rate of uptake of radiolabel by tomato was about twice that by lettuce at the same vapour concentration. Uptake rates were linearly related to external vapour concentration. The relationship between uptake and vapour concentration of 2,4-D for the two species was similar to published values for the butyl and iso-octyl esters. The distribution of herbicide residue in the plant immediately after exposure indicated that the apical leaves of lettuce are particularly active in assimilating vapour, whereas for tomato, leaf position had no influence. Forty days after exposure, both species showed symptoms of toxicity and reduction in shoot dry weight typical of similar doses of 2,4-D esters. It is concluded that the vapour of 2,4-D represents a potential hazard to susceptible plants, and that further work is needed to determine the conditions likely to lead to the production of vapour of the free acids of phenoxyalkanoic herbicides following spraying.     

Chronic ozone exposure alters the growth of leaves, stems and roots of hybrid Populus

Rooted cuttings of hybrid Populus (DN34, Populus deltoides x nigra) were grown outdoors in pots in open-top chambers at Ithaca, NY (74.5 degrees W, 42.5 degrees N), during 1988 and 1989 (experiment 1) and during 1989 and 1990 (experiment 2). Ambient air was passed through charcoal filters to produce a 0.5 times ambient ozone treatment, and ozone generated from oxygen was added to produce one and two times ambient ozone treatments. In experiment 1, treatments were applied for 8-12 h each day for 112 days of the 1988 growing season, then the plants were grown outdoors with ambient ozone in 1989. In experiment 2, treatments were applied for 9 h each day for 98 days of the 1989 growing season, then the plants were grown outdoors with ambient ozone in 1990. Chronic exposure to ozone caused the following changes (statistically significant in one or both experiments at p<0.05): (1) earlier leaf abscission, (2) decreased stem basal diameter, (3) decreased stem mass, (4) decreased internode length, (5) decreased shoot height p=0.005, and (6) decreased leaf size in the growing season following ozone treatment. There was also strong evidence that ozone increased the number of leaves produced p=0.055. Finally, there was some evidence that ozone increased the ratio of shoot mass to root mass p=0.093.     

Response of weeping willows to linear alkylbenzene sulfonate

Linear alkylbenzene sulfonate (LAS) is the most commonly used anionic surfactant in laundry detergents and cleaning agents. LAS compounds are found in surface waters and soils. The short-term acute toxicity of LAS to weeping willows (Salix babylonica L.) was investigated. Willow cuttings were grown in hydroponic solution spiked with LAS at 24.0+/-1 degrees C for 192 h. The normalized relative transpiration of plants was used to determine toxicity. Severe reduction of the transpiration was only found for high doses of LAS (240 mgl(-1)). Chlorophyll contents in leaves of treated plants varied with the dose of LAS, but there was no significant linear correlation. The activities of the enzymes superoxide dismutases (SOD), catalase (CAT), and peroxidase (POD) were quantified at the end of experiments. At higher concentrations of LAS (240 mgl(-1)), the activities of SOD and CAT were decreased. The correlation between the dose of LAS and the POD activity in leaf cells was the highest of all enzyme assays (R(2)=0.5). EC(50) values for a 50% inhibition of the transpiration of the trees were estimated to 374 mgl(-1) (72 h) and 166 mgl(-1) (192 h). Results from this experiment indicated that phytotoxic effects of LAS on willow trees are not expected for normal environmental conditions.     

Alteration in uptake and translocation of essential nutrients in cabbage by excess lead

To observe the tolerance limit of lead phytotoxicity in cabbage (Brassica oleracea L.) var. Golden Aker plants were grown in refined sand with complete nutrient solution for 41 days. On 42nd day, pots with plants were separated into six lots. One lot was allowed to grow as such and was treated as control, in rest of the five lots, lead was applied at 0.1, 0.2, 0.4, 0.5 and 1.0 mM as lead nitrate. At d 75 (34 days after metal exposure), the lead toxicity symptoms as restricted growth was observed on plants at 1.0 mM lead supply. Excess lead (0.5 and 1.0 mM) developed interveinal chlorosis along the margins of young leaves. The affected leaves were reduced in size giving plant a rosette like appearance. Head size was markedly reduced at these (0.5 and 1.0 mM) levels of lead. At 0.5 mM the intensity of symptoms was markedly low. With an increase in lead supply, the concentration of lead and zinc was increased whereas that of P, S, Fe, Mn and Cu were decreased in various parts of cabbage. At 1.0 mM Pb, the concentration of lead was highest in roots and lowest in head. In leaves of cabbage the threshold of toxicity and toxicity values were 150 and 320 microg g-1 dry matter, respectively.     

Growth rate effects, responses of antioxidant enzymes and metabolic fate of the herbicide Propanil in the aquatic plant Lemna minor

Propanil (3,4-dichloropropionanilide) is a selective contact pesticide, recommended for post-emergence use in rice. This herbicide may end up in surface waters and present potential risk for aquatic vascular plants. Therefore, its toxicity was evaluated on Lemna minor L., an aquatic plant regularly used for toxicological studies, during time- and concentration-dependent exposure. Toxicity assessments were based on inhibition of growth of L. minor cultures after 24 days. The obtained results showed that the growth of Lemna was affected by the herbicide. The responses of the guaiacol peroxidase (G-POD) and glutathione S-transferase (GST) involved in the xenobiotic metabolism and antioxidative system were also investigated following Propanil exposure. Our results showed that Propanil has not induced enzymatic antioxidative defenses of L. minor. Both 3,4-dichloroaniline (3,4-DCA) and 3,4-dichloroacetanilide are the major metabolites in this plant. On the contrary, only 3,4-DCA was found in culture media after 4 days. Probably, the enzymatic hydrolysis by acyl acylamidase and the acetylation by acetyl-CoA are the major pathways for these transformation products, respectively. The results of this study showed that the selected aquatic plant has the potential to accumulate and metabolize rice herbicide, like Propanil. Based on these toxicity data this herbicide should impair the establishment of non-target aquatic plants.     

Stress reactions in Vitis vinifera L. following soil application of the herbicide flumioxazin

In order to evaluate the stress effects of flumioxazin (fmx) on grapevine, a non-target plant (Vitis vinifera L.), physiological parameters such as carbohydrate content, water status or nitrogenous metabolites were investigated on fruiting cuttings and plants grown in vineyard. In the leaves of cuttings, the soil-applied herbicide induced stress manifestations including a decrease of the dry weight percentage and the soluble carbohydrate content during the first week after treatment. Thereafter, a decrease of the osmotic potential was observed, as well as a decrease of total protein content and a parallel accumulation of free amino acids, including proline. Altogether, these results suggest that soil-applied fmx induced a stress in grapevines, leading to leaf proteolysis. However, this stress was partially recovered 3 weeks after herbicide application, suggesting that the cuttings were capable to adapt to the fmx exposure. In the vineyard, the flumioxazin effects were still significant 5 months after the treatment, particularly in the CH cv. They included a decrease of the leaf dry weight percentage and soluble carbohydrate content, as well as an increase of the osmotic potential. The decrease of leaf soluble carbohydrates may have dramatic consequences for the berry growth and the reserve constitution. Moreover, treated plants were characterized by a decrease of the free amino acid content and an accumulation of ammonium, while the protein level did not significantly increase, suggesting a degradation of amino acids. The alteration of carbon and nitrogen status after herbicide treatment may affect the grapevine vigour in a long term.     

Phosalone applied to cotton (Gossypium hirsutum L.): its deposition and persistence on foliages, soils and final residues in seeds

Phosalone, O,O-diethyl-S-(6-chloro-1,3-benzoxazol-2(3H)-onyl)methyl phosphorodithioate, was field-applied by ground equipment to cotton (Gossypium hirsutum L.) at the rates of 1050 and 2100 g a.i./ha, respectively, to determine its dissipation on leaves and soils and the residues in seeds at harvest. The insecticide concentrations on cotton leaves and soils were measured periodically for 14 days following its application. It was found that the half lives of the insecticide on cotton leaves at the dosages of 1050 and 2100 g a.i./ha were 6.8 and 6.3 days, respectively. And the half lives on soils for the 2 dosages were 7 and 5.8 days, respectively. The residues remaining in soils at harvest time were 0.072 and 0.121 mg/kg 14 days post-application and the residues in cotton seeds were relatively low (less than 0.02-0.12 mg/kg).     

Uptake and translocation of metals in Spinacia oleracea L. grown on tannery sludge-amended and contaminated soils: effect on lipid peroxidation, morpho-anatomical changes and antioxidants

The plants of Spinacia oleracea L. grown on contaminated soil (CS) and different amendments of tannery sludge (TS) have shown high accumulation of metals in its edible part. The accumulation of toxic metal (Cr) in the leaves of the plants grown on CS was recorded as 40.67 microgg(-1)dw. However, the leaves of the plants grown on 100% TS have accumulated about two times (70.80 microgg(-1)dw) higher Cr than the 10% TS (31.21 microgg(-1)dw). Among growth parameters, the root length was more affected at 90 d than the shoot length, number of leaves and leaf area. The study of scanning electron micrographs showed 29.31% increase in stomatal length in the leaves of the plants grown on CS as compared to garden soil (GS), which served as control, however it decreased in the plants grown on higher amendments of TS. The decrease in MDA content at initial period of exposure and lower amendment was recorded in the leaves, whereas, significant increase (>10% TS onward) was observed with increase in tannery sludge ratio at 90 d as compared to GS. A coordinated increase in all the studied antioxidants (cysteine, non-protein thiol, ascorbic acid, carotenoid contents) was found up to 75 d of growth. At 90 d, most of the antioxidant decreased as compared to 75 d causing oxidative stress as evidenced by increased level of lipid peroxidation and decreased chlorophyll and protein contents. Maximum increase of 181.43% in MDA content and maximum decrease of 53.69% in total chlorophyll content was recorded in the leaves of the plants grown on 100% TS after 90 d of growth. The plants grown on CS have shown an increase in shoot length, number of leaves, leaf area, photosynthetic pigments and protein contents and in all the studied antioxidants. Thus, these plants are able to combat stress involving defense mechanism, resulting in healthy growth of the plants. The results are well coordinated as there is no change in the MDA content as compared to the plants grown on GS. In view of high Cr accumulation in edible part of S. oleracea grown on CS after irrigation with tap water, it is not advisable to use these plants for edible purposes. Summing up, it is recommended that the level of metals in the edible part should be checked instead of healthy growth as deciding parameter for consumption. It is demonstrated through this study that metal enriched plants have detoxification mechanism and grow well on organic matter enriched contaminated soil.     

Uptake of polycyclic aromatic hydrocarbons (PAHs) in salt marsh plants Spartina alterniflora grown in contaminated sediments

Polycyclic aromatic hydrocarbon (PAH) concentrations were measured in Spartina alterniflora plants grown in pots of contaminated sediment, plants grown in native sediment at a marsh contaminated with up to 900 microg/g total PAHs, and from plants grown in uncontaminated control sediment. The roots and leaves of the plants were separated, cleaned, and analyzed for PAHs. PAH compounds were detected at up to 43 microg/g dry weight in the root tissue of plants grown in pots of contaminated soil. PAH compounds were detected at up to 0.2 microg/g in the leaves of plants grown in pots of contaminated soil. Concentrations less than 0.004 microg/g were detected in the leaves of plants grown at a reference site. Root concentration factor (RCF) values ranged from 0.009 to 0.97 in the potted plants, and from 0.004 to 0.31 at the contaminated marsh site. Stem concentration factor (SCF) values ranged from 0.00004 to 0.03 in the potted plants and 0.0002 to 0.04 at the contaminated marsh. No correlation was found between the RCF value and PAH compound or chemical properties such as logKOW. SCF values were higher for the lighter PAHs in the potted plants, but not in the plants collected from the contaminated marsh. PAH concentrations in the roots of the potted plants are strongly correlated with soil concentrations, but there is less correlation for the roots grown in natural sediments. Additional plants were grown directly in PAH-contaminated water and analyzed for alkylated PAH homologs. No difference was found in leaf PAH concentrations between plants grown in contaminated water and control plants.     

Effect of compost and manure amendments on zinc soil speciation, plant content, and translocation in an artificially contaminated soil

The addition of organic matter in soil can modify the bioavailability of heavy metals. A greenhouse pot experiment was carried out using an edible plant species Eruca vesicaria L. Cavalieri grown on an artificially contaminated soil with Zn (665 mg?kg^?1). In this study, the effect of compost at 20 t?ha^?1 (C20) and at 60 t?ha^?1 (C60), manure at 10 t?ha^?1 (M10) and at 30 t?ha^?1 (M30), and chemical fertilizers (NPK) on Zn fate in a soil–plant system was evaluated. At the end of the experiment, the main growth parameters and Zn content in plants were determined. In addition, Zn speciation in the soil was assessed using the original Community Bureau of Reference sequential extraction and diethylene triamine pentaacetic acid extraction. Zinc, though an essential element for plant growth, caused toxicity effects in plants grown on control and manure treatments, while in the compost treatments, plants showed no visual toxicity symptoms. The concentrations of Zn in roots were similar for all treatments, while significant differences were observed for shoots. In fact, in the compost treatments, plants showed the lowest Zn concentration in shoots. Zinc speciation seems not to be affected by the applied treatments. Indeed, Zn plant content and translocation to shoots seems to be affected. Compost amendments significantly reduced Zn content and translocation in comparison to other treatments.     

Sublethal effects of herbicides on the biomass and seed production of terrestrial non-crop plant species,influenced by environment,development stage and assessment date

Guidelines provided by the OECD and EPPO allow the use of single-species tests performed in greenhouses to assess the risk of herbicides to non-target terrestrial plant communities in the field. The present study was undertaken to investigate the use of greenhouse data to determine effects of herbicides with a different mode of action on the biomass, seed production and emergence of field-grown plants. In addition, a single species approach was compared with a mixed species approach. Effects on the biomass of greenhouse and field-grown plants were found to be related at different effect levels, indicating that it might be possible to translate results from greenhouse studies to field situations. However, the use of single-species tests may not be valid. The response of a single plant species to sublethal herbicide dosages differed to the response of the same species grown in a mixture with other species.     

Effects of thiobencarb herbicide to an alga (Nannochloris oculata) and the cladoceran (Daphnia magna)

Chronic toxicity studies were conducted with an algae (Nannochloris oculata) and the cladoceran (Daphnia magna) to determine their relative sensitivities to the thiocarbamate herbicide thiobencarb (S-4-chlorobenzyl diethylthiocarbamate). Most of the algal populations were initially affected by exposure to the herbicide. Thiobencarb concentrations higher than 0.5 mg/L significantly reduced algal densities after 24-h exposure. The 24-h static EC50 in D. magna was 3.01 mg/L. The sublethal effects of 0.3, 0.37, 0.5, 0.75, and 1.5 mg/L of thiobencarb concentrations on the survival, reproduction, and growth of D. magna were monitored for 21 days. The parameters used to determined the effect of the herbicide on D. magna were mean total young per female; mean brood size; days to first brood; intrinsic rate of natural increase (r); growth; and survival. Reproduction was significantly reduced at thiobencarb concentrations of 0.30 mg/L and higher while survival was affected after exposure to 0.75 and 1.5 mg/L of the pesticide. The r value decreased with increasing concentrations of thiobencarb. Growth, as measured by body length, was depressed significantly after exposure to all herbicide concentrations tested.