Michel Couderchet

Toxicity and removal of heavy metals (cadmium, copper, and zinc) by Lemna gibba.

Effects of cadmium, copper, and zinc on the aquatic plant Lemna gibba were determined under controlled conditions; in parallel their removal from the growth medium was followed. The results showed that the three heavy metals affected growth, a physiological stress index defined as the ratio of Chlorophyll to phaeophytin (D665/D665a), and the contents of proline. After 4 days, 10(-3)-10(-1)mg/L Cd inhibited growth by 25-100%, reduced D665/D665a by 35-89%, and increased proline content by 44-567%. Under the same conditions, 10(-4)-10(-1) microg/L Cu inhibited growth by 36-75%, reduced D665/D665a by 19-81%, and increased proline content by 67-288%. Comparable concentrations of Zn had little effect. However, higher concentrations (4, 30, and 50mg/L) inhibited growth by 50-79%. Also, 0.1 and 30 mg/L induced a small reduction of D665/D665a (-3.8% and -22%) and an increase in proline contents (+144% and +177%). When it was observed, proline accumulation was always transient and the maximum was reached after 4 days. Monitoring metal concentration in the medium showed that L. gibba was able to remove metals from the medium. Zn and Cu removal was biphasic, it was rapid during the first 2 days (> 60% reduction) and slow (10-20%) during the following 8 days. For Cd, removal was linear and depended on the initial concentration. It reached approximately 90% after 6 or 8 days for initial concentrations of 10(-1) and 10(-3)mg/L, respectively.

Chitosan oligomers and copper sulfate induce grapevine defense reactions and resistance to gray mold and downy mildew.

ABSTRACT Chitosan (CHN), a deacetylated derivative of chitin, was shown to be efficient in promoting plant defense reactions. CHN oligomers of different molecular weight (MW) and degree of acetylation (DA) triggered an accumulation of phytoalexins, trans- and cis-resveratrol and their derivatives epsilon-viniferin and piceid, in grapevine leaves. Highest phytoalexin production was achieved within 48 h of incubation with CHN at 200 mug/ml with an MW of 1,500 and a DA of 20% (CHN1.5/20), while oligomers with greater MW were less efficient, indicating that a specific MW threshold could be required for phytoalexin response. Treatment of grapevine leaves by highly active CHN1.5/20 also led to marked induction of chitinase and beta-1,3-glucanase activities. CHN1.5/20 applied together with copper sulfate (CuSO(4)) strongly induced phytoalexin accumulation. CuSO(4) alone, especially at low concentrations also elicited a substantial production of phytoalexins in grapevine leaves. Evidence is also provided that CHN1.5/20 significantly reduced the infection of grapevine leaves by Botrytis cinerea and Plasmopara viticola, and in combination with CuSO(4) conferred protection against both pathogens.

Pseudomonas spp.-induced systemic resistance to Botrytis cinerea is associated with induction and priming of defence responses in grapevine

Non-pathogenic rhizobacteria Pseudomonas spp. can reduce disease in plant tissues through induction of a defence state known as induced systemic resistance (ISR). This resistance is based on multiple bacterial determinants, but nothing is known about the mechanisms underlying rhizobacteria-induced resistance in grapevine. In this study, the ability of Pseudomonas fluorescens CHA0 and Pseudomonas aeruginosa 7NSK2 to induce resistance in grapevine against Botrytis cinerea is demonstrated. Both strains also triggered an oxidative burst and phytoalexin (i.e. resveratrol and viniferin) accumulation in grape cells and primed leaves for accelerated phytoalexin production upon challenge with B. cinerea. Treatment of cell cultures with crude cell extracts of bacteria strongly enhanced oxidative burst, but resulted in comparable amounts of phytoalexins and resistance to B. cinerea to those induced by living bacteria. This suggests the production of bacterial compounds serving as inducers of disease resistance. Using other strains with different characteristics, it is shown that P. fluorescens WCS417 (Pch-deficient), P. putida WCS358 (Pch- and SA-deficient) and P. fluorescens Q2-87 (a DAPG producer) were all capable of inducing resistance to an extent similar to that induced by CHA0. However, in response to WCS417 (Pch-negative) the amount of H2O2 induced is less than for the CHA0. WCS417 induced low phytoalexin levels in cells and lost the capacity to prime for phytoalexins in the leaves. This suggests that, depending on the strain, SA, pyochelin, and DAPG are potentially effective in inducing or priming defence responses. The 7NSK2 mutants, KMPCH (Pch- and Pvd-negative) and KMPCH-567 (Pch-, Pvd-, and SA-negative) induced only partial resistance to B. cinerea. However, the amount of H2O2 triggered by KMPCH and KMPCH-567 was similar to that induced by 7NSK2. Both mutants also led to a low level of phytoalexins in grapevine cells, while KMPCH slightly primed grapevine leaves for enhanced phytoalexins. This highlights the importance of SA, pyochelin, and/or pyoverdin in priming phytoalexin responses and induced grapevine resistance by 7NSK2 against B. cinerea.

Effect of oxyfluorfen and diuron alone and in mixture on antioxidative enzymes of Scenedesmus obliquus

Oxyfluorfen and diuron, herbicides commonly used in champagne vineyards, are known to generate reactive oxygen species (ROS) which induce a shift of the balance between prooxidative and antioxidative reactions. Against this oxidative stress, plants have developed enzymatic defenses such as superoxide dismutase, peroxidases, and catalase to limit lipid peroxidation, DNA damage, and protein degradation. These antioxidative enzyme activities were used as biomarkers in the present study to evaluate the toxic effect of oxyfluorfen and diuron on the microalgae Scenedesmus obliquus. Enzymatic activities were measured in the presence of these compounds alone and in mixture after a 24-h exposure to analyze the interactions between these herbicides. Catalase, ascorbate peroxidase, glutathione reductase, and glutathione S-transferase activities were investigated after the exposure of the microalgae to herbicide concentrations responsible for 25, 50, and 75% of growth inhibition. Antioxidative enzymes were significantly stimulated by oxyfluorfen, while no significant variation of catalase and ascorbate peroxidase activity was observed with diuron. However, this herbicide stimulated glutathione reductase and glutathione S-transferase of Scenedesmus obliquus. Growth inhibition test with oxyfluorfen and diuron in mixture showed an antagonistic interaction. At the enzyme level, antagonism was observed only with catalase. According to our data, diuron effect was not caused by an increased stimulation of algal antioxidative enzymes by the photosystem II herbicide.

Phytotoxicity of diuron alone and in combination with copper or folpet on duckweed (Lemna minor).

The photosystem II-herbicide diuron is widely used for weed control in Champagnes vineyards. Its important use and its relative persistence make it of particular interest for ecotoxicological studies. Toxicity of diuron was assessed on Lemna minor L., a representative aquatic macrophyte regularly used for toxicological studies. Toxicity assessments were based on inhibition of growth and total chlorophyll content of L. minor cultures after 7 days. Growth was inhibited and IC(50) and IC(90) were, respectively, 25 and 60 microg l(-1), but chlorophyll content of L. minor increased in response to the herbicide. When diuron was combined with copper, growth inhibition of L. minor depended on the concentrations of both chemicals. For some concentrations, combination of these chemicals resulted in a slight (but non-significant) antagonism. Additivity was observed for all other mixtures. When diuron was combined with folpet, growth and chlorophyll content of L. minor only depended on the concentration of the herbicide. Diuron was also found to prevent the copper-induced decrease of chlorophyll content when it was combined with this metal. A multifactorial model was found more appropriate to characterize interactions between pesticides than Abotts model.

Effects of procymidone, fludioxonil and pyrimethanil on two non-target aquatic plants

Procymidone, fludioxonil, and pyrimethanil are widely used to control the pathogenic fungus Botrytis cinerea in Champagnes vineyards. These fungicides may end up in surface waters and present potential risks for aquatic vascular plants and algae. Therefore, their toxicity was evaluated on Lemna minor and Scenedesmus acutus in six-day or 48-h tests, respectively. Based on growth and chlorophyll (Chl) content of L. minor and S. acutus cultures, the results showed that the alga was the most sensitive to the fungicides. Among the fungicides, pyrimethanil was the most toxic for L. minor, its nominal IC50 was 46.16 mg l(-1) and that of the other two was >100 mg l(-1). In contrast, pyrimethanil appeared the least toxic for S. acutus at low concentration, nominal IC50 were 22.81, 4.85, and 4.55 mg l(-1) for pyrimethanil, fludioxonil, and procymidone, respectively. Fate of the fungicides in the media was also investigated and acute toxicity of the agrochemicals is discussed in regard to concentration in the culture media. Poor solubility of procymidone and fludioxonil appeared to be partly responsible for the low toxicity of these fungicides. Based on these toxicity data and the concentrations found in ponds collecting vineyard runoff water, these pesticides should not impair the establishment of pioneer plants.

Phytoremediation of fungicides by aquatic macrophytes: Toxicity and removal rate

The rate of removal of two fungicides (dimethomorph and pyrimethanil) from water by five macrophyte species (L. minor, S. polyrhiza, C. aquatica, C. palustris and E. canadensis) was assessed in laboratory tests. In order to assure that these studies were performed with healthy plants the effects of the fungicides on chlorophyll fluorescence were studied as well. At exposure concentrations of 600microgL(-1) the effects of the fungicides on chlorophyll fluorescence were minor, so that this initial concentration level was selected for the fungicide removal rate tests. The removal yields during the 4-d test periods varied from 10% to 18% and 7% to 12% for dimethomorph and pyrimethanil, respectively. The maximum removal rate during the 4-d test period was 48microgg(-1) fresh weight (FW) for dimethomorph and 33microgg(-1) FW for pyrimethanil. L. minor and S. polyrhiza showed the highest removal efficiency for the two fungicides.

Biological Activity of Two Stereoisomers of the N-Thienyl Chloroacetamide Herbicide Dimethenamid

Due to the presence of an asymmetrically substituted C atom, dimethenamid [2-chloro-N-(2,4-dimethyl-3-thienyl)-N-(2-methoxy-1-methylethyl)acetamide], a recently introduced N-thienyl chloroacetamide herbicide, exists as two stereoisomers (S and R) having differing herbicidal activities as demonstrated with a selection of weeds and Lemna minor. The activity of the two isomers was investigated in greater detail with the green alga Scenedesmus acutus and compared to that of alachlor [2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide]. As with alachlor, the S isomer (5 μM) strongly inhibited algal growth and fatty acid desaturation while the R isomer had no effect. In short-term experiments (up to 5·5 h), the S isomer and alachlor (100 μM) inhibited [14C]acetate uptake and its incorporation into fatty acids in the same manner, while the R isomer did not. Incorporation of [14C]acetate into a non-lipid fraction of the algae was strongly inhibited by alachlor and the S isomer (100 μM) and only slightly by the R isomer. A 50% inhibition of incorporation of [14C]oleic acid into the same non-lipid fraction was attained with less than 10-7 M of the S isomer while 10-5 M of the R form of dimethenamid achieved only a 40% inhibition. The same stereospecificity of the compound on growth, fatty acid desaturation, acetate uptake and oleic acid incorporation provides strong evidence that dimethenamid may act upon a primary, specific target in lipid metabolism. Furthermore, the comparable biological activities of dimethenamid and alachlor indicate that this target is common to both N-phenyl and N-thienyl chloroacetamide herbicides.

Pigments as biomarkers of exposure to the vineyard herbicide flazasulfuron in freshwater algae

Weed control in Champagne vineyards has long relied on the use of diuron and substituted triazines; these compounds are now being replaced by flazasulfuron, a sulfonylurea that is used at a much lower dosage. The vineyards of Champagne are planted on steep slopes and runoff is important, and even though low doses of these herbicides are used, they may present some potential risk for freshwater ecosystems. Therefore, the effects of the sulfonylurea herbicide, flazasulfuron (formulated as Katana) was investigated on the unicellular green alga Scenedesmus obliquus. The pigment content of the algal suspensions was followed as a biomarker of exposure to the herbicide. The results demonstrate that flazasulfuron induced a reduction in chlorophyll content at concentrations of 10 microg/L, while the increase of pigment content in the culture was reduced with the lowest concentration tested (0.1 microg/L). Among the three pigments tested, chlorophyll a appeared to be the most sensitive biomarker. In the algal medium, flazasulfuron was slowly degraded (DT(50) approximately 8 days) in a compound that was tentatively identified. The toxicity of this herbicide for the algae was comparable to that of older herbicides which are used at a much higher rate. Therefore, we may speculate that even if flazasulfuron comes into contact with freshwater ecosystems, its effects on algae will be less deleterious than that of traditional herbicides.

A specific and sensitive assay to quantify the herbicidal activity of chloroacetamides

Based on a recently discovered property of chloroacetamide herbicides - the inhibition of the incorporation of oleic acid into sporopollenin of Scenedesmus acutus - a rapid quantitative test was developed for chloroacetamide-type herbicidal activity. In this test, algal cells are incubated for 3 h with [ 14 C]oleic acid, saponified and the lipids (including non-saponifiable ones) extracted and discarded. The radioactivity incorporated into the residual non-lipid fraction is determined, and inhibition of this incorporation is used as a marker of chloroacetamide-type activity. Twenty-two agrochemical compounds were screened in this assay, which was found to be very sensitive, a 50% inhibition being reached with submicromolar herbicide concentrations. It is specific to chloroacetamides and related amides, since all these herbicides tested were potent inhibitors, while other herbicides were not. Highest inhibition was shown by cafenstrole followed by butachlor, fluthiamid, metazachlor, alachlor, dimethenamid, metolachlor and mefenacet. For these herbicides (with the exception of butachlor) sensitivity in the test was positively correlated (r = 0.984) with their phytotoxic effect on the alga.