Activity and metabolism of cyano-oxime derivatives in various strains of Botrytis cinerea

The fungitoxicity of cymoxanil, 4 cyano-oxime analogs (in which the acetylurea group was replaced with various groups: amide, ester, propargyl, and cyanomethyl-amide), and 2 cymoxanil-metabolites was studied against various strains of Botrytis cinerea. The fungicidal effect was measured on germ-tube elongation and mycelial growth. Cymoxanil and the analogs bearing the amide and ester groups showed the best anti-botrytis activity. The strains studied can be classified into 3 groups according to the germ-tube sensitivity to cyano-oximes. These groups fitted well with the 3 phenotypes of sensitivity to cymoxanil previously characterized in our laboratory: CyaS₁ (highly sensitive phenotype); CyaS₂ (moderately sensitive phenotype); and CyaR (tolerant phenotype). The bio-transformations of all the cyano-oximes were monitored in the culture-medium of the different strains using HPLC- and IP-HPTLC-methods. HPLC-studies showed that cymoxanil and the analogs bearing the groups amide and ester were quickly metabolized in the culture-medium of the CyaS₁ strain. Moreover, these studies allowed us to correlate disappearance of these cyano-oximes, to their fungicidal activity towards the CyaS₁ strain. This suggests that cyano-oximes and particularly cymoxanil are probably activated in a fungitoxic compound. IP-HPTLC-studies strengthened the precedent results and allowed us to correlate disappearance of cyano-oximes studied with appearance of two acids metabolites of hydrolysis. These metabolites could be the actual active-principles.     

Fungicidal activity of N-(2-cyano-2-methoximinoacetyl)amino acids and their derivatives

More than 20 N-(2-cyano-2-methoximinoacetyl)amino acids and derivatives were synthesised and tested for antifungal activity against grape downy mildew, caused by Plasmopara viticola (de Bary) Berl. & de Toni, and rape downy mildew, Peronospora parasitica Fr. Two of the compounds containing a free carboxylic acid group, a moiety which has been shown to confer phloem mobility on compounds, showed high activity, especially against P. parasitica, in protectant tests. These results indicate that there is no incompatibility between the acid function and fungicidal activity. A number of the esters showed activity comparable with that of cymoxanil in the protectant tests, and the tert-butyl esters of the methionine derivative and its sulfone were more than ten times as active as the commercial compound.     

Synthesis of 2-amino-6-oxocyclohexenylsulfonamides and their activity against Botrytis cinerea

BACKGROUND: With the objective of exploring the fungicidal activity of 2‐oxocyclohexylsulfonamides (2), a series of novel 2‐amino‐6‐oxocyclohexenylsulfonamides (6 to 23) were synthesised, and their fungicidal activities against Botrytis cinerea Pers. were evaluated in vitro and in vivo. RESULTS: The compounds were characterised by IR, ¹H NMR and elemental analysis. Bioassay results of mycelial growth showed that compounds 6 to 23 had a moderate antifungal activity against B. cinerea. N‐(2‐methylphenyl)‐2‐(2‐methylphenylamino)‐4,4‐dimethyl‐6‐oxocyclohexenylsulfonamide (13) and N‐(2‐chlorophenyl)‐2‐(2‐chlorophenylamino)‐6‐oxocyclohexenylsulfonamide (21) showed best antifungal activities, with EC₅₀ values of 8.05 and 10.56 µg mL^?1 respectively. Commercial fungicide procymidone provided an EC₅₀ value of 0.63 µg mL^?1. The conidial germination assay showed that most of compounds 6 to 23 possessed excellent inhibition of spore germination and germ‐tube elongation of conidia of B. cinerea. For in vivo control of B. cinerea colonising cucumber leaves, the compound N‐cyclohexyl‐2‐(cyclohexylamino)‐4,4‐dimethyl‐6‐oxocyclohexenylsulfonamide (19) showed a better control effect than the commercial fungicide procymidone. CONCLUSION: The present work demonstrated that 2‐amino‐6‐oxocyclohexenylsulfonamides can be used as possible new lead compounds for further developing novel fungicides against B. cinerea. Copyright © 2011 Society of Chemical Industry     

Sensitivity ofPhytophthora Infestans to cymoxanil

The efficacy of cymoxanil, Pulsan (oxadixyl + mancozeb + cymoxanil, 1:7:0.4) and Sandocur-M (oxadixyl + mancozeb + cymoxanil, 1:7:2) in controlling late blight was examined in potatoes (cv. ‘Alpha’). Nine oxadixyl-sensitive and seven oxadixyl-resistant isolates ofPhytophthora infestons from six countries were tested. ED₉₀ values of cymoxanil ranged between 64 and 467 mg a.i./l. No relationship was observed between sensitivity to cymoxanil and sensitivity to oxadixyl. Isolate S6A (1305) from California was the most sensitive to cymoxanil (ED₉₀ = 64 mg/l), whereas isolate R11 from Israel was the least sensitive (ED₉₀ = 467 mg/l). Most isolates from Israel were less sensitive (ED₉₀ = 151–467 mg/l) than isolates from Switzerland, the Netherlands, Ireland, Mexico or California (ED₉₀ = 64–152 mg/l). Three-way mixtures of cymoxanil with mancozeb and oxadixyl were more inhibitory than cymoxanil alone, regardless of the isolate. Sandocur-M was, for most isolates, more effective than Pulsan. The correlation coefficient between sensitivity to cymoxanil and sensitivity to Sandocur-M was 0.67, while that between cymoxanil and Pulsan was 0.76.     

Metabolism of metalaxyl in cell suspension cultures of Lactuca sativa L. and Vitis vinifera L

The metabolic fate of metalaxyl in suspension cultures of Lactuca sativa has been investigated as a function of explant source, culture age, and nutrient medium quality. In all cases, the fungicide was the subject of diverse metabolic attack yielding products which arose from O- and N-dealkylation, alkyl and aryl hydroxylation, and ester hydrolysis. Substantial amounts of metabolites were found in the culture filtrates although products arising from aryl hydroxylation and combined O-/N-dealkylation accumulated exclusively as cellular conjugates. Cultures newly isolated from the cotyledon, hypocotyl, and root of seedlings displayed very little variation in the rate and qualitative nature of breakdown and in the relative distribution of metabolites among the polar conjugate fraction, unconjugated cellular fraction, and culture filtrate. Similarly, metabolism proceeded independently of changes in nutrient medium composition. A suspension culture initiated from 4-year-old hypocotyl callus produced less identifiable metabolites and had an impaired ability to perform alkyl hydroxylation, O-dealkylation, and ester hydrolysis. A greater proportion of metabolites occurred as polar conjugates in the 4-year-old hypocotyl cells than that in the newly isolated hypocotyl line. The metabolic profile seen in Vitis vinifera was similar to that in L. sativa and a time-course study indicated some interconversion of metabolites and a change in the ratio of two atropisomers formed by alkyl hydroxylation.     

Metabolism of [14C]cymoxanil in grapes,potatoes and tomatoes

Foliar-applied [¹⁴]cymoxanil, 1-(2-cyano-2-methoxyimino-[2-¹⁴C]acetyl)-3-ethylurea was rapidly metabolised in grapes, tomatoes and potatoes, Furthermore, the metabolism of this fungicide was unusual in that the metabolites were found to be naturally occurring compounds, with glycine as the major metabolite. Significant levels of radioactivity were found in other amino-acids, sugars, starch, fatty acids and lignin, indicating incorporation of carbon-14 via the various metabolic pathways.     

Nonspecific toxins as components of a host‐specific culture filtrate from Fusarium oxysporum f. sp. cubense race 1

Bananas and plantains (Musa spp.) are among the most important crops in the world providing staple food for hundreds of millions of people. However, banana production has been devastated by fungal infestations caused by Fusarium oxysporum f. sp. cubense (Foc). Despite the fact that there is very little known on the role of microbial metabolites in the molecular mechanism of Foc infections, it has been proposed that the toxins fusaric acid and beauvericin produced by Foc play an important role during pathogenesis. The aim of this contribution was to study the toxic components of culture filtrates (CF) of Foc and to isolate the extracellular microbial metabolites involved in the plant response. An in vitro bioassay was used to evaluate the production of phytotoxic metabolites as well as the specificity of culture from a strain of Foc belonging to VCG 01210 (race 1). A host‐specific CF was obtained and the phytotoxic compounds characterized as fusaric acid, beauvericin and fumonisin B1. Despite the presence of these nonspecific toxins, a water‐soluble extract from the CF induced protection to the main phytotoxic fraction, measured by lesion area. This hydrophilic fraction induced a fast and strong response of just jasmonic acid (JA)‐dependent defence genes rather than salicylic acid (SA)‐ and ethylene (ET)‐response genes in resistant cultivars. Extracellular proteins isolated from CF of Foc provide an important source for further investigations on the molecular basis of the interaction between Foc and banana.     

The termiticidal activity of Sextonia rubra (Mez) van der Werff (Lauraceae) extract and its active constituent rubrynolide

BACKGROUND: Termites are degradation agents that inflict severe damage on wood. Some long‐lasting Amazonian trees can resist these insects by producing toxic secondary metabolites. These metabolites could potentially replace synthetic termiticidal products which are becoming more restricted to use. RESULTS: Sextonia rubra is resistant to termite‐induced degradation. It has been demonstrated that this species naturally produces an ethyl‐acetate‐soluble termiticidal metabolite, rubrynolide, to protect its wood. Assays in the presence of tropical and invasive termites established that both rubrynolide and crude ethyl acetate extract from S. rubra wood can be used as a treatment for the protection of sensitive woods against termites. CONCLUSION: Rubrynolide and S. rubra extract are promising candidates for the replacement of synthetic termiticides. Copyright © 2011 Society of Chemical Industry     

Sensitivity of European Plasmopara viticola populations to cymoxanil

Between 1995 and 1997, 278 grape downy mildew (Plasmopara viticola) populations originating from European vineyards were characterised for their sensitivity to cymoxanil in a leaf-disc assay. The sensitivity profile revealed a wide distribution, with minimum inhibitory concentrations (MIC) ranging from 10 to more than 800 mg litre⁻¹. EC₅₀ values ranged from 1 to more than 800 mg litre⁻¹ with an average of 125 mg litre⁻¹. The sensitivity distribution was stable between 1995 and 1997. Surprisingly, populations from Portugal appeared significantly more sensitive than those from France or Italy, which could not be linked to differential cymoxanil usage in these countries. P viticola populations collected outside Europe and never exposed to cymoxanil appeared significantly more sensitive than exposed European populations, with an average EC₅₀ value of 10 mg litre⁻¹. The level of sensitivity of European P viticola populations was relatively unaffected by the number of cymoxanil applications made during a season or by the number of years of cymoxanil use. No link was found between the level of sensitivity in the leaf-disc assay and the level of performance of the cymoxanil mixtures used in the fields where the populations originated. Specific field trials conducted in Italy and Portugal have shown that the performance of cymoxanil-based mixtures remained good even on populations of the grape downy mildew fungus characterised as less sensitive in the leaf-disc assay. While there are no baseline sensitivity data for pre-commercialisation P viticola populations, the results of our study suggest that a shift in sensitivity (12.5-fold) may have occurred in some areas since introduction of cymoxanil on grapes nearly 20 years ago. Because cymoxanil is never used alone, it is difficult to determine whether or not practical resistance is occurring in European vineyards. © 1999 Society of Chemical Industry     

Synergism between fungicides with different mechanisms of action against acylalanine-resistant strains of Phytophthora spp.1

The authors have studied the efficacy of two systemic compounds, cymoxanil and propamocarb, used as partners of acylalanines, in limiting mycelial growth of acylalanine-sensitive and resistant strains of Phytophthora capsici and P. nicotianae var. parasitica. The fungicidal activity of the single components was compared to that of the different mixtures, at various ratios. The degree of synergy was calculated using the method of Wadley. The results obtained indicate synergistic interactions of various intensities: synergistic factors (SF) up to seven were found. The highest synergistic effects were observed in the case of resistant Phytophthora strains.     

The metabolism of [14C] cymoxanil in the rat

A rat, given a single oral dose of [¹⁴C] cymoxanil, 1-(2-cyano-2-methoxyimino-[2-¹⁴C]-acetyl)-3-ethylurea, eliminated 91% of the radioactivity within 72 h. The urine contained 71%, the faeces 11%, and the expired air about 7% of the radiolabel; no ¹⁴C residue was found in the internal organs. Greater than 70% of the radioactivity in the urine was identified. The major metabolite was characterised as glycine, both free and conjugated, as hippuric acid and phenylaceturic acid [N-(phenylacetyl)-glycine], and probably in the form of polypeptides of low molecular weight. The other metabolites identified included 2-cyano-2-methoxyiminoacetic acid, 2-cyano-2-hydroxyiminoacetic acid and 1-ethylimidazolidine-2, 4, 5-trione. The minor metabolites included succinic acid and 2-oxoglutaric acid which indicated reincorporation of metabolic ¹⁴C. Cymoxanil, as such, was not detected in the urine.     

METABOLISM OF MONURON IN EXCISED LEAVES OF CORN AND BEAN PLANTS*

Summary. The metabolism of monuron in excised leaves of bean (Phaseolus vulgaris L., var. Black Valentine) and corn (Zea mays L., var. Batam Cross) were studied with carbonyl-¹⁴C-labelled monuron. The metabolic conversions of monuron in both plant species were exponential and followed first order reaction kinetics. The metabolism of monuron can be divided into two major pathways: demethylation and hydroxylation. At a monuron concentration of 16 ppm the hydroxylation was dominant in the bean leaves. As the concentration of monuron was increased the participation of the hydroxylation pathway became less probably due to the inhibition of enzymes involved by monuron. The demethylation pathway was not affected by higher monuron concentration. In the corn leaf, however, sequential demethylation was always the major pathway of monuron transformation and there was no inhibitory effect observed on either pathways as the concentration of monuron increased. The following radioactive metabolites were found in the alcohol extract of bean and com leaves receiving carbonyl-¹⁴C-labelled monuron: N′-(4-chlorophenyl)-N-methylurea, p-chlorophenylurea, two polypeptide complexes of monuron, one polypeptide complex of N′-(4-chlorophenyl)-N-methylurea and β-D-glucosides of N-(2-hydroxy-4-chlorophenyl) urea, N′-(2-hydroxy-4-chlorophenyl) N-methylurea, and N′-(2-hydroxy-4-chlorophenyl)-N,N-dimethylurea. In addition, one minor radioactive peak was found only in the alcohol extract of corn leaves, which yielded four unidentified radioactive metabolites after acid or enzyme hydrolysis with β-glucosidase. Métabolisme du monuron dans des feuilles excisés de maïs et de haricot     

The metabolism of the herbicide flamprop-methyl in rats and some comparative data for dogs,mice and rabbits

[¹⁴C]Flamprop-methyl administered orally to rats (3-4 mg kg^?1 body weight) was excreted mostly via the faeces (78.7 and 61.6% in males and females, respectively). Elimination was rapid and 90% of the dose of ¹⁴C was excreted in faeces and urine 0-48 h after dosing. The distribution of ¹⁴C between faeces and urine was different in males and females. No expired [¹⁴C]carbon dioxide was detected and less than 2% of the dose remained in the animals 4 days after dosing. The predominant metabolic pathway was hydrolysis of the ester bond to afford the carboxylic acid which was excreted unchanged and as its glucuronide conjugate. Aromatic hydroxylation occurred at the para- and meta-positions of the N-benzoyl ring. N-(3)-Chloro- 4-fluorophenyl-N-(3,4-dihydroxybenzoyl)-DL -alaninate was also formed. This hydroxylated form of flamprop-methyl was partially O-methylated at the 3-hydroxy group. Flamprop-methyl was also metabolised and eliminated rapidly by dogs, mice and rabbits. The last of these three species afforded very little aromatic hydroxylation and also differed from the others in that the metabolites were eliminated mostly in the urine. Aromatic hydroxylation lay in the order: male rat = female rat > dog= mouse>rabbit (female).     

Bioactivity of compounds from Acmella oleracea against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and selectivity to two non-target species

BACKGROUND: Tropical plants are recognised sources of bioactive compounds that can be used for pest control. The objective of this study was to evaluate the biological activity of compounds present in Acmella oleracea (Asteracea) against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), which is the main pest of tomato crops in Latin America. The selectivity of these compounds to the predator Solenopsis saevissima (Smith) (Hymenoptera: Formicidae) and to the pollinator Tetragonisca angustula (Latr.) (Hymenoptera: Apidae: Meliponinae) was also of interest. RESULTS: A bioassay screening with hexane and ethanol extracts from 23 plants was performed. The hexane extract of A. oleraceae was the most active of the extracts and was selected for further study. The following three alkamides were isolated from a hexane extract of the aerial parts of A. oleracea : spilanthol, (E)‐N‐isobutylundeca‐2‐en‐8,10‐diynamide and (R, E)‐N‐(2‐methylbutyl)undeca‐2‐en‐8,10‐diynamide. All of the isolated compounds showed insecticidal activity, with spilanthol being the most active (LD₅₀ = 0.13 µg mg^?1) against T. absoluta . The alkamides were selective to both beneficial species studied. CONCLUSION: The crude hexane extract of A. oleraceae showed high insecticidal activity and can be used to control T. absoluta in organic or conventional crops. Quantification of LD₅₀ values of isolated compounds against T. absoluta showed that alkamides could serve as potent insecticides for T. absoluta control programmes. Spilanthol was the main alkamide active isolated. This alkamide is the most promising as it has the highest insecticidal activity and is selective to non‐target organisms. Copyright © 2011 Society of Chemical Industry     

Mineralisation of the herbicide linuron by Variovorax sp. strain RA8 isolated from Japanese river sediment using an ecosystem model (microcosm)

BACKGROUND: Linuron is a globally used phenylurea herbicide, and a large number of studies have been made on the microbial degradation of the herbicide. However, to date, the few bacteria able individually to mineralise linuron have been isolated only from European agricultural soils. An attempt was made to isolate linuron‐mineralising bacteria from Japanese river sediment using a uniquely designed river ecosystem model (microcosm) treated with ¹⁴C‐ring‐labelled linuron (approximately 1 mg L^?1). RESULTS: A linuron‐mineralising bacterium that inhabits river sediment was successfully isolated. The isolate belongs to the genera Variovorax and was designated as strain RA8. Strain RA8 gradually used linuron in basal salt medium (5.2 mg L^?1) with slight growth. In 15 days, approximately 25% of ¹⁴C‐linuron was mineralised to ¹⁴CO₂, with 3,4‐dichloroaniline as an intermediate. Conversely, in 100‐fold diluted R2A broth, strain RA8 rapidly mineralised ¹⁴C‐linuron (5.5 mg L^?1) and more than 70% of the applied radioactivity was released as ¹⁴CO₂ within 3 days, and a trace amount of 3,4‐dichloroaniline was detected. Additionally, the isolate also degraded monolinuron, metobromuron and chlorobromuron, but not diuron, monuron or isoproturon. CONCLUSION: Although strain RA8 can grow on linuron, some elements in the R2A broth seemed significantly to stimulate its growth and ability to degrade. The isolate strictly recognised the structural difference between N‐methoxy‐N‐methyl and N,N‐dimethyl substitution of various phenylurea herbicides. Copyright © 2010 Society of Chemical Industry     

In-vitro metabolism of a mixture of atrazine and simazine by the soluble fraction (105000g) from goose,pig and sheep liver-homogenates

The soluble fraction (105 000g) from goose, pig and sheep liver-homogenates was found to contain an enzyme which metabolised a mixture of atrazine and simazine in in-vitro incubations by a combination of hydrolysis and partial N-dealkylation. Complete dealkylation was not observed as shown by the removal of only one alkyl group, but not both, from the compounds containing chlorine or hydroxyl-groups attached to the triazine ring.     

疫霉菌对霜脲氰抗性遗传及对霜脲氰和甲霜灵的交互抗性

就疫霉菌对霜脲氰抗性的产生和遗传，以及疫霉菌对霜脲氰和甲霜灵的交互抗性进行了研究。结果表明，苎麻疫霉容易对霜脲氰产生抗药性突变，但抗性在连续３代单游动孢子分离过程中逐渐丧失。大雄疫霉对霜脲氰不易产生抗性突变，恶疫霉、大雄疫霉和苎麻疫霉对甲霜灵和霜脲氰不存在交互抗性     

Allelopathy and the active metabolites of the endophytic fungus,Alternaria J46, from Platycladus orientalis

The endophytic fungus, Alternaria J46, was isolated from the stem of the medicinal plant, Platycladus orientalis. A suspension of Alternaria J46 mycelial segments and the culture filtrates of the fungi exhibited marked seed germination inhibition against the monocot wheat, large crabgrass, bromegrass, rice and barnyardgrass and weak inhibition against the dicot redroot pigweed and morning glory, but it was safe for use on soybean, rape, cucumber, tomato, lettuce and radish crops. It is possible to use J46 culture filtrates in order to prevent monocot weeds in dicot cropland. Three active metabolites were isolated from an extract of the fungus cultures and elucidated as 3‐acetyl‐5‐sec‐butyltetramic acid (1, tenuazonic acid), 3‐acetyl‐5‐iso‐butyltetramic acid (2, vivotoxin II) and cyclo‐(L‐leucyl‐L‐proline) (3). Among these three compounds, compounds 1 and 2 showed significant phytotoxic effects on the seed germination of large crabgrass, while compound 3 exhibited weak activity, and all were safe for lettuce at 100 μg mL^?1. Accordingly, compounds 1 and 2 were the main active metabolites that were responsible for endophytic fungus Alternaria J46's strong seed germination inhibition against monocotyledons.     

Biological control of red rot in sugarcane by native pyoluteorin‐producing Pseudomonas putida strain NH‐50 under field conditions and its potential modes of action

BACKGROUND: Rhizobacteria have a good potential to suppress soilborne diseases, but their efficacy against sugarcane pests is rarely reported. Bacterial strains isolated from the rhizosphere of sugarcane were evaluated for their potential to suppress red rot disease on two susceptible varieties, Co‐1148 and SPF‐234, under field conditions. The strains were also characterised for the production of secondary metabolites associated with their antagonistic activity. RESULTS: One out of four strains, the Pseudomonas putida strain NH‐50 (EU627168), reduced disease severity by 44–60% in different field trials. This potent antagonistic strain produced pyoluteorin antibiotic, as confirmed by high‐performance liquid chromatography (HPLC). The PltB gene involved in pyoluteorin synthesis was amplified from the P. putida strain NH‐50 and sequenced. The extracellular metabolites and volatile and diffusible antibiotics secreted by the tested strains inhibited mycelial growth of Glomerella tucumensis (Speg.) Arx & E Mull in vitro by 7–55%. CONCLUSION: The pyoluteorin‐producing bacteria P. putida strain NH‐50 significantly reduced disease severity on both sugarcane varieties, irrespective of fungal inoculation, i.e. either inoculated through stem or through soil. This strain also possesses other plant growth characteristics and can be used as a biopesticide for sugarcane crop. Copyright © 2011 Society of Chemical Industry     

Effects of cultural conditions on fungal biomass,blastospore yields and toxicity of fungal secreted proteins in batch cultures of Metarhizium anisopliae (Ascomycota: Hypocreales)

BACKGROUND: Recently, two fungal proteins with apparent molecular masses of 11 and 15 kDa and insecticidal activity against Ceratitis capitata (Wied.) have been purified from the crude soluble protein extract (CSPE) secreted by the entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin (strain EAMa 01/58‐Su) in Adamek's liquid medium. The feasibility of culturing this strain in fermentation facilities in order to harvest and formulate the insecticidal proteins for C. capitata control is mainly dependent on the ability to produce high concentrations of the active proteins at a reasonable cost. RESULTS: These studies report that, in batch cultures of EAMa 01/58‐Su strain, the carbon (C) and nitrogen (N) ratios and sources are important considerations with respect to fungal biomass production, blastospore yield and secretion of insecticidal proteins against C. capitata adults. The data indicate that the type and concentration of N source in the medium influence the production of insecticidal protein and thus the toxicity of the CSPEs. The electrophoretic analysis suggests that the monomer of 11 kDa plays an important role in the insecticidal effect described. Concerning biomass production, no clear differences were found between media with different C and N sources and C:N ratios in total biomass production at day 7. Conversely, important differences were found among the media in terms of blastospore yields. CONCLUSIONS: By optimising the culture media, the insecticidal effect of the CSPE against C. capitata can be improved. In the CSPE from G₄₀:P₂₀ (40 g L^?1 glucose and 20 g L^?1 peptone in dH₂O), the LC₅₀ and the LT₅₀ were 7 and 4.5 times lower than in the CSPE obtained from Adamek. Copyright © 2010 Society of Chemical Industry