Analysis Of The Ergosterol Biosynthesis Pathway Cloning, Molecular Characterization And Phylogeny Of Lanosterol 14 α-demethylase (erg11) Gene Of Moniliophthora Perniciosa.

The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches' broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.

Mango stem-end rot (Botryosphaeria dothidea) disease control by partial-pressure infiltration of fungicides

The in vitro efficacy of several fungicides against Botryosphaeria dothidea (syn. Dothiorella dominicana) and their in vivo efficacy in controlling mango cv. Kensington Pride stem-end rot on partial-pressure infiltration v. dip treatment of green mature fruit was evaluated. In vitro sensitivity of B. dothidea to Benlate (benomyl), Sportak (prochloraz) and Scala (pyrimethanil) at 10 dilutions of the manufacturer's recommended rate was first determined at typical cold (13degreesC) and shelf (23degreesC) storage temperatures. The effectiveness of partial-pressure infiltration and conventional hot (52degreesC) or cold (26degreesC) dipping of fruit after harvest was then evaluated using the commercially recommended rate for each fungicide. In vitro, Benlate and Sportak prevented the growth of B. dothidea at both storage temperatures and at all concentrations, while Scala partially controlled growth of the pathogen. Benlate was the most effective fungicide for stem-end rot control. Sportak and Scala resulted in stem-end rot control when applied by partial-pressure infiltration, but not as dips. Partial-pressure infiltration holds promise for enhancing the efficacy of otherwise less effective but alternative fungicides for control of stem-end rot diseases.

Chemical treatment of papaya seeds aiming at long-term storage and control of damping off

Damping off is a nursery disease of great economic importance in papaya and seed treatment may be an effective measure to control. The aim of this work was to evaluate the quality of papaya seeds treated with fungicides and stored under two environmental and packaging conditions. Additionally, the efficiency of fungicide treatments in the control of damping-off caused by Rhizoctonia solani was evaluated. Papaya seeds were treated with the fungicides Captan, Tolylfluanid and the mixture Tolylfluanid + Captan (all commercial wettable powder formulations). Seeds of the control group were not treated. The seeds were stored for nine months in two conditions: packed in aluminum coated paper and kept at 7 ± 1ºC and in permeable kraft paper and kept in non-controlled environment. At the beginning of the storage and every three months the seed quality (germination and vigor tests), emergence rate index, height, dry mass and damping of plants in pre and post-emergence (in contaminated substrate and mycelia-free substrate) were analyzed. Both storage conditions as well as the fungicide treatments preserved the germination and seed vigor. In the infested substrate, seedling emergence was favored by fungicides, but in post-emergence, fungicides alone did not control the damping off caused by R. solani. Symptoms of damping off were not observed in the clean substrate. The results showed that the fungicide treatments may be used to pretreat papaya seed for long-term storage and commercialization.

Development of a SPME-GC-ECD methodology for selected pesticides in must and wine samples

A method for the determination of some pesticide residues in must and wine samples was developed using solid-phase microextraction (SPME) and gas chromatography – electron capture detection (GC/ECD). The procedure only needs dilution as sample pre-treatment and is therefore simple, fast and solvent-free. Eight fungicides (vinclozolin, procymidone, iprodione, penconazole, fenarimol, folpet, nuarimol and hexaconazole), one insecticide (chlorpyriphos) and two acaricides (bromopropylate and tetradifon) can be quantified. Good linearity was observed for all the compounds in the range 5–100 µg/L. The reproducibility of the measurements was found acceptable (with RSD’s below 20%). Detection limits of 11 µg/L, on average, are sufficiently below the proposed maximum residue limits (MRL’s) for these compounds in wine. The analytical method was applied to the determination of these compounds in Portuguese must and wine samples from the Demarcated Region of Alentejo, where any residues could be detected.

Determination of 24 pesticide residues in fortified wines by solid-phase microextraction and gas chromatography tandem mass spectrometry

The present work describes a solid-phase microextraction (SPME) gas chromatography_tandem mass spectrometry (MS/MS) method to quantify 24 pesticides in fortified white wine and fortified red wine. In this study “fortified wine” refers to a wine in which fermentation is arrested before completion by alcohol distillate addition, allowing sugar and alcoholic contents to be higher (around 80-100 g/L total sugars and 19-22% alcohol strength (v/v)). The analytical method showed good linearity, presenting correlation coefficients (R2) ≥ 0.989 for all compounds. Limits of detection (LOD) and quantitation (LOQ) in the ranges of 0.05-72.35 and 0.16-219.23 μg/L, respectively, were obtained. LOQs are below the maximum residue levels (MRL) set by European Regulation for grapes. The proposed method was applied to 17 commercial fortified wines. The analyzed pesticides were not detected in the wines tested.

Storage of organochlorine inseticides in hepatosplenic schistosomiasis

This paper reports on the findings of a survey on OCI storage in people in Bahia, Brasil. In this survey 11 samples of adipose tissue were taken from people who underwent splenectomy for hepato splenic schistosomiasis. In these patients, total DDT averaged 10.66 ppm as compared to 4,83 ppm of total DDT found in presumably healthy people. The difference was statistically significant. It is considered that the increased DDT storage in patients with hepatosplenic schistosomiass may be the result of a reduced metabolzation of p,p'-DDT and reduced excretion of its metabolites. Another possíbility is the increased exposure to organochlorine nsectedes by hepatosplenc patients who frequently come from irrigation zones, where these compounds are more used in intensive cultivatíon.

Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine.

Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R (2) > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.

Toxicokinetics of captan and folpet biomarkers in dermally exposed volunteers

To better assess biomonitoring data in workers exposed to captan and folpet, the kinetics of ring metabolites [tetrahydrophthalimide (THPI), phthalimide (PI) and phthalic acid] were determined in urine and plasma of dermally exposed volunteers. A 10  mg kg(-1) dose of each fungicide was applied on 80  cm(2) of the forearm and left without occlusion or washing for 24  h. Blood samples were withdrawn at fixed time periods over the 72  h following application and complete urine voids were collected over 96  h post-dosing, for metabolite analysis. In the hours following treatment, a progressive increase in plasma levels of THPI and PI was observed, with peak levels being reached at 24  h for THPI and 10  h for PI. The ensuing elimination phase appeared monophasic with a mean elimination half-life (t(½) ) of 24.7 and 29.7 h for THPI and PI, respectively. In urine, time courses PI and phthalic acid excretion rate rapidly evolved in parallel, and a mean elimination t(½) of 28.8 and 29.6  h, respectively, was calculated from these curves. THPI was eliminated slightly faster, with a mean t(½) of 18.7  h. Over the 96  h period post-application, metabolites were almost completely excreted, and on average 0.02% of captan dose was recovered in urine as THPI while 1.8% of the folpet dose was excreted as phthalic acid and 0.002% as PI, suggesting a low dermal absorption fraction for both fungicides. This study showed the potential use of THPI, PI and phthalic acid as key biomarkers of exposure to captan and folpet.

Toxicokinetics of captan and folpet biomarkers in orally exposed volunteers

The time courses of key biomarkers of exposure to captan and folpet was assessed in accessible biological matrices of orally exposed volunteers. Ten volunteers ingested 1 mg kg(-1) body weight of captan or folpet. Blood samples were withdrawn at fixed time periods over the 72 h following ingestion and complete urine voids were collected over 96 h post-dosing. The tetrahydrophthalimide (THPI) metabolite of captan along with the phthalimide (PI) and phthalic acid metabolites of folpet were then quantified in these samples. Plasma levels of THPI and PI increased progressively after ingestion, reaching peak values ~10 and 6 h post-dosing, respectively; subsequent elimination phase appeared monophasic with a mean elimination half-life (t(½) ) of 15.7 and 31.5 h, respectively. In urine, elimination rate time courses of PI and phthalic acid evolved in parallel, with respective t(½) of 27.3 and 27.6 h; relatively faster elimination was found for THPI, with mean t(½) of 11.7 h. However, phthalic acid was present in urine in 1000-fold higher amounts than PI. In the 96 h period post-treatment, on average 25% of folpet dose was excreted in urine as phthalic acid as compared with only 0.02% as PI. The corresponding value for THPI was 3.5%. Overall, THPI and PI appear as interesting biomarkers of recent exposure, with relatively short half-lives; their sensitivity to assess exposure in field studies should be further verified. Although not a metabolite specific to folpet, the concomitant use of phthalic acid as a major biomarker of exposure to folpet should also be considered. Copyright © 2011 John Wiley & Sons, Ltd.

Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin.

The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.

Influence of fungicide seed treatment on soybean nodulation and grain yield

Biological N2 fixation is a major factor contributing to the increased competitiveness of Brazilian soybeans on the international market. However, the contribution of this process may be limited by adverse conditions to symbiotic bacteria, such as fungicide seed treatments. This study aimed to evaluate the effects of the fungicides carbendazim + thiram and carboxin + thiram on soybean nodulation, plant growth and grain yield. Two field experiments were carried out in the Cerrado region of the State of Roraima, in a soil with a low organic matter content and no soybean bradyrhizobia. In 2005, seeds were treated with fungicide carbendazim + thiram and commercial inoculants containing the Bradyrhizobium elkanii strains SEMIA 5019 and SEMIA 587 and B. japonicum strains SEMIA 5079 and SEMIA 5080. In 2006, soybean seeds were treated with the fungicides carbendazim + thiram or carboxin + thiram and inoculated separately with each one of the four strains. The plants were evaluated for number of nodules and dry weight, shoot dry weight and total N accumulated in shoots 35 days after plant emergence, while grain yield and N grain content were determined at harvest. Both fungicides reduced soybean nodulation, especially in the presence of B. elkanii strains. The fungicide carbendazim + thiram reduced nodulation by about 50 % and grain yield by more than 20 % (about 700 kg ha-1), in the treatment inoculated with of strain SEMIA 587.

In-furrow inoculation of soybean as alternative to fungicide and micronutrient seed treatment

Soybean is a major grain crop in Brazil, and yields can be considerably improved by inoculation with selected Bradyrhizobium strains. However, the incompatibility between inoculation and seed treatments with fungicides and micronutrients represents a major barrier to the achievement of high rates of biological N2 fixation. Inoculation practices that can alleviate the negative effects of agrochemicals must therefore be found and in-furrow inoculation seems to be an attractive alternative. This study reports the results of seven field experiments conducted in three growing seasons in Brazil; three in soils previously cropped with inoculated soybean (> 10(4) cells g-1 of soil of Bradyrhizobium), and four in areas where the crop was sown for the first time (< 10² cells g-1 of soil of Bradyrhizobium). The compatibility with fungicides and micronutrients was compared in seeds inoculated with peat or liquid inoculants, or treated with different doses of liquid inoculant in-furrow. In areas with established Bradyrhizobium populations, seed-applied agrochemicals did generally not affect nodulation, but also did not increase yields, while inoculation always increased N grain accumulation or yield, and N fertilizer decreased both nodulation and yield. Where soybean was sown for the first time, the seed treatment with agrochemicals affected nodulation when applied together with peat or liquid inoculant. In-furrow inoculation alleviated the effects of seed treatment with agrochemicals; the best performance was achieved with high Bradyrhizobium cell concentrations, with up to 2.5 million cells seed-1.

A detailed urinary excretion time course study of captan and folpet biomarkers in workers for the estimation of dose, main route-of-entry and most appropriate sampling and analysis strategies

Captan and folpet are two fungicides largely used in agriculture, but biomonitoring data are mostly limited to measurements of captan metabolite concentrations in spot urine samples of workers, which complicate interpretation of results in terms of internal dose estimation, daily variations according to tasks performed, and most plausible routes of exposure. This study aimed at performing repeated biological measurements of exposure to captan and folpet in field workers (i) to better assess internal dose along with main routes-of-entry according to tasks and (ii) to establish most appropriate sampling and analysis strategies. The detailed urinary excretion time courses of specific and non-specific biomarkers of exposure to captan and folpet were established in tree farmers (n = 2) and grape growers (n = 3) over a typical workweek (seven consecutive days), including spraying and harvest activities. The impact of the expression of urinary measurements [excretion rate values adjusted or not for creatinine or cumulative amounts over given time periods (8, 12, and 24 h)] was evaluated. Absorbed doses and main routes-of-entry were then estimated from the 24-h cumulative urinary amounts through the use of a kinetic model. The time courses showed that exposure levels were higher during spraying than harvest activities. Model simulations also suggest a limited absorption in the studied workers and an exposure mostly through the dermal route. It further pointed out the advantage of expressing biomarker values in terms of body weight-adjusted amounts in repeated 24-h urine collections as compared to concentrations or excretion rates in spot samples, without the necessity for creatinine corrections.

Toxicokinetic modeling of folpet fungicide and its ring-biomarkers of exposure in humans.

A human in vivo toxicokinetic model was built to allow a better understanding of the toxicokinetics of folpet fungicide and its key ring biomarkers of exposure: phthalimide (PI), phthalamic acid (PAA) and phthalic acid (PA). Both PI and the sum of ring metabolites, expressed as PA equivalents (PAeq), may be used as biomarkers of exposure. The conceptual representation of the model was based on the analysis of the time course of these biomarkers in volunteers orally and dermally exposed to folpet. In the model, compartments were also used to represent the body burden of folpet and experimentally relevant PI, PAA and PA ring metabolites in blood and in key tissues as well as in excreta, hence urinary and feces. The time evolution of these biomarkers in each compartment of the model was then mathematically described by a system of coupled differential equations. The mathematical parameters of the model were then determined from best fits to the time courses of PI and PAeq in blood and urine of five volunteers administered orally 1 mg kg(-1) and dermally 10 mg kg(-1) of folpet. In the case of oral administration, the mean elimination half-life of PI from blood (through feces, urine or metabolism) was found to be 39.9 h as compared with 28.0 h for PAeq. In the case of a dermal application, mean elimination half-life of PI and PAeq was estimated to be 34.3 and 29.3 h, respectively. The average final fractions of administered dose recovered in urine as PI over the 0-96 h period were 0.030 and 0.002%, for oral and dermal exposure, respectively. Corresponding values for PAeq were 24.5 and 1.83%, respectively. Finally, the average clearance rate of PI from blood calculated from the oral and dermal data was 0.09 ± 0.03 and 0.13 ± 0.05 ml h(-1) while the volume of distribution was 4.30 ± 1.12 and 6.05 ± 2.22 l, respectively. It was not possible to obtain the corresponding values from PAeq data owing to the lack of blood time course data.