Evidence of non-DDD pathway in the anaerobic degradation of DDT in tropical soil.

DDT transformation to DDD in soil is the most commonly reported pathway under anaerobic conditions. A few instances of DDT conversion to products other than DDD/DDE have been reported under aerobic conditions and hardly any under anaerobic conditions. In particular, few reports exist on the anaerobic degradation of DDT in African tropical soils, despite DDT contamination arising from obsolete pesticide stockpiles in the continent as well as new contamination from DDT use for mosquito and tsetse fly control. Moreover, the development of possible remediation strategies for contaminated sites demands adequate understanding of different soil processes and their effect on DDT persistence, hence necessitating the study. The aim of this work was to study the effect of simulated anaerobic conditions and slow-release carbon sources (compost) on the dissipation of DDT in two tropical clay soils (paddy soil and field soil) amenable to periodic flooding. The results showed faster DDT dissipation in the field soil but higher metabolite formation in the paddy soil. To explain this paradox, the levels of dissolved organic carbon and carbon mineralization (CH4 and CO2) were correlated with p,p-DDT and p,p-DDD concentrations. It was concluded that DDT underwent reductive degradation (DDD pathway) in the paddy soil and both reductive (DDD pathway) and oxidative degradation (non-DDD pathway) in the field soil.

Large variation in glyphosate mineralization in 21 different agricultural soils explained by soil properties.

Glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) have frequently been detected in surface water and groundwaters. Since adequate glyphosate mineralization in soil may reduce its losses to environment, improved understanding of site specific factors underlying pesticide mineralization in soils is needed. The aim of this study was to investigate the relationship between soil properties and glyphosate mineralization. To establish a sound basis for resilient correlations, the study was conducted with a large number of 21 agricultural soils, differing in a variety of soil parameters, such as soil texture, soil organic matter content, pH, exchangeable ions etc. The mineralization experiments were carried out with 14C labelled glyphosate at a soil water tension of -15 kPa and at a soil density of 1.3 g cm-3 at 20 ±â€¯1 °C for an incubation period of 32 days. The results showed that the mineralization of glyphosate in different agricultural soils varied to a great extent, from 7 to 70% of the amount initially applied. Glyphosate mineralization started immediately after application, the highest mineralization rates were observed within the first 4 days in most of the 21 soils. Multiple regression analysis revealed exchangeable acidity (H+ and Al3+), exchangeable Ca2+ ions and ammonium lactate extractable K to be the key soil parameters governing glyphosate mineralization in the examined soils. A highly significant negative correlation between mineralized glyphosate and NaOH-extractable residues (NaOH-ER) in soils strongly suggests that NaOH-ER could be used as a simple and reliable parameter for evaluating the glyphosate mineralization capacity. The NaOH-ER were composed of glyphosate, unknown 14C-residues, and AMPA (12%-65%, 3%-34%, 0%-11% of applied 14C, respectively). Our results highlighted the influential role of soil exchangeable acidity, which should therefore be considered in pesticide risk assessments and management to limit efficiently the environmental transfers of glyphosate.

Bacteria utilizing plant-derived carbon in the rhizosphere of Triticum aestivum change in different depths of an arable soil.

Root exudates shape microbial communities at the plant-soil interface. Here we compared bacterial communities that utilize plant-derived carbon in the rhizosphere of wheat in different soil depths, including topsoil, as well as two subsoil layers up to 1 m depth. The experiment was performed in a greenhouse using soil monoliths with intact soil structure taken from an agricultural field. To identify bacteria utilizing plant-derived carbon, 13 C-CO2 labelling of plants was performed for two weeks at the EC50 stage, followed by isopycnic density gradient centrifugation of extracted DNA from the rhizosphere combined with 16S rRNA gene-based amplicon sequencing. Our findings suggest substantially different bacterial key players and interaction mechanisms between plants and bacteria utilizing plant-derived carbon in the rhizosphere of subsoils and topsoil. Among the three soil depths, clear differences were found in 13 C enrichment pattern across abundant operational taxonomic units (OTUs). Whereas, OTUs linked to Proteobacteria were enriched in 13 C mainly in the topsoil, in both subsoil layers OTUs related to Cohnella, Paenibacillus, Flavobacterium showed a clear 13 C signal, indicating an important, so far overseen role of Firmicutes and Bacteriodetes in the subsoil rhizosphere.

Enhanced degradation of isoproturon in an agricultural soil by a Sphingomonas sp. strain and a microbial consortium.

Isoproturon (IPU) degradation in an agricultural soil inoculated with an isolated IPU-degrader strain (Sphingomonas sp. strain AK1, IS) or a microbial consortium (MC) harboring this strain, with or without carrier material, were investigated in soil microcosm experiments during 46 days. Effect of the carrier material and inoculation size on IPU-degradation efficacy of the inoculants were studied. Mineralization, extractable residues and non-extractable residues of 14C-labeled IPU were analyzed. The low IPU mineralization in untreated soil (7.0%) was enhanced to different extents by inoculation of IS (17.4%-46.0%) or MC (58.9%-67.5%). Concentrations of IPU residues in soils amended with MC (0.002-0.095 µg g dry soil-1) were significantly lower than in soils amended with IS (0.02-0.67 µg g dry soil-1) and approximately 10 times lower than in the uninoculated soil (0.06-0.80 µg g dry soil-1). Less extractable residues and non-extractable residues were detected in soil with higher IPU mineralization. Inoculation size (as indicated by the volume of liquid cultures or by the number of carrier particles) determined the IPU-removal efficacy of IS in soil, but this effect was less pronounced for MC. The low sorption of IPU to soil and the decreasing IPU-mineralizing rates suggested incapability of IS to establish the IPU-mineralizing function in the soil. The thorough removal of IPU and persistent IPU-mineralizing activity of soil inoculated with MC indicated a high persistence of IPU-metabolic trait. Our results showed that microbial consortia might be more efficient than single degrader strains to enhance clean-up of organic chemicals in soil.

Persistence and dioxin-like toxicity of carbazole and chlorocarbazoles in soil.

Halogenated carbazoles have recently been detected in soil and water samples, but their environmental effects and fate are unknown. Eighty-four soil samples obtained from a site with no recorded history of pollution were used to assess the persistence and dioxin-like toxicity of carbazole and chlorocarbazoles in soil under controlled conditions for 15 months. Soil samples were divided into two temperature conditions, 15 and 20 °C, both under fluctuating soil moisture conditions comprising 19 and 44 drying-rewetting cycles, respectively. This was characterized by natural water loss by evaporation and rewetting to -15 kPa. Accelerated solvent extraction (ASE) and cleanup were performed after incubation. Identification and quantification were done using high-resolution gas chromatogram/mass spectrometer (HRGC/MS), while dioxin-like toxicity was determined by ethoxyresorufin-O-deethylase (EROD) induction in H4IIA rat hepatoma cells assay and multidimensional quantitative structure-activity relationships (mQSAR) modelling. Carbazole, 3-chlorocarbazole and 3,6-dichlorocarbazole were detected including trichlorocarbazole not previously reported in soils. Carbazole and 3-chlorocarbazole showed significant dissipation at 15 °C but not at 20 °C incubating conditions indicating that low temperature could be suitable for dissipation of carbazole and chlorocarbazoles. 3,6-Dichlorocarbazole was resistant at both conditions. Trichlorocarbazole however exhibited a tendency to increase in concentration with time. 3-Chlorocarbazole, 3,6-dibromocarbazole and selected soil extracts exhibited EROD activity. Dioxin-like toxicity did not decrease significantly with time, whereas the sum chlorocarbazole toxic equivalence concentrations (∑TEQ) did not contribute significantly to the soil assay dioxin-like toxicity equivalent concentrations (TCDD-EQ). Carbazole and chlorocarbazoles are persistent with the latter also toxic in natural conditions.

Energy-dependent uptake of benzo[a]pyrene and its cytoskeleton-dependent intracellular transport by the telluric fungus Fusarium solani.

In screening indigenous soil filamentous fungi for polycyclic aromatic hydrocarbons (PAHs) degradation, an isolate of the Fusarium solani was found to incorporate benzo[a]pyrene (BaP) into fungal hyphae before degradation and mineralization. The mechanisms involved in BaP uptake and intracellular transport remain unresolved. To address this, the incorporation of two PAHs, BaP, and phenanthrene (PHE) were studied in this fungus. The fungus incorporated more BaP into cells than PHE, despite the 400-fold higher aqueous solubility of PHE compared with BaP, indicating that PAH incorporation is not based on a simple diffusion mechanism. To identify the mechanism of BaP incorporation and transport, microscopic studies were undertaken with the fluorescence probes Congo Red, BODIPY®493/503, and FM®4-64, targeting different cell compartments respectively fungal cell walls, lipids, and endocytosis. The metabolic inhibitor sodium azide at 100 mM totally blocked BaP incorporation into fungal cells indicating an energy-requirement for PAH uptake into the mycelium. Cytochalasins also inhibited BaP uptake by the fungus and probably its intracellular transport into fungal hyphae. The perfect co-localization of BaP and BODIPY reveals that lipid bodies constitute the intracellular storage sites of BaP in F. solani. Our results demonstrate an energy-dependent uptake of BaP and its cytoskeleton-dependent intracellular transport by F. solani.

Assessment of the viability of Cryptosporidium parvum oocysts with the induction ratio of hsp70 mRNA production in manure.

Determining the presence of viable Cryptosporidium parvum oocysts in complex environmental matrices in hygiene control can prevent the contamination of water resources and food with this pathogen. This study assessed the induction ratio of hsp70 mRNA production by heat shock in different oocysts as a marker of viability. Using different procedures for (m)RNA extraction directly from manure and reverse transcription real-time qPCR, this study found slightly increased hsp70 mRNA contents in viable oocysts that were heat shock induced at 45°C for 20 min compared to not induced oocysts (1.6 fold induction in average). Prolonging the heat shock treatment to 2h did not further increase the copy numbers. Heat shock by consecutive stimuli, such as freezing and then heating, did not yield significantly higher copy numbers than the 45°C treatment. There was a certain background level of hsp70 mRNA in viable oocysts that were not exposed to heat shock, indicating a constitutive production of the transcripts in the oocysts. The production of hsp70 mRNA induced by heat shock in oocysts aged for 9 months that exhibited reduced viability was lower than in fresher oocysts (induction ratio<1.2). No production of hsp70 mRNA by heat shock was detected in 12 months old oocysts that were not viable in the excystation test. Oocysts inactivated at 75°C for 30 min were not able to respond to heat shock, and low amount of copies were occasionally measured only in total RNA extracts, but not in mRNA extracts that were purified directly with an oligo (dT)25 based system. The induction ratio of hsp70 mRNA varied according to the viability of the organisms in a sample. Copy numbers of ß-tubulin mRNA in viable oocysts were lower than hsp70 mRNA, therefore the latter is more suitable to detect low numbers of oocysts by RT-qPCR.

Enhanced degradation of 14C-HCB in two tropical clay soils using multiple anaerobic-aerobic cycles.

The aim of the study was to induce and enhance the degradation of hexachlorobenzene (HCB), a highly-chlorinated persistent organic pollutant, in two ecologically different tropical soils: a paddy soil (PS) and a non-paddy soil (FS). The degradation of HCB was enhanced using two anaerobic-aerobic cycles in model laboratory experiments. There was greater degradation of HCB in the PS (half-life of 224 days) relative to the FS (half-life of 286 days). It was further shown that soils amended with compost had higher metabolite concentrations relative to the non-amended soils. In the first cycle, there was little degradation of HCB in both soils. However, in the second cycle, there was enhanced mineralization in the PS under aerobic conditions, with the compost-treated samples showing higher mineralization. There was also extensive volatilization in both soils. The metabolite pattern revealed that the increased mineralization and volatilization was due to the formation of lower chlorinated benzenes.

Abundance and diversity of ammonia-oxidizing prokaryotes in the root-rhizosphere complex of Miscanthus × giganteus grown in heavy metal-contaminated soils.

Mine wastes have been considered as a source of heavy metal (HM) contamination in the environment and negatively impact many important ecosystem services provided by soils. Plants like Miscanthus, which tolerate high HM concentrations in soil, are often used for phytoremediation and provide the possibility to use these soils at least for the production of energy crops. However, it is not clear if plant growth at these sites is limited by the availability of nutrients, mainly nitrogen, as microbes in soil might be affected by the contaminant. Therefore, in this study, we investigated in a greenhouse experiment the response of ammonia-oxidizing microbes in the root-rhizosphere complex of Miscanthus × giganteus grown in soils with different levels of long-term arsenic (As) and lead (Pb) contamination. Quantitative PCR of the ammonia monooxigenease gene (amoA) was performed to assess the abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) at two different points of plant growth. Furthermore, bulk soil samples before planting were analyzed. In addition, terminal restriction fragment length polymorphism (T-RFLP) analysis was used to investigate the diversity of archaeal amoA amplicons. Whereas high concentrations of As and Pb in soil (83 and 15 g/kg, respectively) resulted independent from plant growth in a clear reduction of AOA and AOB compared to the control soils with lower HM contents, in soils with contamination levels of 10 g/kg As and 0.2 g/kg Pb, only AOB were negatively affected in bulk soil samples. Diversity analysis of archaeal amoA genes revealed clear differences in T-RFLP patterns in response to the degree of HM contamination. Therefore, our results could clearly prove the different response patterns of AOA and AOB in HM-contaminated soils and the development of archaeal amoA phylotypes which are more tolerant towards HMs in soil samples from the areas that were impacted the most by mining waste, which could contribute to functional redundancy of ammonia-oxidizing microbes in soils and stability of nitrification pattern.

Incorporation of carbon from decomposing litter of two pioneer plant species into microbial communities of the detritusphere.

Initial ecosystems are characterized by a low availability of nutrients and a low soil organic matter content. Interactions of plants and microorganisms in such environments, particularly in relation to litter decomposition, are very important for further ecosystem development. In a litter decomposition study using an initial substrate from a former mining area, we applied the litter of two contrasting pioneer plant species (legume vs. pasture plants), Lotus corniculatus and Calamagrostis epigejos, which are commonly observed in the study area. Litter decomposition was investigated and carbon (C) translocation from litter into soil microorganisms was described by following (13) C from labelled plant litter materials into the fraction of phospholipid fatty acids. Labile C compounds of both plant litter types were easily degraded during the first 4 weeks of litter decomposition. In contrast to climax ecosystems, where the importance of fungi for litter degradation has been shown in many studies, in our experiment, data clearly indicate an outcompetition of fungi by Gram-positive bacteria as soon as available nitrogen is limited in the detritusphere.

Effects of genetically modified starch metabolism in potato plants on photosynthate fluxes into the rhizosphere and on microbial degraders of root exudates.

A high percentage of photosynthetically assimilated carbon is released into soil via root exudates, which are acknowledged as the most important factor for the development of microbial rhizosphere communities. As quality and quantity of root exudates are dependent on plant genotype, the genetic engineering of plants might also influence carbon partitioning within the plant and thus microbial rhizosphere community structure. In this study, the carbon allocation patterns within the plant-rhizosphere system of a genetically modified amylopectin-accumulating potato line (Solanum tuberosum L.) were linked to microbial degraders of root exudates under greenhouse conditions, using (13)C-CO(2) pulse-chase labelling in combination with phospholipid fatty acid (PLFA) analysis. In addition, GM plants were compared with the parental cultivar as well as a second potato cultivar obtained by classical breeding. Rhizosphere samples were obtained during young leaf developmental and flowering stages. (13)C allocation in aboveground plant biomass, water-extractable organic carbon, microbial biomass carbon and PLFA as well as the microbial community structure in the rhizosphere varied significantly between the natural potato cultivars. However, no differences between the GM line and its parental cultivar were observed. Besides the considerable impact of plant cultivar, the plant developmental stage affected carbon partitioning via the plant into the rhizosphere and, subsequently, microbial communities involved in the transformation of root exudates.

Impact of soil water regime on degradation and plant uptake behaviour of the herbicide isoproturon in different soil types.

The environmental fate of the worldwide used herbicide isoproturon was studied in four different, undisturbed lysimeters in the temperate zone of Middle Europe. To exclude climatic effects due to location, soils were collected at different regions in southern Germany and analyzed at a lysimeter station under identical environmental conditions. (14)C-isoproturon mineralization varied between 2.59% and 57.95% in the different soils. Barley plants grown on these lysimeters accumulated (14)C-pesticide residues from soil in partially high amounts and emitted (14)CO(2) in an extent between 2.01% and 13.65% of the applied (14)C-pesticide. Plant uptake and (14)CO(2) emissions from plants were inversely linked to the mineralization of the pesticide in the various soils: High isoproturon mineralization in soil resulted in low plant uptake whereas low isoproturon mineralization in soil resulted in high uptake of isoproturon residues in crop plants and high (14)CO(2) emission from plant surfaces. The soil water regime was identified as an essential factor that regulates degradation and plant uptake of isoproturon whereby the intensity of the impact of this factor is strongly dependent on the soil type.

Comparison of barley succession and take-all disease as environmental factors shaping the rhizobacterial community during take-all decline.

The root disease take-all, caused by Gaeumannomyces graminis var. tritici, can be managed by monoculture-induced take-all decline (TAD). This natural biocontrol mechanism typically occurs after a take-all outbreak and is believed to arise from an enrichment of antagonistic populations in the rhizosphere. However, it is not known whether these changes are induced by the monoculture or by ecological rhizosphere conditions due to a disease outbreak and subsequent attenuation. This question was addressed by comparing the rhizosphere microflora of barley, either inoculated with the pathogen or noninoculated, in a microcosm experiment in five consecutive vegetation cycles. TAD occurred in soil inoculated with the pathogen but not in noninoculated soil. Bacterial community analysis using terminal restriction fragment length polymorphism of 16S rRNA showed pronounced population shifts in the successive vegetation cycles, but pathogen inoculation had little effect. To elucidate rhizobacterial dynamics during TAD development, a 16S rRNA-based taxonomic microarray was used. Actinobacteria were the prevailing indicators in the first vegetation cycle, whereas the third cycle-affected most severely by take-all-was characterized by Proteobacteria, Bacteroidetes, Chloroflexi, Planctomycetes, and Acidobacteria. Indicator taxa for the last cycle (TAD) belonged exclusively to Proteobacteria, including several genera with known biocontrol traits. Our results suggest that TAD involves monoculture-induced enrichment of plant-beneficial taxa.

Abundance of microbes involved in nitrogen transformation in the rhizosphere of Leucanthemopsis alpina (L.) Heywood grown in soils from different sites of the Damma glacier forefield.

Glacier forefields are an ideal playground to investigate the role of development stages of soils on the formation of plant-microbe interactions as within the last decades, many alpine glaciers retreated, whereby releasing and exposing parent material for soil development. Especially the status of macronutrients like nitrogen differs between soils of different development stages in these environments and may influence plant growth significantly. Thus, in this study, we reconstructed major parts of the nitrogen cycle in the rhizosphere soil/root system of Leucanthemopsis alpina (L.) HEYWOOD: as well as the corresponding bulk soil by quantifying functional genes of nitrogen fixation (nifH), nitrogen mineralisation (chiA, aprA), nitrification (amoA AOB, amoA AOA) and denitrification (nirS, nirK and nosZ) in a 10-year and a 120-year ice-free soil of the Damma glacier forefield. We linked the results to the ammonium and nitrate concentrations of the soils as well as to the nitrogen and carbon status of the plants. The experiment was performed in a greenhouse simulating the climatic conditions of the glacier forefield. Samples were taken after 7 and 13 weeks of plant growth. Highest nifH gene abundance in connection with lowest nitrogen content of L. alpina was observed in the 10-year soil after 7 weeks of plant growth, demonstrating the important role of associative nitrogen fixation for plant development in this soil. In contrast, in the 120-year soil copy numbers of genes involved in denitrification, mainly nosZ were increased after 13 weeks of plant growth, indicating an overall increased microbial activity status as well as higher concentrations of nitrate in this soil.

Degradation capacity of a 1,2,4-trichlorobenzene mineralizing microbial community for traces of organochlorine pesticides.

A soil-borne microbial community isolated from a contaminated site was previously shown to mineralize 1,2,4-trichlorobenzene (1,2,4-TCB) under aerobic conditions. The key degrader in this community was identified as Bordetella sp. F2. The objective of the study was to test the capacity of the microbial community to degrade a complex mixture of 27 organochlorine compounds and pesticides (OCPs) commonly detected in the environment. The hypothesis was that the microbes would utilize the OCPs as carbon sources at the low concentrations of these compounds, found in natural waters and soil solution. The study was carried out in liquid culture and the changes in concentration of the OCPs were monitored using GC-MS. Data analysis was done using a multivariate analysis method similar to Principal Response Curve (PRC) analysis. Contrary to expectations, the data analysis showed a general trend where higher concentrations were observed in the microbially treated samples relative to the controls. The observed trend was attributed to decreased volatilization due to sorption of the chemicals by microbes since most of the compounds in the cocktail had high Kow values. Nevertheless, when using adequate statistical methods for analysing the very complex data set, correlation of Kow and K(H) values with the loadings of the PRCs showed that three chlorinated mono-aromatics - pentachlorobenzene, pentachloroanisole and octachloroanisole - were amenable to degradation. This provided indications that the community could hold promise for the degradation of higher-chlorinated mono-aromatic OCPs.

A comparative study of most probable number (MPN)-PCR vs. real-time-PCR for the measurement of abundance and assessment of diversity of alkB homologous genes in soil.

The utilization of quantitative PCR (qPCR) approaches such as MPN-qPCR and real-time-qPCR for in situ assessment of functional genes yields substantial quantitative and qualitative differences. We show this by targeting the alkB gene related to biodegradation of aliphatic alkanes in a set of environmental samples with differing hydrocarbon content.

Batch experiments versus soil pore water extraction--what makes the difference in isoproturon (bio-)availability?

Two approaches to determine pesticide (bio-)availability in soils (i) batch experiments with "extraction with an excess of water" (EEW) and (ii) the recently introduced "soil pore water (PW) extraction" of pesticide incubated soil samples have been compared with regard to the sorption behavior of the model compound isoproturon in soils. A significant correlation between TOC and adsorbed pesticide amount was found when using the EEW approach. In contrast, there was no correlation between TOC and adsorbed isoproturon when using the in situ PW extraction method. Furthermore, sorption was higher at all concentrations in the EEW method when comparing the distribution coefficients (K(d)) for both methods. Over all, sorption in incubated soil samples at an identical water tension (-15 kPa) and soil density (1.3 g cm(-3)) appears to be controlled by a complex combination of sorption driving soil parameters. Isoproturon bioavailability was found to be governed in different soils by binding strength and availability of sorption sites as well as water content, whereas the dominance of either one of these factors seems to depend on the individual composition and characteristics of the respective soil sample. Using multiple linear regression analysis we obtained furthermore indications that the soil pore structure is affected by the EEW method due to disaggregation, resulting in a higher availability of pesticide sorption sites than in undisturbed soil samples. Therefore, it can be concluded that isoproturon sorption is overestimated when using the EEW method, which should be taken into account when using data from this approach or similar batch techniques for risk assessment analysis.

Quantification of key genes steering the microbial nitrogen cycle in the rhizosphere of sorghum cultivars in tropical agroecosystems.

The effect of agricultural management practices on geochemical cycles in moderate ecosystems is by far better understood than in semiarid regions, where fertilizer availability and climatic conditions are less favorable. We studied the impact of different fertilizer regimens in an agricultural long-term observatory in Burkina Faso at three different plant development stages (early leaf development, flowering, and senescence) of sorghum cultivars. Using real-time PCR, we investigated functional microbial communities involved in key processes of the nitrogen cycle (nitrogen fixation, ammonia oxidation, and denitrification) in the rhizosphere. The results indicate that fertilizer treatments and plant development stages combined with environmental factors affected the abundance of the targeted functional genes in the rhizosphere. While nitrogen-fixing populations dominated the investigated communities when organic fertilizers (manure and straw) were applied, their numbers were comparatively reduced in urea-treated plots. In contrast, ammonia-oxidizing bacteria (AOB) increased not only in absolute numbers but also in relation to the other bacterial groups investigated in the urea-amended plots. Ammonia-oxidizing archaea exhibited higher numbers compared to AOB independent of fertilizer application. Similarly, denitrifiers were also more abundant in the urea-treated plots. Our data imply as well that, more than in moderate regions, water availability might shape microbial communities in the rhizosphere, since low gene abundance data were obtained for all tested genes at the flowering stage, when water availability was very limited.

Evaluation of two methods for quantification of hsp70 mRNA from the waterborne pathogen Cryptosporidium parvum by reverse transcription real-time PCR in environmental samples.

We optimized and evaluated two mRNA extraction methods to quantify induced hsp70 mRNA from viable and injured Cryptosporidium parvum oocysts by reverse transcription quantitative real-time PCR (RT-qPCR) in raw and treated manure. Methods based on guanidinium isothiocyanate/phenol/chloroform (GITC-PC) purification and direct mRNA extraction with magnetic oligo(dT)25-coated beads were evaluated for applicability and sensitivity. Both methods proved to be suitable for processing manure samples. With washed manure samples and oocyst disruption by bead beating for 165 s in time intervals with cumulative pooling of the lysate fractions, optimum RT-qPCR results were achieved. On average, 2.6 times more hsp70 mRNA was detected with the oligo(dT)25 method in comparison to the GITC-PC based method using fresh oocysts, whereas less mRNA was detected in aged oocysts. For fresh oocysts, analytical and method detection limits for the oligo(dT)25 based method were 1.7 cDNA copies/qPCR reaction and 5150 oocysts/mL manure, and for the GITC-PC based method 17 cDNA copies/qPCR reaction and 4950 oocysts/mL, respectively. In 12 months old oocysts with reduced viability, mRNA was occasionally detected only by the GITC-PC based method. Failure of or reduced detection with the oligo(dT)25 based method was apparently a result of weakened oocyst walls leading to quicker release of mRNA and therefore mRNA shredding by bead beating in the relatively long stretch between the capture sequence and the RT-qPCR target sites.