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Importance: Synthetic nitrification inhibitors are routinely used with nitrogen fertilizers to reduce nitrogen losses from agroecosystems, despite having drawbacks like poor efficiency, cost, and entry into the food chain. Plant-derived BNIs constitute a more environmentally conducive alternative. Knowledge on the activity of BNIs to soil nitrifiers is largely based on bioassays with a single Nitrosomonas europaea strain which does not constitute a dominant member of the community of ammonia-oxidizing microorganisms (AOM) in soil. We determined the activity of several plant-derived molecules reported as having activity, including the recently discovered maize-isolated BNI, zeanone, and its natural analog, 2-methoxy-1,4-naphthoquinone, on a range of ecologically relevant AOM and one nitrite-oxidizing bacterial culture, expanding our knowledge on the intrinsic inhibition potential of BNIs toward AOM and highlighting the necessity for a deeper understanding of the effect of BNIs on the overall soil microbiome integrity before their further use in agricultural settings., Competing Interests: The authors declare no conflict of interest.

Dicyandiamide (DCD) and nitrapyrin (NP) are nitrification inhibitors (NIs) used in agriculture for over 40 years. Recently, ethoxyquin (EQ) was proposed as a novel potential NI, acting through its derivative quinone imine (QI). Still, the specific activity of these NIs on the different groups of ammonia-oxidizing microorganisms (AOM), and mostly their effects on other soil microbiota remain unknown. We determined the impact of QI, and comparatively of DCD and NP, applied at two doses (regular versus high), on the function, diversity, and dynamics of target (AOM), functionally associated (nitrite-oxidizing bacteria-NOB), and off-target prokaryotic and fungal communities in two soils mainly differing in pH (5.4 versus 7.9). QI was equally effective to DCD but more effective than NP in inhibiting nitrification in the acidic soil, while in the alkaline soil QI was less efficient than DCD and NP. This was attributed to the higher activity of QI toward AOA prevailing in the acidic soil. All NIs induced significant effects on the composition of the AOB community in both soils, unlike AOA, which were less responsive. Beyond on-target effects, we noted an inhibitory effect of all NIs on the abundance of NOB in the alkaline soil, with Nitrobacter being more sensitive than Nitrospira . QI, unlike the other NIs, induced significant changes in the composition of the bacterial and fungal communities in both soils. Our findings have serious implications for the efficiency and future use of NIs on agriculture and provide unprecedented evidence for the potential off-target effects of NIs on soil microbiota. IMPORTANCE NIs could improve N use efficiency and decelerate N cycling. Still, we know little about their activity on the distinct AOM groups and about their effects on off-target soil microorganisms. Here, we studied the behavior of a new potent NI, QI, compared to established NIs. We show that (i) the variable efficacy of NIs across soils with different pH reflects differences in the inherent specific activity of the NIs to AOA and AOB; (ii) beyond AOM, NIs exhibit negative effects on other nitrifiers, like NOB; (iii) QI was the sole NI that significantly affected prokaryotic and fungal diversity. Our findings (i) highlight the need for novel NI strategies that consider the variable sensitivity of AOM groups to the different NIs (ii) identify QI as a potent AOA inhibitor, and (iii) stress the need for monitoring NIs' impact on off-target soil microorganisms to ensure sustainable N fertilizers use and soil ecosystem functioning.

Nitrification inhibitors (NIs) applied to soil reduce nitrogen fertilizer losses from agr o -ecosystems. NIs that are currently registered for use in agriculture appear to selectively inhibit ammonia-oxidizing bacteria (AOB), while their impact on other nitrifiers is limited or unknown. Ethoxyquin (EQ), a fruit preservative shown to inhibit ammonia-oxidizers (AO) in soil, is rapidly transformed to 2,6-dihydro-2,2,4-trimethyl-6-quinone imine (QI), and 2,4-dimethyl-6-ethoxy-quinoline (EQNL). We compared the inhibitory potential of EQ and its derivatives with that of dicyandiamide (DCD), nitrapyrin (NP), and 3,4-dimethylpyrazole-phosphate (DMPP), NIs that have been used in agricultural settings. The effect of each compound on the growth of AOB ( Nitrosomonas europaea, Nitrosospira multiformis ), ammonia-oxidizing archaea (AOA; " Candidatus Nitrosocosmicus franklandus," " Candidatus Nitrosotalea sinensis"), and a nitrite-oxidizing bacterium (NOB; Nitrobacter sp. NHB1), all being soil isolates, were determined in liquid culture over a range of concentrations by measuring nitrite production or consumption and qPCR of amoA and nxrB genes, respectively. The degradation of NIs in the liquid cultures was also determined. In all cultures, EQ was transformed to the short-lived QI (major derivative) and the persistent EQNL (minor derivative). They all showed significantly higher inhibition activity of AOA compared to AOB and NOB isolates. QI was the most potent AOA inhibitor (EC 50 = 0.3-0.7 μM) compared to EQ (EC 50 = 1-1.4 μM) and EQNL (EC 50 = 26.6-129.5 μM). The formation and concentration of QI in EQ-amended cultures correlated with the inhibition patterns for all isolates suggesting that it was primarily responsible for inhibition after application of EQ. DCD and DMPP showed greater inhibition of AOB compared to AOA or NOB, with DMPP being more potent (EC 50 = 221.9-248.7 μM vs EC 50 = 0.6-2.1 μM). NP was the only NI to which both AOA and AOB were equally sensitive with EC 50s of 0.8-2.1 and 1.0-6.7 μM, respectively. Overall, EQ, QI, and NP were the most potent NIs against AOA, NP, and DMPP were the most effective against AOB, while NP, EQ and its derivatives showed the highest activity against the NOB isolate. Our findings benchmark the activity range of known and novel NIs with practical implications for their use in agriculture and the development of NIs with broad or complementary activity against all AO., (Copyright © 2020 Papadopoulou, Bachtsevani, Lampronikou, Adamou, Katsaouni, Vasileiadis, Thion, Menkissoglu-Spiroudi, Nicol and Karpouzas.)

Auxotrophy to amino acids and vitamins is a common feature in the bacterial world shaping microbial communities through cross-feeding relations. The amino acid auxotrophy of pollutant-degrading bacteria could hamper their bioremediation potential, however, the underlying mechanisms of auxotrophy remain unexplored. We employed genome sequence-based metabolic reconstruction to identify potential mechanisms driving the amino acid auxotrophy of a Sphingomonas haloaromaticamans strain degrading the fungicide ortho -phenylphenol (OPP) and provided further verification for the identified mechanisms via in vitro bacterial assays. The analysis identified potential gaps in the biosynthesis of isoleucine, phenylalanine and tyrosine, while methionine biosynthesis was potentially effective, relying though in the presence of B12. Supplementation of the bacterium with the four amino acids in all possible combinations rescued its degrading capacity only with methionine. Genome sequence-based metabolic reconstruction and analysis suggested that the bacterium was incapable of de novo biosynthesis of B12 (missing genes for the construction of the corrin ring) but carried a complete salvage pathway for corrinoids uptake from the environment, transmembrane transportation and biosynthesis of B12. In line with this the bacterium maintained its degrading capacity and growth when supplied with environmentally relevant B12 concentrations (i.e., 0.1 ng ml -1 ). Using genome-based metabolic reconstruction and in vitro testing we unraveled the mechanism driving the auxotrophy of a pesticide-degrading S. haloaromaticamans . Further studies will investigate the corrinoids preferences of S. haloaromaticamans for optimum growth and OPP degradation., (Copyright © 2020 Perruchon, Vasileiadis, Papadopoulou and Karpouzas.)

Pesticides are key stressors of soil microorganisms with reciprocal effects on ecosystem functioning. These effects have been mainly attributed to the parent compounds, while the impact of their transformation products (TPs) has been largely overlooked. We assessed in a meadow soil (soil A) the transformation of iprodione and its toxicity in relation to (i) the abundance of functional microbial groups, (ii) the activity of key microbial enzymes, and (iii) the diversity of bacteria, fungi, and ammonia-oxidizing microorganisms (AOM) using amplicon sequencing. 3,5-Dichloroaniline (3,5-DCA), the main iprodione TP, was identified as a key explanatory factor for the persistent reduction in enzymatic activities and potential nitrification (PN) and for the observed structural changes in the bacterial and fungal communities. The abundances of certain bacterial ( Actinobacteria , Hyphomicrobiaceae , Ilumatobacter , and Solirubrobacter ) and fungal ( Pichiaceae ) groups were negatively correlated with 3,5-DCA. A subsequent study in a fallow agricultural soil (soil B) showed limited formation of 3,5-DCA, which concurred with the lack of effects on nitrification. Direct 3,5-DCA application in soil B induced a dose-dependent reduction of PN and NO 3 - -N, which recovered with time. In vitro assays with terrestrial AOM verified the greater toxicity of 3,5-DCA over iprodione. " Candidatus Nitrosotalea sinensis" Nd2 was the most sensitive AOM to both compounds. Our findings build on previous evidence on the sensitivity of AOM to pesticides, reinforcing their potential utilization as indicators of the soil microbial toxicity of pesticides in pesticide environmental risk analysis and stressing the need to consider the contribution of TPs in the toxicity of pesticides on the soil microbial community. IMPORTANCE Pesticide toxicity on soil microorganisms is an emerging issue in pesticide risk assessment, dictated by the pivotal role of soil microorganisms in ecosystem services. However, the focus has traditionally been on parent compounds, while transformation products (TPs) are largely overlooked. We tested the hypothesis that TPs can be major contributors to the soil microbial toxicity of pesticides using iprodione and its main TP, 3,5-dichloroaniline, as model compounds. We demonstrated, by measuring functional and structural endpoints, that 3,5-dichloroaniline and not iprodione was associated with adverse effects on soil microorganisms, with nitrification being mostly affected. Pioneering in vitro assays with relevant ammonia-oxidizing bacteria and archaea verified the greater toxicity of 3,5-dichloroaniline. Our findings are expected to advance environmental risk assessment, highlighting the potential of ammonia-oxidizing microorganisms as indicators of the soil microbial toxicity of pesticides and stressing the need to consider the contribution of TPs to pesticide soil microbial toxicity., (Copyright © 2018 American Society for Microbiology.)

Pesticides are intentionally applied to agricultural fields for crop protection. They can harm non-target organisms such as soil microorganisms involved in important ecosystem functions with impacts at the global scale. Within the frame of the pesticide registration process, the ecotoxicological impact of pesticides on soil microorganisms is still based on carbon and nitrogen mineralization tests, despite the availability of more extensive approaches analyzing the abundance, activity or diversity of soil microorganisms. In this study, we used a high-density DNA microarray (PhyloChip) and 16S rDNA amplicon next-generation sequencing (NGS) to analyze the impact of the organophosphate insecticide chlorpyrifos (CHL), the phenyl-urea herbicide isoproturon (IPU), or the triazole fungicide tebuconazole (TCZ) on the diversity and composition of the soil bacterial community. To our knowledge, it is the first time that the combination of these approaches are applied to assess the impact of these three pesticides in a lab-to-field experimental design. The PhyloChip analysis revealed that although no significant changes in the composition of the bacterial community were observed in soil microcosms exposed to the pesticides, significant differences in detected operational taxonomic units (OTUs) were observed in the field experiment between pesticide treatments and control for all three tested pesticides after 70 days of exposure. NGS revealed that the bacterial diversity and composition varied over time. This trend was more marked in the microcosm than in the field study. Only slight but significant transient effects of CHL or TCZ were observed in the microcosm and the field study, respectively. IPU was not found to significantly modify the soil bacterial diversity or composition. Our results are in accordance with conclusions of the Environmental Food Safety Authority (EFSA), which concluded that these three pesticides may have a low risk toward soil microorganisms.

Diphenylamine (DPA) is a common soil and water contaminant. A Pseudomonas putida strain, recently isolated from a wastewater disposal site, was efficient in degrading DPA. Thorough knowledge of the metabolic capacity, genetic stability and physiology of bacteria during biodegradation of pollutants is essential for their future industrial exploitation. We employed genomic, proteomic, transcription analyses and plasmid curing to (i) identify the genetic network of P. putida driving the microbial transformation of DPA and explore its evolution and origin and (ii) investigate the physiological response of bacterial cells during degradation of DPA. Genomic analysis identified (i) two operons encoding a biphenyl (bph) and an aniline (tdn) dioxygenase, both flanked by transposases and (ii) two operons and several scattered genes encoding the ortho -cleavage of catechol. Proteomics identified 11 putative catabolic proteins, all but BphA1 up-regulated in DPA- and aniline-growing cells, and showed that the bacterium mobilized cellular mechanisms to cope with oxidative stress, probably induced by DPA and its derivatives. Transcription analysis verified the role of the selected genes/operons in the metabolic pathway: DPA was initially transformed to aniline and catechol by a biphenyl dioxygenase (DPA-dioxygenase); aniline was then transformed to catechol which was further metabolized via the ortho -cleavage pathway. Plasmid curing of P. putida resulted in loss of the DPA and aniline dioxygenase genes and the corresponding degradation capacities. Overall our findings provide novel insights into the evolution of the DPA degradation pathway and suggests that the degradation capacity of P. putida was acquired through recruitment of the bph and tdn operons via horizontal gene transfer.

The application of the fungicide thiabendazole (TBZ) in fruit packaging plants (FPP) results in the production of effluents which are often disposed in adjacent field sites. These require remediation to prevent further environmental dispersal of TBZ. We assessed the bioaugmentation potential of a newly isolated TBZ-degrading bacterial consortium in a naturally contaminated soil (NCS) exhibiting a natural gradient of TBZ levels (12000, 400, 250 and 12 mg kg -1 ). The effect of aging on bioaugmentation efficacy was comparatively tested in a soil with similar physicochemical properties and soil microbiota, which was artificially, contaminated with the same TBZ levels (ACS). The impact of bioaugmentation and TBZ on the bacterial diversity in the NCS was explored via amplicon sequencing. Bioaugmentation effectively removed TBZ from both soils at levels up to 400 mg kg -1 but failed at the highest contamination level (12000 mg kg -1 ). Dissipation of TBZ in bioaugmented samples showed a concentration-dependent pattern, while aging of TBZ had a slight effect on bioaugmentation efficiency. Bioaugmentation had no impact on the soil bacterial diversity, in contrast to TBZ contamination. Soils from the hotspots of TBZ contamination (12000 mg kg -1 ) showed a drastically lower α-diversity driven by the dominance of β- and γ-proteobacteria at the expense of all other bacterial phyla, especially Actinobacteria. Overall, bioaugmentation with specialized microbial inocula could be an effective solution for the recovery of disposal sites contaminated with persistent chemicals like TBZ., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Ortho-phenylphenol (OPP) is a fungicide contained in agro-industrial effluents produced by fruit-packaging plants. Within the frame of developing bio-strategies to detoxify these effluents, an OPP-degrading Sphingomonas haloaromaticamans strain was isolated. Proteins/genes with a putative catabolic role and bacterium adaptation mechanisms during OPP degradation were identified via genomic and proteomic analysis. Transcription analysis of all putative catabolic genes established their role in the metabolism of OPP. The formation of key transformation products was verified by chromatographic analysis. Genomic analysis identified two orthologous operons encoding the ortho-cleavage of benzoic acid (BA) (ben/cat). The second ben/cat operon was located in a 92-kb scaffold along with (i) an operon (opp) comprising genes for the transformation of OPP to BA and 2-hydroxypenta-2,4-dienoate (and genes for its transformation) and (ii) an incomplete biphenyl catabolic operon (bph). Proteomics identified 13 up-regulated catabolic proteins when S. haloaromaticamans was growing on OPP and/or BA. Transcription analysis verified the key role of the catabolic operons located in the 92-kb scaffold, and flanked by transposases, on the transformation of OPP by S. haloaromaticamans. A flavin-dependent monoxygenase (OppA1), one of the most up-regulated proteins in the OPP-growing cells, was isolated via heterologous expression and its catabolic activity was verified in vitro.

Citrus fruit-packaging plants (FPP) produce large wastewater volumes with high loads of fungicides like ortho-phenylphenol (OPP) and imazalil (IMZ). No methods are in place for the treatment of those effluents and biobeds appear as a viable alternative. We employed a column study to investigate the potential of spent mushroom substrate (SMS) of Pleurotus ostreatus, either alone or in mixture with straw and soil plus a mixture of straw /soil to retain and dissipate IMZ and OPP. The role of P. ostreatus on fungicides dissipation was also investigated by studying in parallel the performance of fresh mushroom substrate of P. ostreatus (FMS) and measuring lignolytic enzymatic activity in the leachates. All substrates effectively reduced the leaching of OPP and IMZ which corresponded to 0.014-1.1% and 0.120-0.420% of their initial amounts respectively. Mass balance analysis revealed that FMS and SMS/Straw/Soil (50/25/25 by vol) offered the most efficient removal of OPP and IMZ from wastewaters respectively. Regardless of the substrate, OPP was restricted in the top 0-20cm of the columns and was bioavailable (extractable with water), compared to IMZ which was less bioavailable (extractable with acetonitrile) but diffused at deeper layers (20-50, 50-80cm) in the SMS- and Straw/Soil-columns. PLFAs showed that fungal abundance was significantly lower in the top layer of all substrates from where the highest pesticide amounts were recovered suggesting an inhibitory effect of fungicides on total fungi in the substrates tested. Our data suggest that biobeds packed with SMS-rich substrates could ensure the efficient removal of IMZ and OPP from wastewaters of citrus FPP., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Pesticides generate transformation products (TPs) when they are released into the environment. These TPs may be of ecotoxicological importance. Past studies have demonstrated how difficult it is to predict the occurrence of pesticide TPs and their environmental risk. The monitoring approaches mostly used in current regulatory frameworks target only known ecotoxicologically relevant TPs. Here, we present a novel combined approach which identifies and categorizes known and unknown pesticide TPs in soil by combining suspect screening time-of-flight mass spectrometry with in silico molecular typology. We used an empirical and theoretical pesticide TP library for compound identification by both non-target and target time-of-flight (tandem) mass spectrometry, followed by structural proposition through a molecular structure correlation program. In silico molecular typology was then used to group TPs according to common molecular descriptors and to indirectly elucidate their environmental parameters by analogy to known pesticide compounds with similar molecular descriptors. This approach was evaluated via the identification of TPs of the triazole fungicide tebuconazole occurring in soil during a field dissipation study. Overall, 22 empirical and 12 yet unknown TPs were detected, and categorized into three groups with defined environmental properties. This approach combining suspect screening time-of-flight mass spectrometry with molecular typology could be extended to other organic pollutants and used to rationalize the choice of TPs to be investigated towards a more comprehensive environmental risk assessment scheme., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Thiabendazole (TBZ), imazalil (IMZ), ortho-phenylphenol (OPP), diphenylamine (DPA), and ethoxyquin (EQ) are used in fruit-packaging plants (FPP) with the stipulation that wastewaters produced by their application would be depurated on site. However, no such treatment systems are currently in place, leading FPP to dispose of their effluents in agricultural land. We investigated the dissipation of those pesticides and their impact on soil microbes known to have a key role on ecosystem functioning. OPP and DPA showed limited persistence (50% dissipation time [DT50], 0.6 and 1.3 days) compared to TBZ and IMZ (DT50, 47.0 and 150.8 days). EQ was rapidly transformed to the short-lived quinone imine (QI) (major metabolite) and the more persistent 2,4-dimethyl-6-ethoxyquinoline (EQNL) (minor metabolite). EQ and OPP exerted significant inhibition of potential nitrification, with the effect of the former being more persistent. This was not reflected in the abundance (determined by quantitative PCR [qPCR]) of the amoA gene of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Considering the above discrepancy and the metabolic pattern of EQ, we further investigated the hypothesis that its metabolites and not only EQ were toxic to ammonia oxidizers. Potential nitrification, amoA gene abundance, and amoA gene transcripts of AOB and AOA showed that QI was probably responsible for the inhibition of nitrification. Our findings have serious ecological and practical implications for soil productivity and N conservation in agriculturally impacted ecosystems and stress the need to include metabolites and RNA-based methods when the soil microbial toxicity of pesticides is assessed., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Phospholipid fatty acid (PLFA) analysis is a robust method for characterizing soil microbial communities. We determined the effects of extraction solvent (chloroform vs dichloromethane) and buffer (phosphate vs citrate) on the yield and the profile of PLFAs extracted from an acidic (pH 5.5) and an alkaline (pH 8.6) soil following two 2-h sequential extractions. The yield and the profile of the PLFAs obtained separately by the first and the second 2-h extraction were compared to identify the impact of sequential extractions on the PLFA extraction efficiency. Chloroform-citrate and a 2 × 2-h extraction maximized PLFA yields in both soils. Multivariate analysis of the data showed that the choice of the extraction mixture did not significantly influence the profile of the PLFAs obtained by the first 2-h extraction, whereas it had a profound effect on the profile of the PLFAs obtained by the second 2-h extraction. Most PLFAs were extracted during the first extraction except 18:2ω6,9 and 22:0 which were almost equally extracted by the two sequential extractions. The choice of organic solvent significantly influenced the profile of the PLFAs extracted; their yield increased with chloroform with the exception of 18:2ω6,9 and 22:0 which were favored by dichloromethane. Overall, a 2 × 2-h extraction with chloroform/methanol/citrate is expected to provide maximum PLFA yields.

Background: Patients on long-term maintenance hemodialysis (HD) are at high risk of developing cardiovascular disease and suffering various cardiovascular complications during HD., Hypothesis: The purpose of this study was to evaluate the influence of changing loading conditions on the myocardial performance index (MPI) in patients on long-term HD and to specify an optimal level of fluid loss during HD that would maintain stable global cardiac function., Methods: The study consisted of 52 patients with end-stage renal failure (ESRF), mean age 56±11.7 y, range: 25-80 y, on regular HD. For each patient a complete echocardiographic-Doppler examination was performed before and after HD. Systolic and diastolic parameters of left ventricular function were measured, and the myocardial performance index (MPI) was calculated., Results: The MPI was significantly prolonged after HD (0.47±0.15 before HD versus 0.59±0.16 after HD, p < 0.001). Mean change in body weight during HD was 2.1±0.86 kg. The MPI did not change significantly in patients with intradialytic weight loss up to 1.75 kg., Conclusions: The MPI value seems to be independent of acute preload changes only when fluid loss is less than 1.75 kg. A 1.75-kg fluid loss during HD seems to be the optimal goal. In ESRF patients on HD, the MPI seems to be a good indicator of global left ventricular function and potentially a valuable aid in the effort to maintain optimal fluid balance., (Copyright © 2010 Wiley Periodicals, Inc.)

Background: Early diagnosis of heart transplant rejection is mandatory, since even mild rejection can rapidly progress to more severe rejection. Noninvasive diagnosis of heart transplant rejection still remains a challenge., Hypothesis: The purpose of the study was to determine a possible association between myocardial performance index (MPI) and biopsy score of the heart transplant., Methods: This is a retrospective cohort analysis of 99 complete Doppler echocardiographic studies from 24 consecutive patients (23 men) performed within 24 h of endomyocardial biopsy. Mean age of the cohort was 50 +/- 9 years and mean time from transplantation was 19 +/- 21 months (1-81). All patients were in sinus rhythm. Myocardial performance index was calculated as the ratio of isovolumic contraction time plus isovolumic relaxation time divided by ejection time. Left ventricular dimensions, left ventricular mass, ejection fraction, and a number of Doppler indices (E-point velocity, A-point velocity, deceleration time, and deceleration slope) were also measured. The International Society for Heart and Lung Transplantation (ISHLT) grading system was used for the classification of endomyocardial biopsies., Results: Myocardial performance index was significantly prolonged (0.60 +/- 0.13, 0.68 +/- 0.08, 0.75 +/- 0.20, in biopsy scores 0, IA, and IB, respectively; p<0.001). Isovolumic contraction time was significantly prolonged; isovolumic relaxation time was not significantly changed. Ejection time and deceleration time were significantly shortened. Multivariate stepwise regression analysis revealed that MPI and deceleration time were the only independent predictors of biopsy score (r=0.48, F=10.53, p<0.0001)., Conclusion: Myocardial performance index seems to be a useful adjunct in the follow-up of cardiac transplant patients. These preliminary data suggest that a larger study may be indicated to clarify the relevance of myocardial performance index.