The effect of natural products used as pesticides on the soil microbiota: OECD 216 nitrogen transformation test fails to identify effects that were detected via q-PCR microbial abundance measurement.

BACKGROUND: Natural products present an environmentally attractive alternative to synthetic pesticides which have been implicated in the off-target effect. Currently, the assessment of pesticide toxicity on soil microorganisms relies on the OECD 216 N transformation assay (OECD stands for the Organisation Economic Co-operation and Development, which is a key international standard-setting organisation). We tested the hypotheses that (i) the OECD 216 assay fails to identify unacceptable effects of pesticides on soil microbiota compared to more advanced molecular and standardized tests, and (ii) the natural products tested (dihydrochalcone, isoflavone, aliphatic phenol, and spinosad) are less toxic to soil microbiota compared to a synthetic pesticide compound (3,5-dichloraniline). We determined the following in three different soils: (i) ammonium (NH4 +) and nitrate (NO3 -) soil concentrations, as dictated by the OECD 216 test, and (ii) the abundance of phylogenetically (bacteria and fungi) and functionally distinct microbial groups [ammonia-oxidizing archaea (AOA) and bacteria (AOB)] using quantitative polymerase chain reaction (q-PCR). RESULTS: All pesticides tested exhibited limited persistence, with spinosad demonstrating the highest persistence. None of the pesticides tested showed clear dose-dependent effects on NH4 + and NO3 - levels and the observed effects were <25% of the control, suggesting no unacceptable impacts on soil microorganisms. In contrast, q-PCR measurements revealed (i) distinct negative effects on the abundance of total bacteria and fungi, which were though limited to one of the studied soils, and (ii) a significant reduction in the abundance of both AOA and AOB across soils. This reduction was attributed to both natural products and 3,5-dichloraniline. CONCLUSION: Our findings strongly advocate for a revision of the current regulatory framework regarding the toxicity of pesticides to soil microbiota, which should integrate advanced and well-standardized tools. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Anthelminthic Veterinary Medicines Interactions with the Soil Microbiota.

Anthelminthics (AHs) are used to control gastrointestinal nematodes (GINs) in productive animals. They are rapidly excreted by animals, ending up in soil through direct deposition of animal dung or application of animal excreta as manures. Most environmental research on AHs has focused on their toxicity to aquatic organisms and soil fauna while their interactions with the soil microbiota, a key component of a functioning soil ecosystem, have been overlooked. In this article, we summarize current knowledge on the interactions of Ahs with the soil (micro) biota, we highlight recent evidence for the toxicity of AHs on soil microorganisms and discuss those results in the frame of the current environmental risk assessment (ERA) of veterinary medicines.

Assessing the activity of different plant-derived molecules and potential biological nitrification inhibitors on a range of soil ammonia- and nitrite-oxidizing strains.

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.

Isolation of soil bacteria able to degrade the anthelminthic compound albendazole.

Anthelmintic (AHs) veterinary drugs constitute major environmental contaminants. The use of AH-contaminated fecal material as manures in agricultural settings constitutes their main route of environmental dispersal. Once in soils, these compounds induce toxic effects to soil fauna and soil microbiota, both having a pivotal role in soil ecosystem functioning. Therefore, it is necessary to identify mitigation strategies to restrict the environmental dispersal of AHs. Bioaugmentation of AH-contaminated manures or soils with specialized microbial inocula constitutes a promising remediation strategy. In the present study, we aimed to isolate microorganisms able to actively transform the most widely used benzimidazole anthelminthic albendazole (ABZ). Enrichment cultures in minimal growth media inoculated with a soil known to exhibit rapid degradation of ABZ led to the isolation of two bacterial cultures able to actively degrade ABZ. Two oxidative products of ABZ, ABZSO and ABZSO2, were detected at low amounts along its degradation. This suggested that the oxidation of ABZ is not a major transformation process in the isolated bacteria which most probably use other biotic pathways to degrade ABZ leading to the formation of products not monitored in this study. Full length sequencing of their 16S rRNA gene and phylogenetic analysis assigned both strains to the genus Acinetobacter. The sequences were submitted in GeneBank NCBI, database with the accession numbers OP604271 to OP604273. Further studies will employ omic tools to identify the full transformation pathway and the associated genetic network of Acinetobacter isolates, information that will unlock the potential use of these isolates in the bioaugmentation of contaminated manures.

Vineyard-mediated factors are still operative in spontaneous and commercial fermentations shaping the vinification microbial community and affecting the antioxidant and anticancer properties of wines.

The grapevine and vinification microbiota have a strong influence on the characteristics of the produced wine. Currently we have a good understanding of the role of vineyard-associated factors, like cultivar, vintage and terroir in shaping the grapevine microbiota. Notwithstanding, their endurance along the vinification process remains unknown. Thus, the main objective of our study was to determine how these factors influence (a) microbial succession during fermentation (i.e., bacterial and fungal) and (b) the antioxidant, antimutagenic and anticancer potential of the produced wines. These were evaluated under different vinification strategies (i.e., spontaneous V1, spontaneous with preservatives V2, commercial V3), employed at near full-scale level by local wineries, for two cultivars (Roditis and Sideritis), two terroir types, and two vintages. Cultivar and vintage were strong and persistent determinants of the vinification microbiota, unlike terroir whose effect became weaker from the vineyard, and early fermentation stages, where non-Saccharomyces yeasts, filamentous fungi (i.e., Aureobasidium, Cladosporium, Lachancea, Alternaria, Aspergillus, Torulaspora) and acetic acid bacteria (AAB) (Gluconobacter, Acetobacter, Komagataeibacter) dominated, to late fermentation stages where Saccharomyces and Oenococcus become prevalent. Besides vineyard-mediated factors, the vinification process employed was the strongest determinant of the fungal community compared to the bacterial community were effects varied per cultivar. Vintage and vinification type were the strongest determinants of the antioxidant, antimutagenic and anticancer potential of the produced wines. Further analysis identified significant positive correlations between members of the vinification microbiota like the yeasts Torulaspora debrueckii and Lachancea quebecensis with the anticancer and the antioxidant properties of wines in both cultivars. These findings could be exploited towards a microbiota-modulated vinification process to produce high-quality wines with desirable properties and enhanced regional identity.

Investigation of the Critical Biomass of Acclimated Microbial Communities to High Ammonia Concentrations for a Successful Bioaugmentation of Biogas Anaerobic Reactors with Ammonia Inhibition.

This study aimed to investigate the role of the bioaugmented critical biomass that should be injected for successful bioaugmentation for addressing ammonia inhibition in anaerobic reactors used for biogas production. Cattle manure was used as a feedstock for anaerobic digestion (AD). A mixed microbial culture was acclimated to high concentrations of ammonia and used as a bioaugmented culture. Different volumes of bioaugmented culture were injected in batch anaerobic reactors under ammonia toxicity levels i.e., 4 g of NH4+-N L-1. The results showed that injecting a volume equal to 65.62% of the total working reactor volume yielded the best methane production. Specifically, this volume of bioaugmented culture resulted in methane production rates of 196.18 mL g-1 Volatile Solids (VS) and 245.88 mL g-1 VS after 30 and 60 days of AD, respectively. These rates were not significantly different from the control reactors (30d: 205.94 mL CH4 g-1 VS and 60d: 230.26 mL CH4 g-1 VS) operating without ammonia toxicity. Analysis of the microbial community using 16S rRNA gene sequencing revealed the dominance of acetoclastic methanogen members from the genus Methanosaeta in all reactors.

Interactions of anthelmintic veterinary drugs with the soil microbiota: Toxicity or enhanced biodegradation?

Anthelmintic (AH) compounds are used to control gastrointestinal nematodes (GINs) in livestock production. They are only partially metabolized in animals ending in animal excreta whose use as manures leads to AH dispersal in agricultural soils. Once in soil, AHs interact with soil microorganisms, with the outcome being either detrimental, or beneficial. We aimed to disentangle the mechanisms of these complex interactions. Two soils previously identified as « fast ¼ or « slow¼, regarding the degradation of albendazole (ABZ), ivermectin (IVM), and eprinomectin (EPM), were subjected to repeated applications at two dose rates (1, 2 mg kg-1and 10, 20 mg kg-1). We hypothesized that this application scheme will lead to enhanced biodegradation in «fast ¼ soils and accumulation and toxicity in «slow ¼ soils. Repeated application of ABZ resulted in different transformation pathways in the two soils and a clear acceleration of its degradation in the «fast ¼ soil only. In contrast residues of IVM and EPM accumulated in both soils. ABZ was the sole AH that induced a consistent reduction in the abundance of total fungi and crenarchaea. In addition, inhibition of nitrification and reduction in the abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) by all AHs was observed, while commamox bacteria were less responsive. Amplicon sequencing analysis showed dose-depended shifts in the diversity of bacteria, fungi, and protists in response to AHs application. ABZ presented the most consistent effect on the abundance and diversity of most microbial groups. Our findings provide first evidence for the unexpected toxicity of AHs on key soil microbial groups that might have to be considered in a regulatory context.

Above- and below-ground microbiome in the annual developmental cycle of two olive tree varieties.

The olive tree is a hallmark crop in the Mediterranean region. Its cultivation is characterized by an enormous variability in existing genotypes and geographical areas. As regards the associated microbial communities of the olive tree, despite progress, we still lack comprehensive knowledge in the description of these key determinants of plant health and productivity. Here, we determined the prokaryotic, fungal and arbuscular mycorrhizal fungal (AMF) microbiome in below- (rhizospheric soil, roots) and above-ground (phyllosphere and carposphere) plant compartments of two olive varieties 'Koroneiki' and 'Chondrolia Chalkidikis' grown in Southern and Northern Greece respectively, in five developmental stages along a full fruit-bearing season. Distinct microbial communities were supported in above- and below-ground plant parts; while the former tended to be similar between the two varieties/locations, the latter were location specific. In both varieties/locations, a seasonally stable root microbiome was observed over time; in contrast the plant microbiome in the other compartments were prone to changes over time, which may be related to seasonal environmental change and/or to plant developmental stage. We noted that olive roots exhibited an AMF-specific filtering effect (not observed for bacteria and general fungi) onto the rhizosphere AMF communities of the two olive varieties/locations/, leading to the assemblage of homogenous intraradical AMF communities. Finally, shared microbiome members between the two olive varieties/locations include bacterial and fungal taxa with putative functional attributes that may contribute to olive tree tolerance to abiotic and biotic stress.

Accelerated dissipation, soil microbial toxicity and dispersal of antimicrobial resistance in soils repeatedly exposed to tiamulin, tilmicosin and sulfamethoxazole.

The application of manures leads to the contamination of agricultural soils with veterinary antibiotics (VAs). These might exert toxicity on the soil microbiota and threaten environmental quality, and public health. We obtained mechanistic insights about the impact of three VAs, namely, sulfamethoxazole (SMX), tiamulin (TIA) and tilmicosin (TLM), on the abundance of key soil microbial groups, antibiotic resistance genes (ARGs) and class I integron integrases (intl1). In a microcosm study, we repeatedly treated two soils (differing in pH and VA dissipation capacity) with the studied VAs, either directly or via fortified manure. This application scheme resulted in accelerated dissipation of TIA, but not of SMX, and accumulation of TLM. Potential nitrification rates (PNR), and the abundance of ammonia-oxidizing microorganism (AOM) were reduced by SMX and TIA, but not by TLM. VAs strongly impacted the total prokaryotic and AOM communities, whereas manure addition was the main determinant of the fungal and protist communities. SMX stimulated sulfonamide resistance, while manure stimulated ARGs and horizontal gene transfer. Correlations identified opportunistic pathogens like Clostridia, Burkholderia-Caballeronia-Paraburkholderia, and Nocardioides as potential ARG reservoirs in soil. Our results provide unprecedented evidence about the effects of understudied VAs on soil microbiota and highlight risks posed by VA-contaminated manures. ENVIRONMENTAL IMPLICATION: The dispersal of veterinary antibiotics (VAs) through soil manuring enhances antimicrobial resistance (AMR) development and poses a threat to the environment and the public health. We provide insights about the impact of selected VAs on their: (i) microbially-mediated dissipation in soil; (ii) ecotoxicity on the soil microbial communities; (iii) capacity to stimulate AMR. Our results (i) demonstrate the effects of VAs and their application-mode on the bacterial, fungal, and protistan communities, and on the soil ammonia oxidizers; (ii) describe natural attenuation processes against VA dispersal, (iii) depict potential soil microbial AMR reservoirs, essential for the development of risk assessment strategies.

Veterinary drug albendazole inhibits root colonization and symbiotic function of the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Arbuscular mycorrhizal fungi (AMF) are plant symbionts that have a pivotal role in maintaining soil fertility and nutrient cycling. However, these microsymbionts may be exposed to organic pollutants like pesticides or veterinary drugs known to occur in agricultural soils. Anthelminthics are veterinary drugs that reach soils through the application of contaminated manures in agricultural settings. Their presence might threaten the function of AMF, considered as sensitive indicators of the toxicity of agrochemicals to the soil microbiota. We determined the impact of the anthelminthic compounds albendazole and ivermectin on the establishment and functionality of the symbiosis between the model-legume Lotus japonicus and the AMF Rhizophagus irregularis. Our analyses revealed negative effects of albendazole on the development and functionality of arbuscules, the symbiotic organelle of AMF, at a concentration of 0.75 µg g-1. The impairment of the symbiotic function was verified by the reduced expression of genes SbtM1, PT4 and AMT2;2 involved in arbuscules formation, P and N uptake, and the lower phosphorus shoot content detected in the albendazole-treated plants. Our results provide first evidence for the toxicity of albendazole on the colonization capacity and function of R. irregularis at concentrations that may occur in agricultural soils systematically amended with drug-containing manures.

Repeated applications of fipronil, propyzamide and flutriafol affect soil microbial functions and community composition: A laboratory-to-field assessment.

Pesticides play an important role in conventional agriculture by controlling pests, weeds, and plant diseases. However, repeated applications of pesticides may have long lasting effects on non-target microorganisms. Most studies have investigated the short-term effects of pesticides on soil microbial communities at the laboratory scale. Here, we assessed the ecotoxicological impact of fipronil (insecticide), propyzamide (herbicide) and flutriafol (fungicide) on (i) soil microbial enzymatic activities, (ii) potential nitrification, (iii) abundance of the fungal and bacterial community and key functional genes (nifH, amoA, chiA, cbhl and phosphatase) and (iii) diversity of bacteria, fungi, ammonia oxidizing bacteria (AOB) and archaea (AOA) after repeated pesticide applications in laboratory and field experiments. Our results showed that repeated applications of propyzamide and flutriafol affected the soil microbial community structure in the field and had significant inhibitory effects on enzymatic activities. The abundances of soil microbiota affected by pesticides recovered to levels similar to the control following a second application, suggesting that they might be able to recover from the pesticide effects. However, the persistent pesticide inhibitory effects on soil enzymatic activities suggests that the ability of the microbial community to cope with the repeated application was not accompanied by functional recovery. Overall, our results suggest that repeated pesticide applications may influence soil health and microbial functionalities and that more information should be collected to inform risk-based policy development.

Impact of twenty pesticides on soil carbon microbial functions and community composition.

Pesticides are known to affect non-targeted soil microorganisms. Still, studies comparing the effect of multiple pesticides on a wide range of microbial endpoints associated with carbon cycling are scarce. Here, we employed fluorescence enzymatic assay and real-time PCR to evaluate the effect of 20 commercial pesticides, applied at their recommended dose and five times their recommended dose, on soil carbon cycling related enzymatic activities (α-1,4-glucosidase, ß-1,4-glucosidase, ß-d-cellobiohydrolase and ß-xylosidase), and on the absolute abundance of functional genes (cbhl and chiA), in three different South Australian agricultural soils. The effects on cellulolytic and chitinolytic microorganisms, and the total microbial community composition were determined using shotgun metagenomic sequencing in selected pesticide-treated and untreated samples. The application of insecticides significantly increased the cbhl and chiA genes absolute abundance in the acidic soil. At the community level, insecticide fipronil had the greatest stimulating effect on cellulolytic and chitinolytic microorganisms, followed by fungicide metalaxyl-M and insecticide imidacloprid. A shift towards a fungal dominated microbial community was observed in metalaxyl-M treated soil. Overall, our results suggest that the application of pesticides might affect the soil carbon cycle and may disrupt the formation of soil organic matter and structure stabilisation.

Nutritional inter-dependencies and a carbazole-dioxygenase are key elements of a bacterial consortium relying on a Sphingomonas for the degradation of the fungicide thiabendazole.

Thiabendazole (TBZ), is a persistent fungicide/anthelminthic and a serious environmental threat. We previously enriched a TBZ-degrading bacterial consortium and provided first evidence for a Sphingomonas involvement in TBZ transformation. Here, using a multi-omic approach combined with DNA-stable isotope probing (SIP) we verified the key degrading role of Sphingomonas and identify potential microbial interactions governing consortium functioning. SIP and amplicon sequencing analysis of the heavy and light DNA fraction of cultures grown on 13 C-labelled versus 12 C-TBZ showed that 66% of the 13 C-labelled TBZ was assimilated by Sphingomonas. Metagenomic analysis retrieved 18 metagenome-assembled genomes with the dominant belonging to Sphingomonas, Sinobacteriaceae, Bradyrhizobium, Filimonas and Hydrogenophaga. Meta-transcriptomics/-proteomics and non-target mass spectrometry suggested TBZ transformation by Sphingomonas via initial cleavage by a carbazole dioxygenase (car) to thiazole-4-carboxamidine (terminal compound) and catechol or a cleaved benzyl ring derivative, further transformed through an ortho-cleavage (cat) pathway. Microbial co-occurrence and gene expression networks suggested strong interactions between Sphingomonas and a Hydrogenophaga. The latter activated its cobalamin biosynthetic pathway and Sphingomonas its cobalamin salvage pathway to satisfy its B12 auxotrophy. Our findings indicate microbial interactions aligning with the 'black queen hypothesis' where Sphingomonas (detoxifier, B12 recipient) and Hydrogenophaga (B12 producer, enjoying detoxification) act as both helpers and beneficiaries.

The Effects of Quinone Imine, a New Potent Nitrification Inhibitor, Dicyandiamide, and Nitrapyrin on Target and Off-Target Soil Microbiota.

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.

Biodegradation of anthelmintics in soils: does prior exposure of soils to anthelmintics accelerate their dissipation?

Anthelmintics (AHs) control animal infections with gastrointestinal nematodes. They reach soil through animal faeces deposited on soils or through manuring. Although soil constitutes a major AH sink, we know little about the mechanisms controlling their soil dissipation. We employed studies with fumigated and non-fumigated soils collected from 12 sheep farms with a variable record of albendazole (ABZ), ivermectin (IVM) and eprinomectin (EPM) use. From each farm, we collected soils from inside small ruminant barn facilities (series A, high exposure) and the associated grazing pastures (series B, low exposure). We asked the following questions: (a) What is the role of soil microorganisms in AH dissipation? (b) Does repeated exposure of soils to AHs lead to their accelerated biodegradation? (c) Which soil physicochemical properties control AH dissipation? Soil fumigation significantly retarded ABZ (DT50 1.9 and 4.33 days), IVM (34.5 and 108.7 days) and EPM dissipation (30 and 121 days) suggesting a key role of soil microorganisms in AH dissipation. No significant acceleration in AH dissipation was evident in soils from units with a record of the administration of AHs or in soil series A vs series B, suggesting that the level of prior exposure was not adequate to induce their enhanced biodegradation. Significant positive and negative correlations of soil total organic carbon (TOC) and ABZ and IVM dissipation, respectively, were observed. Soil adsorption of AHs increased in the order IVM > ABZ > EPM. TOC controlled soil adsorption of IVM and EPM, but not of ABZ, in support of the contrasting effect of TOC on IVM and ABZ dissipation.

Using biobeds for the treatment of fungicide-contaminated effluents from various agro-food processing industries: Microbiome responses and mobile genetic element dynamics.

Agro-food processing industries generate large amounts of pesticide-contaminated effluents that pose a significant environmental threat if managed improperly. Biopurification systems like biobeds could be utilized for the depuration of these effluents although direct evidence for their efficiency are still lacking. We employed a column leaching experiment with pilot biobeds to (i) assess the depuration potential of biobeds against fungicide-contaminated effluents from seed-producing (carboxin, metalaxyl-M, fluxapyroxad), bulb-handling (thiabendazole, fludioxonil and chlorothalonil) and fruit-packaging (fludioxonil, imazalil) industries, (ii) to monitor microbial succession via amplicon sequencing and (iii) to determine the presence and dynamics of mobile genetic elements like intl1, IS1071, IncP-1 and IncP-1ε often associated with the transposition of pesticide-degrading genes. Biobeds could effectively retain (adsorbed but extractable with organic solvents) and dissipate (degraded and/or not extractable with organic solvents) the fungicides that were contained in the agro-industrial effluents with 93.1-99.98% removal efficiency in all cases. Lipophilic substances like fluxapyroxad were mostly retained in the biobed while more polar substances like metalaxyl-M and carboxin were mostly dissipated or showed higher leaching potential like metalaxyl-M. Biobeds supported a bacterial and fungal community that was not affected by fungicide application but showed clear temporal patterns in the different biobed horizons. This was most probably driven by the establishment of microaerophilic conditions upon water saturation of biobeds, as supported by the significant increase in the abundance of facultative or strict anaerobes like Chloroflexi/Anaerolinae, Acidibacter and Myxococcota. Wastewater application did not affect the dynamics of mobile genetic elements in biobeds whose abundance (intl1, IS1071, IncP-1ε) showed significant increases with time. Our findings suggest that biobeds could effectively decontaminate fungicide-contaminated effluents produced by agro-food industries and support a rather resilient microbial community.

Isolation, characterization and industrial application of a Cladosporium herbarum fungal strain able to degrade the fungicide imazalil.

Imazalil (IMZ) is an imidazole fungicide commonly used by fruit-packaging plants (FPPs) to control fungal infections during storage. Its application leads to the production of pesticide-contaminated wastewaters, which, according to the European Commission, need to be treated on site. Considering the lack of efficient treatment methods, biodepuration systems inoculated with tailored-made inocula specialized on the removal of such persistent fungicides appear as an appropriate solution. However, nothing is known about the biodegradation of IMZ. We aimed to isolate and characterize microorganisms able to degrade the recalcitrant fungicide IMZ and eventually to test their removal efficiency under near practical bioengineering conditions. Enrichment cultures from a soil receiving regular discharges of effluents from a FPP, led to the isolation of a Cladosporium herbarum strain, which showed no pathogenicity on fruits, a trait essential for its biotechnological exploitation in FPPs. The fungus was able to degrade up to 100 mg L-1 of IMZ. However, its degrading capacity and growth was reduced at increasing IMZ concentrations in a dose-dependent manner, suggesting the involvement of a detoxification rather than an energy-gain mechanism in the dissipation of IMZ. The isolate could tolerate and gradually degrade the fungicides fludioxonil (FLD) and thiabendazole (TBZ), also used in FPPs and expected to coincide alongside IMZ in FPP effluents. The capacity of the isolate to remove IMZ in a practical context was evaluated in a benchtop immobilized-cell bioreactor fed with artificial IMZ-contaminated wastewater (200 mg L-1). The fungal strain established in the reactor, completely dominated the fungal community and effectively removed >96% of IMZ. The bioreactor also supported a diverse bacterial community composed of Sphingomonadales, Burkholderiales and Pseudomonadales. Our study reports the isolation of the first IMZ-degrading microorganism with high efficiency to remove IMZ from agro-industrial effluents under bioengineering conditions.

Pesticide effects on nitrogen cycle related microbial functions and community composition.

The extensive application of pesticides in agriculture raises concerns about their potential negative impact on soil microorganisms, being the key drivers of nutrient cycling. Most studies have investigated the effect of a single pesticide on a nutrient cycling in single soil type. We, for the first time, investigated the effect of 20 commercial pesticides with different mode of actions, applied at their recommended dose and five times their recommended dose, on nitrogen (N) microbial cycling in three different agricultural soils from southern Australian. Functional effects were determined by measuring soil enzymatic activities of ß-1,4-N-acetyliglucosaminidase (NAG) and l-leucine aminopeptidase (LAP), potential nitrification (PN), and the abundance of functional genes involved in N cycling (amoA and nifH). Effects on nitrifiers diversity were determined with amplicon sequencing. Overall, the pesticides effect on N microbial cycling was dose-independent and soil specific. The fungicides flutriafol and azoxystrobin, the herbicide chlorsulfuron and the insecticide fipronil induced a significant reduction in PN and ß-1,4-N-acetylglucosaminidase activity (P < 0.05) (NAG) in the alkaline loam soil with low organic carbon content i.e. a soil with properties which typically favors pesticide bioavailability and therefore potential toxicity. For the nitrifier community, the greatest pesticide effects were on the most dominant Nitrososphaeraceae (ammonia-oxidizing archaea; AOA) whose abundance increased significantly compared to the less dominant AOA and other nitrifiers. The inhibiting effects were more evident in the soil samples treated with fungicides. By testing multiple pesticides in a single study, our findings provide crucial information that can be used for pesticide hazard assessment.

Bioprocess performance, transformation pathway, and bacterial community dynamics in an immobilized cell bioreactor treating fludioxonil-contaminated wastewater under microaerophilic conditions.

Fludioxonil is a post-harvest fungicide contained in effluents produced by fruit packaging plants, which should be treated prior to environmental dispersal. We developed and evaluated an immobilized cell bioreactor, operating under microaerophilic conditions and gradually reduced hydraulic retention times (HRTs) from 10 to 3.9 days, for the biotreatment of fludioxonil-rich wastewater. Fludioxonil removal efficiency was consistently above 96%, even at the shortest HRT applied. A total of 12 transformation products were tentatively identified during fludioxonil degradation by using liquid chromatography coupled to quadrupole time-of-flight Mass spectrometry (LC-QTOF-MS). Fludioxonil degradation pathway was initiated by successive hydroxylation and carbonylation of the pyrrole moiety and disruption of the oxidized cyanopyrrole ring at the NH-C bond. The detection of 2,2-difluoro-2H-1,3-benzodioxole-4-carboxylic acid verified the decyanation and deamination of the molecule, whereas its conversion to the tentatively identified compound 2,3-dihydroxybenzoic acid indicated its defluorination. High-throughput amplicon sequencing revealed that HRT shortening led to reduced α-diversity, significant changes in the ß-diversity, and a shift in the bacterial community composition from an initial activated sludge system typical community to a community composed of bacterial taxa like Clostridium, Oligotropha, Pseudomonas, and Terrimonas capable of performing advanced degradation and/or aerobic denitrification. Overall, the immobilized cell bioreactor operation under microaerophilic conditions, which minimizes the cost for aeration, can provide a sustainable solution for the depuration of fludioxonil-contaminated agro-industrial effluents.