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.

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.

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.

Insights into the Function and Horizontal Transfer of Isoproturon Degradation Genes (pdmAB) in a Biobed System.

Biobeds, designed to minimize pesticide point source contamination, rely mainly on biodegradation processes. We studied the interactions of a biobed microbial community with the herbicide isoproturon (IPU) to explore the role of the pdmA gene, encoding the large subunit of an N-demethylase responsible for the initial demethylation of IPU, via quantitative PCR (qPCR) and reverse transcription-PCR (RT-qPCR) and the effect of IPU on the diversity of the total bacterial community and its active fraction through amplicon sequencing of DNA and RNA, respectively. We further investigated the localization and dispersal mechanisms of pdmAB in the biobed packing material by measuring the abundance of the plasmid pSH (harboring pdmAB) of the IPU-degrading Sphingomonas sp. strain SH (previously isolated from the soil used in the biobed) compared with the abundance of the pdmA gene and metagenomic fosmid library screening. pdmA abundance and expression increased concomitantly with IPU mineralization, verifying its major role in IPU transformation in the biobed system. DNA- and RNA-based 16S rRNA gene sequencing analysis showed no effects on bacterial diversity. The pdmAB-harboring plasmid pSH showed a consistently lower abundance than pdmA, suggesting the localization of pdmAB in replicons other than pSH. Metagenomic analysis identified four pdmAB-carrying fosmids. In three of these fosmids, the pdmAB genes were organized in a well-conserved operon carried by sphingomonad plasmids with low synteny with pSH, while the fourth fosmid contained an incomplete pdmAB cassette localized in a genomic fragment of a Rhodanobacter strain. Further analysis suggested a potentially crucial role of IS6 and IS256 in the transposition and activation of the pdmAB operon.IMPORTANCE Our study provides novel insights into the interactions of IPU with the bacterial community of biobed systems, reinforces the assumption of a transposable nature of IPU-degrading genes, and verifies that on-farm biobed systems are hot spots for the evolution of pesticide catabolic traits.

Expanding the use of biobeds: Degradation and adsorption of pesticides contained in effluents from seed-coating, bulb disinfestation and fruit-packaging activities.

Agro-food industries that use pesticides constitute significant point sources for the contamination of natural water resources. Despite that, little is known about the treatment of their pesticide-contaminated effluents. Biobeds could be a possible solution for the depuration of these effluents. In this context, we explored the degradation and adsorption of pesticides used in seed-coating (carboxin (CBX), metalaxyl-M (MET-M), fluxapyroxad (FLX), fludioxonil (FLD)), bulb-dipping (chlorothalonil (CHT), thiabendazole (TBZ), FLD) and fruit-packaging activities (FLD) in a biomixture, used as biobed packing material, and in soil. The degradation of pesticides was tested individually and in mixtures relevant to their industrial use, while FLD was also tested at different concentrations (10, 20, and 150 mg kg-1) representing its use in the different industries. CBX, FLD, and CHT, when applied individually, and all other pesticides when applied in mixtures, degraded more rapidly in biomixture than in soil. In most cases pesticides application in mixtures retarded their degradation. This was more pronounced in soil than in biomixture, especially for MET-M and FLD. CHT had the most prominent inhibitory effect on the degradation of TBZ and FLD. FLD degradation showed a dose-dependent pattern (DT50 42.4 days at 10 mg kg-1 and 107.6 days at 150 mg kg-1). All pesticides showed higher adsorption affinity in the biomixture (Kf = 3.23-123.3 g mL-1) compared to soil (Kf = 1.15-31.2 g mL-1). We provide initial evidence for the potential of the tested biomixture to remove pesticides contained in effluents produced by different agro-industrial activities. Tests in full-scale biobeds packed with this biomixture will unravel their full depuration potential for the treatment of these agro-industrial effluents.

Isolation of a bacterial consortium able to degrade the fungicide thiabendazole: the key role of a Sphingomonas phylotype.

Thiabendazole (TBZ) is a fungicide used in fruit-packaging plants. Its application leads to the production of wastewaters requiring detoxification. In the absence of efficient treatment methods, biological depuration of these effluents could be a viable alternative. However, nothing is known regarding the microbial degradation of the recalcitrant and toxic to aquatics TBZ. We report the isolation, via enrichment cultures from a polluted soil, of the first bacterial consortium able to rapidly degrade TBZ and use it as a carbon source. Repeated efforts using various culture-dependent approaches failed to isolate TBZ-degrading bacteria in axenic cultures. Denaturating gradient gel electrophoresis (DGGE) and cloning showed that the consortium was composed of α-, ß- and γ-Proteobacteria. Culture-independent methods including antibiotics-driven selection with DNA/RNA-DGGE, q-PCR and stable isotope probing (SIP)-DGGE identified a Sphingomonas phylotype (B13) as the key degrading member. Cross-feeding studies with structurally related chemicals showed that ring substituents of the benzimidazole moiety (thiazole or furan rings) favoured the cleavage of the imidazole moiety. LC-MS/MS analysis verified that TBZ degradation proceeds via cleavage of the imidazole moiety releasing thiazole-4-carboxamidine, which was not further transformed, and the benzoyl moiety, possibly as catechol, which was eventually consumed by the bacterial consortium as suggested by SIP-DGGE.

Characterization of the biodegradation, bioremediation and detoxification capacity of a bacterial consortium able to degrade the fungicide thiabendazole.

Thiabendazole (TBZ) is a persistent fungicide used in the post-harvest treatment of fruits. Its application results in the production of contaminated effluents which should be treated before their environmental discharge. In the absence of efficient treatment methods in place, biological systems based on microbial inocula with specialized degrading capacities against TBZ could be a feasible treatment approach. Only recently the first bacterial consortium able to rapidly transform TBZ was isolated. This study aimed to characterize its biodegradation, bioremediation and detoxification potential. The capacity of the consortium to mineralize 14C-benzyl-ring labelled TBZ was initially assessed. Subsequent tests evaluated its degradation capacity under various conditions (range of pH, temperatures and TBZ concentration levels) and relevant practical scenarios (simultaneous presence of other postharvest compounds) and its bioaugmentation potential in soils contaminated with increasing TBZ levels. Finally cytotoxicity assays explored its detoxification potential. The consortium effectively mineralized the benzoyl ring of the benzimidazole moiety of TBZ and degraded spillage level concentrations of the fungicide in aqueous cultures (750 mg L-1) and in soil (500 mg kg-1). It maintained its high degradation capacity in a wide range of pH (4.5-7.5) and temperatures (15-37 °C) and in the presence of other pesticides (ortho-phenylphenol and diphenylamine). Toxicity assays using the human liver cancer cell line HepG2 showed a progressive decrease in cytotoxicity, concomitantly with the biodegradation of TBZ, pointing to a detoxification process. Overall, the bacterial consortium showed high potential for future implementation in bioremediation and biodepuration applications.

Land Spreading of Wastewaters from the Fruit-Packaging Industry and Potential Effects on Soil Microbes: Effects of the Antioxidant Ethoxyquin and Its Metabolites on Ammonia Oxidizers.

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.

The potential of organic substrates based on mushroom substrate and straw to dissipate fungicides contained in effluents from the fruit-packaging industry - Is there a role for Pleurotus ostreatus?

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.

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.

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.

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.

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.

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.

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.

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.

Impact of fungicides on the diversity and function of non-target ammonia-oxidizing microorganisms residing in a litter soil cover.

Litter soil cover constitutes an important micro-ecosystem in sustainable viticulture having a key role in nutrient cycling and serving as a habitat of complex microbial communities. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are known to regulate nitrification in soil while little is known regarding their function and diversity in litter. We investigated the effects of two fungicides, penconazole and cyprodinil, commonly used in vineyards, on the function and diversity of total and active AOB and AOA in a microcosm study. Functional changes measured via potential nitrification and structural changes assessed via denaturating gradient gel electrophoresis (DGGE) at the DNA and RNA levels were contrasted with pesticide dissipation in the litter layer. The latter was inversely correlated with potential nitrification, which was temporarily inhibited at the initial sampling dates (0 to 21 days) when nearly 100 % of the applied pesticide amounts was still present in the litter. Fungicides induced changes in AOB and AOA communities with RNA-DGGE analysis showing a higher sensitivity. AOA were more responsive to pesticide application compared to AOB. Potential nitrification was less sensitive to the fungicides and was restored faster than structural changes, which persisted. These results support the theory of microbial redundancy for nitrification in a stressed litter environment.

On-farm biopurification systems for the depuration of pesticide wastewaters: recent biotechnological advances and future perspectives.

Point source contamination of natural water resources by pesticides constitutes a serious problem and on-farm biopurification systems (BPS) were introduced to resolve it. This paper reviews the processes and parameters controlling BPS depuration efficiency and reports on recent biotechnological advances which have been used for enhancing BPS performance. Biomixture composition and water management are the two factors which either individually or through their interactions control the depuration performance of BPS. Which process (biodegradation or adsorption) will dominate pesticides dissipation in BPS depends on biomixture composition and the physicochemical properties of the pesticides. Biotechnological interventions such as augmentation with pesticide-degrading microbes or pesticide-primed matrices have resulted in enhanced biodegradation performance of BPS. Despite all these advancement in BPS research, there are still several issues which should be resolved to facilitate their full implementation. Safe handling and disposal of the spent biomixture is a key practical issue which needs further research. The use of BPS for the depuration of wastewaters from post-farm activities such as postharvest treatment of fruits should be a priority research issue considering the lack of alternative treatment systems. However, the key point hampering optimization of BPS is the lack of fundamental knowledge on BPS microbiology. The use of advanced molecular and biochemical methods in BPS would shed light into this issue in the future.