Effects of Systemic Pesticides Imidacloprid and Metalaxyl on the Phyllosphere of Pepper Plants

Microbes inhabiting the phyllosphere of crops are exposed to pesticides applied either directly onto plant foliage or indirectly through soil. Although, phyllosphere microbiology has been rapidly evolving, little is still known regarding the impact of pesticides on the epiphytic microbial community and especially on fungi. We determined the impact of two systemic pesticides (metalaxyl and imidacloprid), applied either on foliage or through soil, on the epiphytic fungal and bacterial communities via DGGE and cloning. Both pesticides induced mild effects on the fungal and the bacterial communities. The only exception was the foliage application of imidacloprid which showed a more prominent effect on the fungal community. Cloning showed that the fungal community was dominated by putative plant pathogenic ascomycetes (Erysiphaceae and Cladosporium), while a few basidiomycetes were also present. The former ribotypes were not affected by pesticides application, while selected yeasts (Cryptococcus) were stimulated by the application of imidacloprid suggesting a potential role in its degradation. A less diverse bacterial community was identified in pepper plants. Metalaxyl stimulated an Enterobacteriaceae clone which is an indication of the involvement of members of this family in fungicide degradation. Further studies will focus on the isolation of epiphytic microbes which appear to be stimulated by pesticides application

Repeated application of diluted olive mill wastewater induces changes in the structure of the soil microbial community

Repeated application of diluted olive mill wastewater (OMW) is a common disposal method which allows the application of large amounts of OMW and improves the organic matter and nutrient status of Mediterranean soils. However, there is lack of information regarding the effects of this practice on the soil microbial community. A study was carried out to investigate these effects on the structure of the bacterial and fungal community of a loamy sand (LS) and a sandy loam (SL) soil, using denaturating gradient gel electrophoresis (DGGE) fingerprinting. OMW was daily applied as aqueous solutions of 0, 2 and 4%, in the presence or absence of nitrogen fertilization, for a three-month period. Multivariate analysis of the DGGE profiles showed that OMW applications resulted in marked changes in the fungal community in both soils, while nitrogen fertilization diminished these effects. Small effects were evident for the bacteria only in the LS soil and this was attributed to the higher availability of OMW-derived phenolics in this soil which resulted in a direct impact on bacteria. Nitrogen fertilization alleviated the effects of OMW on the bacterial community. We Suggest that the impact of OMW on the structure of the soil microbial communities was mostly a result of its indirect effect on the soil nutritional status, which becomes enriched in organic substrates and poor in available nitrogen. (C) 2009 Elsevier Masson SAS. All rights reserved

Functional metagenomics on biobed systems : Evolutionary mechanisms of known and isolation of novel pesticide biocatalytic enzymes

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Assessment of the impact of the fumigant dimethyl disulfide on the dynamics of major fungal plant pathogens in greenhouse soils

Soil fumigants constitute a major tool for the control of soil borne fungal plant pathogens in protected crops. Dimethyl disulfide (DMDS) is a novel soil fumigant used either alone or in combination with other fumigants for the control of soil borne pests and diseases. In a commercial greenhouse for tomato production, we evaluated the impact of DMDS, comparatively to the alternative fumigant metam sodium, on the population of the dominant fungal plant pathogens in the study soil via q-PCR. Prior to soil fumigation, estimation of the fungal diversity in the studied soil via clone libraries identified Fusarium oxysporum and Rhizoctonia solani as the most abundant soil borne plant pathogens, while Cladosporium spp., known as opportunistic airborne tomato pathogens, were the most dominant fungi and based on this their dynamics upon fumigation was also studied. DMDS, at two dose rates, induced a drastic reduction in the population of F. oxysporum and R. solani, which lasted for the whole cultivation season. On the contrary, metam sodium exhibited an inhibitory effect on F. oxysporum that was alleviated at 120 d post fumigation. Both DMDS and metam sodium induced only a temporal reduction in the soil population of Cladosporium sp. which recovered by 60 days post fumigation. Our data suggest that DMDS even at the low dose rate (56.4 g m−2) could drastically reduce the population of the major soil borne tomato pathogens F. oxysporum and R. solani. Establishment of population thresholds as determined by q-PCR could represent a valuable tool for the estimation of the risk for disease severity and crop yield losses. © 2016, Koninklijke Nederlandse Planteziektenkundige Vereniging

Olive mill wastewater affects the structure of soil bacterial communities

Land spreading of olive mill wastewater (OMW) has beets proposed as a useful means for its disposal. However, there is no information regarding the impact of OMW soil applications on the structure of bacterial communities. Two soils, a loamy sand (LS) and a sandy loam (SL), were irrigated weekly wills aqueous solutions of 2 and 4% of OMW for a period of 49 days. Fluctuations in key soil physical, chemical and biological properties were measured during the irrigation period. Structural changes in functional bacterial guilds such as actinobacteria and ammonia-oxidizing bacteria (AOB) were determined by denaturating gradient gel electrophoresis (DGGE) and cloning and sequencing. OMW application caused a drastic reduction in mineral-N in both soils indicating net N immobilization. An increase in soil electrical conductivity and a late peak in soil respiration and phenolics were observed only in the LS soil treated with 4% OMW. DGGE analysis showed a clear effect of OMW on the AOB communities in both soils, whereas OMW induced consistent changes in the structure of actinobacteria community in the LS soil only. The alterations in the bacterial communities induced by OMW application are potentially the result of environmental changes including lowered oxidative conditions, strong competition for mineral-N and availability of phenolics, whereas altered C-sources and selective inhibition of other microbial groups by available phenolics might have been particularly important for the actinobacteria. Cloning of the AOB community showed that all clone sequences representing dominant bands Us the DGGE profiles belonged to cluster 3 of Nitrosospira sp. Members of the community were favored in OMW-treated soils, while others were sensitive to OMW. Overall, OMW soil addition induces changes in soil physical and chemical properties and consequent alterations on the structure of the bacterial communities which were found to be soil-dependent. However these changes do not appear to relate to a general toxicity effect. (C) 2010 Elsevier B.V. All rights reserved

Metabolic and evolutionary insights in the transformation of diphenylamine by a Pseudomonas putida strain unravelled by genomic, proteomic, and transcription analysis

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. © 2018 Papadopoulou, Perruchon, Vasileiadis, Rousidou, Tanou, Samiotaki, Molassiotis and Karpouzas

Distribution and function of carbamate hydrolase genes cehA and mcd in soils: The distinct role of soil pH

Synthetic carbamates constitute a significant pesticide group with oxamyl being a leading compound in the nematicide market. Oxamyl degradation in soil is mainly microbially mediated. However, the distribution and function of carbamate hydrolase genes (cehA, mcd, cahA) associated with the soil biodegradation of carbamates is not yet clear. We studied oxamyl degradation in 16 soils from a potato monoculture area in Greece where oxamyl is regularly used. Oxamyl showed low persistence (DT50 2.4-26.7 days). q-PCR detected the cehA and mcd genes in 10 and three soils, respectively. The abundance of the cehA gene was positively correlated with pH, while both cehA abundance and pH were negatively correlated with oxamyl DT50. Amongst the carbamates used in the study region, oxamyl stimulated the abundance and expression only of the cehA gene, while carbofuran stimulated the abundance and expression of both genes. The cehA gene was also detected in pristine soils upon repeated treatments with oxamyl and carbofuran and only in soils with pH ≥7.2, where the most rapid degradation of oxamyl was observed. These results have major implications regarding the maintenance of carbamate hydrolase genes in soils, have practical implications regarding the agricultural use of carbamates, and provide insights into the evolution of cehA. © FEMS 2016. All rights reserve

The Impact of Biofumigation and Chemical Fumigation Methods on the Structure and Function of the Soil Microbial Community

Biofumigation (BIOF) is carried out mainly by the incorporation of brassica plant parts into the soil, and this fumigation activity has been linked to their high glucosinolate (GSL) content GSLs are hydrolyzed by the endogenous enzyme myrosinase to release isothiocyanates (ITCs). A microcosm study was conducted to investigate the effects induced on the soil microbial community by the incorporation of broccoli residues into soil either with (BM) or without (B) added myrosinase and of chemical fumigation, either as soil application of 2-phenylethyl ITC (PITC) or metham sodium (MS). Soil microbial activity was evaluated by measuring fluorescein diacetate hydrolysis and soil respiration. Effects on the structure of the total microbial community were assessed by phospholipid fatty acid analysis, while the impact on important fungal (ascomycetes (ASC)) and bacterial (ammonia-oxidizing bacteria (AOB)) guilds was evaluated by denaturating gradient gel electrophoresis (DGGE). Overall, B, and to a lesser extent BM, stimulated microbial activity and biomass. The diminished effect of BM compared to B was particularly evident in fungi and Gram-negative bacteria and was attributed to rapid ITC release following the myrosinase treatment. PITC did not have a significant effect, whereas an inhibitory effect was observed in the MS-treated soil. DGGE analysis showed that the ASC community was temporarily altered by BIOF treatments and more persistently by the MS treatment, while the structure of the AOB community was not affected by the treatments. Cloning of the ASC community showed that MS application had a deleterious effect on potential plant pathogens like Fusarium, Nectria, and Cladosporium compared to BIOF treatments which did not appear to inhibit them. Our findings indicate that BIOF induces changes on the structure and function of the soil microbial community that are mostly related to microbial substrate availability changes derived from the soil amendment with fresh organic materials

Metabolic pathway and cell adaptation mechanisms revealed through genomic, proteomic and transcription analysis of a Sphingomonas haloaromaticamans strain degrading ortho-phenylphenol

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. © 2017 The Author(s)

Effect of continuous olive mill wastewater applications, in the presence and absence of nitrogen fertilization, on the structure of rhizosphere-soil fungal communities

Olive mill wastewater (OMW) is rich in potentially toxic organics precluding its disposal into water receptors. However, land application of diluted OMW may result in safe disposal and fertilization. In order to investigate the effects of OMW on the structure of soil fungal groups, OMW was applied daily to pepper plants growing in a loamy sand and a sandy loam at two doses for a period of 3 months (total OMW equivalents 900 and 1800 m(3) ha(-1)). Nitrogen (N) fertilization alleviated N scarcity and considerably enhanced plant biomass production; however, when applied in combination with the high OMW dose, it induced plant stress. OMW applications resulted in marked changes in the denaturing gradient gel electrophoresis patterns of soil basidiomycete communities, while concurrent N fertilization reduced these effects. In contrast, the ascomycete communities required N fertilization to respond to OMW addition. Cloning libraries for the basidiomycete communities showed that Cryptococcus yeasts and Ceratobasidium spp. dominated in the samples treated with OMW. In contrast, certain plant pathogenic basidiomycetes such as Thanatephorus cucumeris and Athelia rolfsii were suppressed. The observed changes may be reasonably explained by the capacity of OMW to enrich soils in organic substrates, to induce N immobilization and to directly introduce OMW-derived basidiomycetous yeasts