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

Lab to Field Assessment of the Ecotoxicological Impact of Chlorpyrifos, Isoproturon, or Tebuconazole on the Diversity and Composition of the Soil Bacterial Community

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

Απομόνωση και μελέτη βακτηρίων ικανών να αποδομούν γεωργικά φάρμακα που περιέχονται στα υγρά απόβλητα από συσκευαστήρια φρούτων

Fruits are particularly vulnerable to fungal infestations or other physiological disorders during storage thus diminishing their market value. Post-harvest fruit treatments with fungicides like thiabendazole (TBZ), imazalil (IMZ), and ortho-phenyl-phenol (OPP), and antioxidants like diphenylamine (DPA) are the most effective means to minimize fruit spoilage in storage. Pesticide application methods in fruit-packaging plants are water-based generating at the end of the process large volumes of wastewaters containing high concentrations of pesticide. Direct discharge of these wastewaters into the environment without prior treatment constitutes a serious point-source of contamination. Therefore, a treatment system able to efficiently depurate these wastewaters is required. However, the only system actually patented (CONTROL TEC-ECO® system) is highly costly precluding its market uptake. Thus, a sustainable, cheap, effective, and environmentally friendly method for the depuration of the fruit packaging wastewater is needed. The development of biological treatment facilities based on the degrading ability of microorganisms seems promising. However, very little (OPP, DPA) or nothing (TBZ, IMZ) is known regarding the microbial degradation of those chemicals. Thus the main research aim of this thesis was to isolate and characterize bacteria able to degrade the major pesticides used in the fruit packaging industry (TBZ, IMZ, OPP, and DPA). Enrichment cultures from soils collected from wastewater disposal sites led to the isolation of TBZ, OPP, and DPA-degrading bacteria showing high potential for bioremediation applications, while, despite different attempts, it was not possible to isolate any bacterium able to degrade IMZ.Κατά την αποθηκευσής τους, τα φρούτα είναι ιδιαίτερα ευαίσθητα σε μυκητολογικές προσβολές ή άλλες φυσιολογικές διαταραχές, που περιορίζουν την εμπορικη τους αξία. Οι μετασυλλεκτικές εφαρμογές μυκητοκτόνων όπως τα thiabendazole (TBZ), imazalil (IMZ), και ortho-phenyl-phenol (OPP), αλλά και αντιοξειδωτικών όπως το diphenylamine (DPA) είναι ο πιο αποτελεσματικός τρόπος αντιμετώπισης ανάλογων προβλημάτων που παρουσιάζονται κατά την αποθήκευση. Οι μέθοδοι εφαρμογής των γεωργικών φαρμάκων που χρησιμοποιούνται στα συσκευαστήρια φρούτων βασίζονται στη χρήση νερού και οδηγούν στο τέλος της διαδικασίας στην παραγωγή μεγάλου όγκου υγρών αποβλήτων με υψηλές συγκεντρώσεις γεωργικών φαρμάκων. Η άμεση απόριψη των παραγόμενων υγρών αποβλήτων στο περιβάλλον, χωρίς προηγούμενη επεξεργασία, αποτελεί σοβαρό κίνδυνο σημειακής ρύπανσης. Ως εκ τούτου, απαιτείται η ύπαρξη ενός συστήματος διαχείρισης ικανού να αποτοξικοποιήσει τα υγρά απόβλητα που προέρχονται από τα συσκευαστήρια φρούτων. Ωστόσο, το μόνο κατοχυρωμένο με δίπλωμα ευρεσιτεχνίας σύστημα διαχείρισης υγρών αποβλήτων (CONTROL TEC-ECO® system) χαρακτηρίζεται από ιδιαίτερα υψηλό κόστος αποκλείοντας έτσι τη γενικευμένη χρήση του στην αγορά. Συνεπώς, υπάρχει ανάγκη για μια αειφόρο, φθηνή, αποτελεσματική και φιλική προς το περιβάλλον μέθοδο αποτοξικοποίησης των αποβλήτων που παράγονται από τα συσκευαστήρια φρούτων. Η ανάπτυξη βιολόγικών συστημάτων διαχείρισης με βάση την ικανότητα των μικροοργανισμών να διασπούν τα γεωργικά φάρμακα είναι μια πολλά υποσχόμενη προοπτική για το μέλλον. Ωστόσο, οι γνώσεις μας σχετικά με την μικροβιακή αποτοξικοποίηση των συγκεκριμένων γεωργικών φαρμάκων είναι ελάχιστες (OPP, DPA) έως μηδαμινές (TBZ, IMZ). Για το λόγο αυτό, βασικός ερευνητικός στόχος της παρούσας διδακτορικής διατριβής ήταν η απομόνωση και ο χαρακτηρισμός βακτηρίων ικανών να διασπούν τα κύρια γεωργικά φάρμακα που χρησιμοποιούνται στη βιομηχανία της συσκευασίας των φρούτων (TBZ, IMZ, OPP, and DPA). Εμπλουτισμένες καλλιέργειες από εδάφη που συλλέχθηκαν από σημεία όπου απορίπτονταν υγρά απόβλητα από συσκευαστήρια φρούτων οδήγησαν στην απομόνωση βακτηρίων ικανών να διασπούν ΤΒΖ, ΟΡΡ και DPA, αποδεικνύοντας υψηλό δυναμικό για εφαρμογή τους σε συστήματα αποτοξικοποίησης, ενώ παρά τις όποιες προσπάθειες δεν αποδείχτηκε δυνατή η απομόνωση κάποιου βακτηρίου ικανού να διασπάσει το ΙΜΖ

Functional metagenomic analysis of biobed systems: an invaluable source of genes for the degradation of pesticides

International audienc

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

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

National audienceBiobeds, designed to minimize pesticide point source contamination, mainly rely 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 qPCR and 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 mechanism of pdmAB in the biobed packing material by measuring the abundance of the plasmid pSH (harboring pdmAB) of the IPU-degrading Sphingomonas sp. SH strain (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 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 those, pdmAB were organized in a well-conserved operon carried by sphingomonads' plasmids with low synteny to pSH, while the fourth fosmid contained an incomplete pdmAB cassette localized in a genomic fragment of a Rhodanobacter strain. Further analysis suggested a potential crucial role of IS6 and IS256 in the transposition and activation of the pdmAB operon.Importance: Our study provides novel insights in 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 hotspots for the evolution of pesticide catabolic traits

DataSheet1.pdf

<p>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.</p