Chiara Perruchon

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.

Isolation and characterisation of a Sphingomonas strain able to degrade the fungicide ortho‐phenylphenol

BACKGROUND Ortho-phenylphenol (OPP) is a fungicide used in fruit packaging plants for the control of fungal infestations during storage. Its application leads to the production of large wastewater volumes which according to the European legislation should be treated on site. In spite of this, no efficient treatment systems are currently available, and the development of biological systems based on tailored-made pesticide-degrading inocula for the treatment of these wastewaters is an appealing solution. RESULTS Enrichment cultures from a soil collected from a wastewater disposal site resulted in the isolation of a pure Sphingomonas haloaromaticamans strain P3 able to degrade rapidly OPP and use it as an energy source. Its degrading capacity was dependent on the external supply of amino acids or on the presence of other bacteria that did not contribute to fungicide degradation. The isolated S. haloaromaticamans strain was able to metabolise up to 150 mg L(-1) of OPP within 7 days, in a wide range of pH (4.5-9) and temperatures (4-37 °C), and in the presence of other pesticides (thiabendazole and diphenylamine) co-used in the fruit packaging industry. CONCLUSION Overall, the OPP-degrading bacterium isolated showed high potential for use in future biodepuration treatment systems and bioremediation strategies.

Integrated biodepuration of pesticide-contaminated wastewaters from the fruit-packaging industry using biobeds: Bioaugmentation, risk assessment and optimized management

Wastewaters from fruit-packaging plants contain high loads of toxic and persistent pesticides and should be treated on site. We evaluated the depuration performance of five pilot biobeds against those effluents. In addition we tested bioaugmentation with bacterial inocula as a strategy for optimization of their depuration capacity. Finally we determined the composition and functional dynamics of the microbial community via q-PCR. Practical issues were also addressed including the risk associated with the direct environmental disposal of biobed-treated effluents and decontamination methods for the spent packing material. Biobeds showed high depuration capacity (>99.5%) against all pesticides with bioaugmentation maximizing their depuration performance against the persistent fungicide thiabendazole (TBZ). This was followed by a significant increase in the abundance of bacteria, fungi and of catabolic genes of aromatic compounds catA and pcaH. Bioaugmentation was the most potent decontamination method for spent packing material with composting being an effective alternative. Risk assessment based on practical scenarios (pome and citrus fruit-packaging plants) and the depuration performance of the pilot biobeds showed that discharge of the treated effluents into an 0.1-ha disposal site did not entail an environmental risk, except for TBZ-containing effluents where a larger disposal area (0.2ha) or bioaugmentation alleviated the risk.

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-, β- 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.

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.

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.